http://2007.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Alice.pasini&year=&month=2007.igem.org - User contributions [en]2024-03-29T09:46:09ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T21:03:56Z<p>Alice.pasini: /* Concluding Remarks */</p>
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"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
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"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
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"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
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"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
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''(Aforisma di Ippocrate di Coo)''<br />
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= About Us =<br />
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Welcome to Bologna’s IGEM Wiki!<br />
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[[Image:Teambodef.jpg|center]]<br />
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Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], PhD student in Biochemistry.<br />
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We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
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Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
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= Our Project=<br />
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==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
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'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main feature of this device is to be a “smart” switch: that means a switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the quantitative modification of the value that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
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[[Image:switch_1.jpg|center]]<br />
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Figure1.''The "stupid" switch.''<br />
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So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
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[[Image:switch_2.jpg|center]]<br />
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Figure2.''The "smart" switch.''<br />
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This kind of "smart” switch, the "Schmitt Switch", is largely used in technical applications since it overcomes the instability issue; in fact, the minimal variation able to cause a change in the output must be as large as the difference between the two thresholds. Then noise becomes not so critical.<br />
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[[Image:scaldabagno3.jpg|center]]<br />
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Figure 3.Typical application of the Schmitt Trigger: The boiler.'' Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature controls the heater that determines the temperature), it is able to automatically maintain a warm temperature: the temperature holds stable and so does your mood if you are in the bath tub.''<br />
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The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit aims to reproduce this fundamental property of Schmitt Trigger.<br />
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'''How?''' (AKA '''Technical Requirements''')<br \><br />
To do it we exploited one of such genetic systems, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, ''E. coli'' can survive by metabolizing either glucose or lactose - in the case of lack of glucose. Lactose- or glucose-metabolizing modes are the two stable state of the system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. It is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] constitutively shut off by the presence of the LacI protein, that inhibits the transcription of the downstream genes in the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When IPTG is in the environment, several concurrent processes take place in the cell. IPTG flows across the membrane and after some processing it is able to quench the repressor LacI, thus allowing the transcription of ''lacY'' gene that in turn enhances IPTG uptake and increases its action on the repressor LacI. This represents the positive feedback loop we need.<br />
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[[Image:schema1.jpg|center]]<br />
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Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
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As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac-driven gene expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
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'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
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'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the level of the gene expression.<br />
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'''2.''' <u>''Glucose Sensor''</u> <br \><br />
A second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
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[[Image:introduz.jpg|center]]<br />
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Figure 5. ''Schematic view of our device''<br />
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==Mathematical Model==<br />
Based on the our knowledge on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon] and on the works of Santillàn et al. (2004, 2007), we decided to explore the behavior of a possible synthetic circuit which mimics the genetic circuit by modeling the parts depicted in Figure 6.<br />
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[[Image:modellomath_def.jpg|center]]<br />
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Figure 6. ''The core of the synthetic circuit as modelled in the subsequent equations. P and T represent the Protein and the Transcript of the corresponding genes.''<br />
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===Model Equations===<br />
Let’s start with the state model equations. They represent the variation over time (derivative d/dt) of the intracellular level (in molecules per bacterium, mpb) of:<br />
:1. the transcript of lacY (T);<br />
:2. the membrane permease coded by LacY (P) <br />
:3. IPTG: this is an artificial Lactose equivalent that is un-metabolizable by the cell. This allows us to neglect the effects of the other genomic Lac Operon Genes.<br />
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[[Image:StateIng.jpg|center]]<br />
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These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex, to avoid [[Bologna University/Equations mess | biologist melancholy]].<br />
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The inputs of this system are the extracellular concentrations of Glucose and IPTG, that are processed by the non linear functions described in the table below.<br />
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{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
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! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
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! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
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! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
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! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
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|}<br />
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and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
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Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP: in first approximation, we simply assume:<br />
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[[Image:outputEqn.jpg|center]]<br />
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All the genomic parameters (from Santillàn et al, 2007) are defined [[Bologna_University/Literature Parameters | here]], while the parameters adopted to model the synthetic parts are defined in the table of [[Bologna University/SynthPar| Synthetic Parameters]].<br />
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===Numerical Simulation===<br />
To analyse the circuit’s behaviour under different stimulations we implemented model equations in [[Bologna University/simulink | simulink]] (Mathworks). As model input we impose a periodic change in the extracellular IPTG levels (slow enough to assume that the system is near to the equilibrium for each IPTG value) while Glucose is kept constant. <br />
Several simulations were run by assigning a different value to Glucose. The results are shown in Figure 7: the system shows hysteresis, distinct thresholds depending on the Glucose and, once induced, constant level of expression. Notably, the circuit shows bistability (two stable equilibria) for IPTG values between the thresholds. <br />
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[[Image:Iptg_Gfp3.jpg|center]]<br />
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Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
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Dependence of IPTG thresholds on Glucose are shown in Figure 8: the points that lie between the thresholds are values (of the inputs) at which the system exhibit bistability; that is, we are not able of predict the final state with no information on the initial state. Outside these values, system reaches without any doubts, steady induced or uninduced state.<br />
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[[Image:Stability_plane_def.jpg|center]]<br />
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Figure 8. ''Shows how the 2 thresholds value changes for varying levels of external glucose. All the points that lie between the two curves represent states for which the trascription can occur or not, the points below the blue curve correspond to pLac full repression (trascription off) while points over the red curve correpond to a condition where Plac is partially repressed (trascription on).''<br />
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When the higher threshold is crossed transcripion takes place with an expression level unsensitive to the ITPG stimulus (see Figure 7). In this condition (i.e. IPTG 900 microM), the saturation level depends on the Glucose (see figure 8), and the circuit acts like a glucose sensor.<br />
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[[Image:grafico3.jpg|center]]<br />
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Figure 9.''Shows that the final level of GFP concentration (output of the system) in the fully induced steady state depends on the External Glucose.''<br />
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In conclusion, numerical simulation predicts a circuit functionality which is coherent with our initial speculation, i.e. the circuit presented in Figure 6 can operate like a [[#Introduction | Schmitt Trigger]]. <br />
The values of few parameters were assigned with some uncertainty. In particular, we used a value of LacI that is about 30 times the genomic one for a circuit copy number of 10. Since these values can affect the model prediction we will perform specific experiment to identify this parameters. However, the LacI values was incresed in order to trigger off the system for low values of IPTG. To realize this increase we decided to build a sub-circuit where the LacI gene is under the control of pTet promoter. <br />
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At the same way, the GFP steady state expression decreases with extracellular Glucose (see Figure 9), while we want, for an intuitive use of the sensor a fluorescent signal that increases with Glucose. To obtain this behavior we plan to build a sub-circuit that acts like an inverter, with an RFP under the control of pλ, and to put in the main circuit the cI gene which encode for the protein that regulates pλ.<br />
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==Image Acquisition and Analysis==<br />
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===Image Acquisition System===<br />
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[[Image:Microscopy.jpg|center]]<br />
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Figure 10. Image acquisition system based on the fluorescence microscope.<br />
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For complete description of Acquisition System, see [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here].<br />
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===Image Acquisition===<br />
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Using the experimental set up, we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
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[[Image:batteri1.jpg|center]]<br />
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Figure 11.''Fluorescent bacteria''<br />
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===Image Elaboration===<br />
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Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
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Here are the processing phases:<br />
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*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
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[[Image:batteri2.jpg|center]]<br />
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Figure 12.''Algorithm output.''<br />
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*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
1) ''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
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2) ''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
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This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
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''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
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==Biodevice==<br />
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===Components===<br />
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The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
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[[Image:Tutto3.jpg]]<br />
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''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
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:1.<u>pTetR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for ''LacI'' gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTetR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTetR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of ''LacI'' gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
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:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
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:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
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===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
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[[Image:no_IPTG.jpg]]<br />
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''Figure 14: Project in action: no IPTG in the medium.''<br />
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For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and ''cI'', ''LacY'' and ''GFP'' genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
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[[Image:con_IPTG1.jpg]]<br />
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''Figure 15: Project in action: IPTG in the medium.''<br />
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===Intermediates===<br />
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The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
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-in the first column the link to the Registry;<br />
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-in the second column the components for each part;<br />
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-in the third column the plasmid the part has been cloned in;<br />
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-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
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-in the fifth column cell vitality after transformation;<br />
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-in the sixth column fluo data for each part.<br />
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{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
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| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
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| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
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| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and Pst1 enzymes.<br />
<br />
<br />
<br />
==Components Characterization==<br />
<br />
===Fluorescence intensity dependence on Promoter type===<br />
<br />
The three promoters used in the biodevice ([http://partsregistry.org/Part:BBa_I763004 I763004], [http://partsregistry.org/Part:BBa_I763035 I763035] and [http://partsregistry.org/Part:BBa_I763011 I763011]) have been separately characterized in order to gain useful information for the model. We chose to characterize the behavior of promoters escluding the LacY positive feedback.<br />
<br />
First, the three different intermediate parts have been tested in LB medium to stabilish differences in transcription rates. All of the three intermediates have GFP protein as a reporter to avoid difference in protein synthetis and degradation.<br />
<br />
Mesuarements have been performed at OD value of 1.2. We have taken pictures of two fields from each of the five slides we prepared; then, we calculated the mean value for each slide and finally the mean and the standard deviation on these values. <br />
Results are in figure below:<br />
<br />
[[Image:Istogramma1.jpg|center]]<br />
<br />
<br />
<br />
Fluorescence intensity levels for [http://partsregistry.org/Part:BBa_I763004 I763004] and [http://partsregistry.org/Part:BBa_I763035 I763035] are similar, while [http://partsregistry.org/Part:BBa_I763011 I763011] beams a significantely lower fluorescence (30%).<br />
<br />
<br />
Experiments have been repeated with bacteria grown into a different culture medium (M9 medium), that is the one chosen for testing the Genetic Schmitt Trigger. <br />
Results are in figure below:<br />
<br />
<br />
[[Image:Istogramma21.jpg|center]]<br />
<br />
<br />
In this case, the three promoters produce levels of fluorescence significantely different being pTet the one with the highest transcription activity.<br />
<br />
===Comparison between fluorescence elicited by the two media===<br />
<br />
To highlight the differences between the two media, we have directely compared for each intermediate fluorescence intensity in the two media.<br />
The comparison is shown in figure below:<br />
<br />
[[Image:Istogramma3333.jpg|center]]<br />
<br />
pλ promoter seems to not been influenced by the growth in different media. As expected, pLac promoter seems to have a higher transcription rate in a medium not added with carbon sources (M9 minimal medium). This result strectly agree with the prediction obteined by our mathematical model (20% experimental vs 23% simulation). Surprisingly, pTet promoter increases its activity changing the medium. We are not able to justify this result. Anyway, our mathematical model does not include such dependence and this aspect deserve to be investigated to be clarify.<br />
<br />
<br />
Complete data set, summarized in the table below, is [[Bologna University/Fluo results (Promoter - Medium) | here]].<br />
<br />
<br />
<br />
{| border="0" cellpadding="5" cellspacing="1" align="center"<br />
|-<br />
! style="background:white;" | <br />
! style="background:white;" |<br />
! style="background:#ffdead;" | LB<br />
! style="background:#ffdead;" | M9<br />
|-<br />
| style="background:#ffdead;" | I763004<br />
| style="background:#ffdead;" | pLac-GFP<br />
| style="background:#ffefbe;" | 102.9 ± 13.4<br />
| style="background:#ffefbe;" | 123.2 ± 13.1<br />
|-<br />
| style="background:#ffdead;" | I763035<br />
| style="background:#ffdead;" | pTetR-LacI-GFP<br />
| style="background:#ffefbe;" | 106.6 ± 6.0<br />
| style="background:#ffefbe;" | 138.1 ± 7.7<br />
|-<br />
| style="background:#ffdead;" | I763011<br />
| style="background:#ffdead;" | pλ-GFP<br />
| style="background:#ffefbe;" | 71.6 ± 12.3<br />
| style="background:#ffefbe;" | 81.3 ± 13.9<br />
|-<br />
|}<br />
<br />
===Switch Time Course===<br />
<br />
We also tested the intermediate device pTet-LacI-pLac-GFP [http://partsregistry.org/Part:BBa_I763027 (I763027)] in a high copy plasmid [http://partsregistry.org/Part:pSB1A2 pSB1A2] to verify if it really works as a toggle switch.In this intermediate device only LacY is not included since we would study transcriptional activity without the positive feedback due to the lactose permease.<br />
<br />
Following the [[Bologna University/Fluorescence Test | Fluorescence Test protocol]], bacteria grew in LB medium O/N. The day after we tested output fluorescence intensity in M9 with 1M glucose. We induced them with two different IPTG concentrations (2mM and 8mM) to activate pLac promoter. We measured output fluorescence intensity at regular time intervals and we verified that it incresed esponentially. After 90 minutes we eliminated IPTG induction resuspendig the cells in M9 medium with 1M glucose and we verified that fluorescence output intensity decreased quickly to the initial level.<br />
We could not measure fluorescence output intensity of cells in 8mM IPTG at 140 minutes because cells died.<br />
<br />
Our device seems to work as expected like a switch as the mathematical model predicted.<br />
<br />
<br />
<br />
[[Image:curvetempi2.jpg|center]]<br />
<br />
=Concluding Remarks=<br />
<br />
Despite our preliminar results on the intermediates are confident with the initial device design, a lot of work still remains to do in order to obtain a functional Genetic Schmitt Trigger.<br />
<br />
First of all, we need to insert LacY permease gene downstream pLac promoter. [http://partsregistry.org/Part:BBa_I763032:Experience Preliminar tests] on this part have shown that we can not have a full control of LacY production after IPTG induction.<br />
<br />
Second one, our complete biodevice includes LacI gene under the control of pTet promoter in order to have a sufficient production of the repressor that should fully repress pLac transcription. Our results on the intermediate pTet promoter might jeopardize this choice. <br />
So, we need to reproduce our experiments and to add this aspect in the model in order to verify that hysteresis persists.<br />
<br />
=Acknowledgements=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:[[#Biodevice | '''Biodevice''']]<br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image acquisition and analysis | Image Acquisition and Analysis]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University#Fluorescence intensity dependence on Promoter type | Fluorescence reliance on Promoter and Medium]]<br />
::3. [[#Components Characterization | Components Characterization]]<br />
:[[#Concluding Remarks | '''Concluding Remarks''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T20:52:15Z<p>Alice.pasini: /* Comparison between fluorescence elicited by the two media */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main feature of this device is to be a “smart” switch: that means a switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the quantitative modification of the value that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technical applications since it overcomes the instability issue; in fact, the minimal variation able to cause a change in the output must be as large as the difference between the two thresholds. Then noise becomes not so critical.<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.'' Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature controls the heater that determines the temperature), it is able to automatically maintain a warm temperature: the temperature holds stable and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit aims to reproduce this fundamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
To do it we exploited one of such genetic systems, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, ''E. coli'' can survive by metabolizing either glucose or lactose - in the case of lack of glucose. Lactose- or glucose-metabolizing modes are the two stable state of the system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. It is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] constitutively shut off by the presence of the LacI protein, that inhibits the transcription of the downstream genes in the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When IPTG is in the environment, several concurrent processes take place in the cell. IPTG flows across the membrane and after some processing it is able to quench the repressor LacI, thus allowing the transcription of ''lacY'' gene that in turn enhances IPTG uptake and increases its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
<br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac-driven gene expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the level of the gene expression.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
A second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the our knowledge on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon] and on the works of Santillàn et al. (2004, 2007), we decided to explore the behavior of a possible synthetic circuit which mimics the genetic circuit by modeling the parts depicted in Figure 6.<br />
<br />
[[Image:modellomath_def.jpg|center]]<br />
<br />
Figure 6. ''The core of the synthetic circuit as modelled in the subsequent equations. P and T represent the Protein and the Transcript of the corresponding genes.''<br />
<br />
<br />
===Model Equations===<br />
Let’s start with the state model equations. They represent the variation over time (derivative d/dt) of the intracellular level (in molecules per bacterium, mpb) of:<br />
:1. the transcript of lacY (T);<br />
:2. the membrane permease coded by LacY (P) <br />
:3. IPTG: this is an artificial Lactose equivalent that is un-metabolizable by the cell. This allows us to neglect the effects of the other genomic Lac Operon Genes.<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex, to avoid [[Bologna University/Equations mess | biologist melancholy]].<br />
<br />
The inputs of this system are the extracellular concentrations of Glucose and IPTG, that are processed by the non linear functions described in the table below.<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP: in first approximation, we simply assume:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the genomic parameters (from Santillàn et al, 2007) are defined [[Bologna_University/Literature Parameters | here]], while the parameters adopted to model the synthetic parts are defined in the table of [[Bologna University/SynthPar| Synthetic Parameters]].<br />
<br />
===Numerical Simulation===<br />
To analyse the circuit’s behaviour under different stimulations we implemented model equations in [[Bologna University/simulink | simulink]] (Mathworks). As model input we impose a periodic change in the extracellular IPTG levels (slow enough to assume that the system is near to the equilibrium for each IPTG value) while Glucose is kept constant. <br />
Several simulations were run by assigning a different value to Glucose. The results are shown in Figure 7: the system shows hysteresis, distinct thresholds depending on the Glucose and, once induced, constant level of expression. Notably, the circuit shows bistability (two stable equilibria) for IPTG values between the thresholds. <br />
<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
Dependence of IPTG thresholds on Glucose are shown in Figure 8: the points that lie between the thresholds are values (of the inputs) at which the system exhibit bistability; that is, we are not able of predict the final state with no information on the initial state. Outside these values, system reaches without any doubts, steady induced or uninduced state.<br />
<br />
<br />
[[Image:Stability_plane_def.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds value changes for varying levels of external glucose. All the points that lie between the two curves represent states for which the trascription can occur or not, the points below the blue curve correspond to pLac full repression (trascription off) while points over the red curve correpond to a condition where Plac is partially repressed (trascription on).''<br />
<br />
<br />
When the higher threshold is crossed transcripion takes place with an expression level unsensitive to the ITPG stimulus (see Figure 7). In this condition (i.e. IPTG 900 microM), the saturation level depends on the Glucose (see figure 8), and the circuit acts like a glucose sensor.<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows that the final level of GFP concentration (output of the system) in the fully induced steady state depends on the External Glucose.''<br />
<br />
<br />
In conclusion, numerical simulation predicts a circuit functionality which is coherent with our initial speculation, i.e. the circuit presented in Figure 6 can operate like a [[#Introduction | Schmitt Trigger]]. <br />
The values of few parameters were assigned with some uncertainty. In particular, we used a value of LacI that is about 30 times the genomic one for a circuit copy number of 10. Since these values can affect the model prediction we will perform specific experiment to identify this parameters. However, the LacI values was incresed in order to trigger off the system for low values of IPTG. To realize this increase we decided to build a sub-circuit where the LacI gene is under the control of pTet promoter. <br />
<br />
At the same way, the GFP steady state expression decreases with extracellular Glucose (see Figure 9), while we want, for an intuitive use of the sensor a fluorescent signal that increases with Glucose. To obtain this behavior we plan to build a sub-circuit that acts like an inverter, with an RFP under the control of pλ, and to put in the main circuit the cI gene which encode for the protein that regulates pλ.<br />
<br />
==Image Acquisition and Analysis==<br />
<br />
===Image Acquisition System===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
<br />
<br />
For complete description of Acquisition System, see [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here].<br />
<br />
<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up, we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
<br />
[[Image:batteri1.jpg|center]]<br />
<br />
Figure 11.''Fluorescent bacteria''<br />
<br />
<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:batteri2.jpg|center]]<br />
<br />
<br />
Figure 12.''Algorithm output.''<br />
<br />
<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
1) ''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
<br />
2) ''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
<br />
:1.<u>pTetR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for ''LacI'' gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTetR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTetR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of ''LacI'' gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and ''cI'', ''LacY'' and ''GFP'' genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and Pst1 enzymes.<br />
<br />
<br />
<br />
==Components Characterization==<br />
<br />
===Fluorescence intensity dependence on Promoter type===<br />
<br />
The three promoters used in the biodevice ([http://partsregistry.org/Part:BBa_I763004 I763004], [http://partsregistry.org/Part:BBa_I763035 I763035] and [http://partsregistry.org/Part:BBa_I763011 I763011]) have been separately characterized in order to gain useful information for the model. We chose to characterize the behavior of promoters escluding the LacY positive feedback.<br />
<br />
First, the three different intermediate parts have been tested in LB medium to stabilish differences in transcription rates. All of the three intermediates have GFP protein as a reporter to avoid difference in protein synthetis and degradation.<br />
<br />
Mesuarements have been performed at OD value of 1.2. We have taken pictures of two fields from each of the five slides we prepared; then, we calculated the mean value for each slide and finally the mean and the standard deviation on these values. <br />
Results are in figure below:<br />
<br />
[[Image:Istogramma1.jpg|center]]<br />
<br />
<br />
<br />
Fluorescence intensity levels for [http://partsregistry.org/Part:BBa_I763004 I763004] and [http://partsregistry.org/Part:BBa_I763035 I763035] are similar, while [http://partsregistry.org/Part:BBa_I763011 I763011] beams a significantely lower fluorescence (30%).<br />
<br />
<br />
Experiments have been repeated with bacteria grown into a different culture medium (M9 medium), that is the one chosen for testing the Genetic Schmitt Trigger. <br />
Results are in figure below:<br />
<br />
<br />
[[Image:Istogramma21.jpg|center]]<br />
<br />
<br />
In this case, the three promoters produce levels of fluorescence significantely different being pTet the one with the highest transcription activity.<br />
<br />
===Comparison between fluorescence elicited by the two media===<br />
<br />
To highlight the differences between the two media, we have directely compared for each intermediate fluorescence intensity in the two media.<br />
The comparison is shown in figure below:<br />
<br />
[[Image:Istogramma3333.jpg|center]]<br />
<br />
pλ promoter seems to not been influenced by the growth in different media. As expected, pLac promoter seems to have a higher transcription rate in a medium not added with carbon sources (M9 minimal medium). This result strectly agree with the prediction obteined by our mathematical model (20% experimental vs 23% simulation). Surprisingly, pTet promoter increases its activity changing the medium. We are not able to justify this result. Anyway, our mathematical model does not include such dependence and this aspect deserve to be investigated to be clarify.<br />
<br />
<br />
Complete data set, summarized in the table below, is [[Bologna University/Fluo results (Promoter - Medium) | here]].<br />
<br />
<br />
<br />
{| border="0" cellpadding="5" cellspacing="1" align="center"<br />
|-<br />
! style="background:white;" | <br />
! style="background:white;" |<br />
! style="background:#ffdead;" | LB<br />
! style="background:#ffdead;" | M9<br />
|-<br />
| style="background:#ffdead;" | I763004<br />
| style="background:#ffdead;" | pLac-GFP<br />
| style="background:#ffefbe;" | 102.9 ± 13.4<br />
| style="background:#ffefbe;" | 123.2 ± 13.1<br />
|-<br />
| style="background:#ffdead;" | I763035<br />
| style="background:#ffdead;" | pTetR-LacI-GFP<br />
| style="background:#ffefbe;" | 106.6 ± 6.0<br />
| style="background:#ffefbe;" | 138.1 ± 7.7<br />
|-<br />
| style="background:#ffdead;" | I763011<br />
| style="background:#ffdead;" | pλ-GFP<br />
| style="background:#ffefbe;" | 71.6 ± 12.3<br />
| style="background:#ffefbe;" | 81.3 ± 13.9<br />
|-<br />
|}<br />
<br />
===Switch Time Course===<br />
<br />
We also tested the intermediate device pTet-LacI-pLac-GFP [http://partsregistry.org/Part:BBa_I763027 (I763027)] in a high copy plasmid [http://partsregistry.org/Part:pSB1A2 pSB1A2] to verify if it really works as a toggle switch.In this intermediate device only LacY is not included since we would study transcriptional activity without the positive feedback due to the lactose permease.<br />
<br />
Following the [[Bologna University/Fluorescence Test | Fluorescence Test protocol]], bacteria grew in LB medium O/N. The day after we tested output fluorescence intensity in M9 with 1M glucose. We induced them with two different IPTG concentrations (2mM and 8mM) to activate pLac promoter. We measured output fluorescence intensity at regular time intervals and we verified that it incresed esponentially. After 90 minutes we eliminated IPTG induction resuspendig the cells in M9 medium with 1M glucose and we verified that fluorescence output intensity decreased quickly to the initial level.<br />
We could not measure fluorescence output intensity of cells in 8mM IPTG at 140 minutes because cells died.<br />
<br />
Our device seems to work as expected like a switch as the mathematical model predicted.<br />
<br />
<br />
<br />
[[Image:curvetempi2.jpg|center]]<br />
<br />
=Concluding Remarks=<br />
<br />
<br />
=Acknowledgements=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:[[#Biodevice | '''Biodevice''']]<br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image acquisition and analysis | Image Acquisition and Analysis]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University#Fluorescence intensity dependence on Promoter type | Fluorescence reliance on Promoter and Medium]]<br />
::3. [[#Components Characterization | Components Characterization]]<br />
:[[#Concluding Remarks | '''Concluding Remarks''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/BolognaBologna2007-10-26T20:51:34Z<p>Alice.pasini: /* Switch Time Course */</p>
<hr />
<div>[[Image:BOLOGNA.jpg]]<br />
<br />
<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
<br />
<br />
<br />
<br />
<br />
<br />
= About Us =<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
[[Image:Teambodef.jpg|center]]<br />
<br />
<br />
Our team: [[Bologna_University/FB | Francesca Buganè]], [[Bologna_University/MM| Michela Mirri]], [[Bologna_University/FP | Francesco Pasqualini]], and [[Bologna_University/ST | Silvia Tamarri]], all undergraduate students in Biomedical Engineering; [[Bologna_University/GC | Guido Costa]], undergraduate student in Electronic Engineering; [[Bologna_University/IB | Iros Barozzi]], undergraduate student in Industrial Biotechnology and Bioinformatics and [[Bologna_University/AP | Alice Pasini]], PhD student in Biochemistry.<br />
<br />
We are advised by: [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr Emanuele Giordano], Lecturer in Biochemistry and [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Electronics. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are: [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Bioengineering; [[Bologna_University/FC | Francesca Ceroni]], BiotechD and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Electronic Engineering .<br />
<br />
= Our Project=<br />
<br />
==Introduction==<br />
Our goal is the realization of a genetic circuit able to implement the functionality typical of an electronic device called Schmitt Trigger (as defined by its inventor [http://www.otto-schmitt.org/ Dr Otto H. Schmitt]).<br />
<br />
<br />
<br />
<br />
<br />
'''Pardon me, what do you mean?''' (AKA '''Functional Requirements''') <br \> <br />
The main feature of this device is to be a “smart” switch: that means a switch with memory.<br />
In a "stupid" switch when the input (some environmental condition) crosses a certain threshold the output (some switch properties) changes, for instance from on to off. Often the environmental change is the quantitative modification of the value that describes the environment (temperature, pressure, pH, ect). The "stupid" device switches just for a given value of the input (threshold).<br />
<br />
[[Image:switch_1.jpg|center]]<br />
<br />
Figure1.''The "stupid" switch.''<br />
<br />
<br />
So far so good, however, if the switch input has a value that continually even ''minimally'' changes across the threshold, the device will keep going on and off, wasting energy and leaving the system in an unstable state.<br />
To avoid all this we need a “smart” switch. Basically, this device switches on or off at two different thresholds (High and Low thresholds called ''Ton'' and ''Toff'' respectively) depending on the history of the system. So, according to the state of the device, the threshold for switching will change. <br />
<br />
<br />
[[Image:switch_2.jpg|center]]<br />
<br />
Figure2.''The "smart" switch.''<br />
<br />
<br />
This kind of "smart” switch, the "Schmitt Switch", is largely used in technical applications since it overcomes the instability issue; in fact, the minimal variation able to cause a change in the output must be as large as the difference between the two thresholds. Then noise becomes not so critical.<br />
<br />
[[Image:scaldabagno3.jpg|center]]<br />
<br />
Figure 3.Typical application of the Schmitt Trigger: The boiler.'' Once the water temperature passes the higher threshold, boiler turn off until temperature crosses the lower threshold. Since this system work in closed loop (temperature controls the heater that determines the temperature), it is able to automatically maintain a warm temperature: the temperature holds stable and so does your mood if you are in the bath tub.''<br />
<br />
<br />
The reason why the “smart” switch works that well is due to its [http://en.wikipedia.org/wiki/Hysteresis hysteresis] properties. <br />
Our genetic circuit aims to reproduce this fundamental property of Schmitt Trigger.<br />
<br />
<br />
'''How?''' (AKA '''Technical Requirements''')<br \><br />
To do it we exploited one of such genetic systems, existing in the complex of genes that form the Lac Operon shown in Figure 4.<br />
Namely, ''E. coli'' can survive by metabolizing either glucose or lactose - in the case of lack of glucose. Lactose- or glucose-metabolizing modes are the two stable state of the system. To perform experiments we use IPTG a structural analog of lactose that cannot be metabolized. Thus the input of our system are the external concentrations of Glucose and IPTG (Gluex and IPTGex respectively).<br />
Since lactose metabolism is more energy consuming, usually all the apparatus that takes care of the lactose metabolism is repressed. It is [http://en.wikipedia.org/wiki/Lac_operon the promoter pLac ] constitutively shut off by the presence of the LacI protein, that inhibits the transcription of the downstream genes in the operon ([http://bcs.whfreeman.com/mga2e/pages/bcs-main.asp?s=132&n=003&i=283&v=&o=&ns=0&uid=0&rau=0 this] is how it works). When IPTG is in the environment, several concurrent processes take place in the cell. IPTG flows across the membrane and after some processing it is able to quench the repressor LacI, thus allowing the transcription of ''lacY'' gene that in turn enhances IPTG uptake and increases its action on the repressor LacI. This represents the positive feedback loop we need.<br />
<br />
[[Image:schema1.jpg|center]]<br />
<br />
Figure 4. ''Schematic overview of the Lac operon.''<br\><br />
<br />
<br />
<br />
As it is the system can already perform like a Schmitt trigger, for certain values of the inputs. However there is another mechanism that allows the fine tuning of the system: glucose has a double action both on a molecule called cAMP that it bounds to, inhibiting its action on pLac-driven gene expression (catabolite repression) and on the lactose, since it reduces its uptake (inducer exclusion).<br />
<br />
<br />
'''What for?'''(AKA '''Applications''')<br \><br />
We figured that such a system could be useful for two main purposes.<br />
<br />
'''1.''' <u>''Controlled Gene Expression Trigger''</u> <br \><br />
Manipulating gene expression using most of the current protocols in molecular biology usually relay either on a strong overexpression of a protein or on its total silencing. Once induced, both these two extreme perturbations are generally unmodifiable (so that this approaches, although shedding light on the general role of the candidate gene, are rough indicators of the effect of its real physiological transcriptional level). Our genetic device can limit these drawbacks offering 1) a controlled on-off transition of the protein expression at will (by dosing the extracellular IPTG input) and 2) its graded transcriptional level (playing with the extracellular Glucose concentration), representing a powerful tool for biologists. Moreover, in addition to the ability to control the timing and the extent of the induction, our device has the additional advantage to guarantee that small changes in the extracellular concentration of the inducer IPTG would not affect the stability of the level of the gene expression.<br />
<br />
'''2.''' <u>''Glucose Sensor''</u> <br \><br />
A second application takes advantage of the external glucose dependence. In fact, we know that the expression of the system is stable, but its intensity depends on the glucose. We can reverse the perspective and observe the intensity of the expression to infer the abundance of glucose in the medium.<br />
<br />
[[Image:introduz.jpg|center]]<br />
<br />
Figure 5. ''Schematic view of our device''<br />
<br />
==Mathematical Model==<br />
Based on the our knowledge on the [http://en.wikipedia.org/wiki/Lac_operon Lac operon] and on the works of Santillàn et al. (2004, 2007), we decided to explore the behavior of a possible synthetic circuit which mimics the genetic circuit by modeling the parts depicted in Figure 6.<br />
<br />
[[Image:modellomath_def.jpg|center]]<br />
<br />
Figure 6. ''The core of the synthetic circuit as modelled in the subsequent equations. P and T represent the Protein and the Transcript of the corresponding genes.''<br />
<br />
<br />
===Model Equations===<br />
Let’s start with the state model equations. They represent the variation over time (derivative d/dt) of the intracellular level (in molecules per bacterium, mpb) of:<br />
:1. the transcript of lacY (T);<br />
:2. the membrane permease coded by LacY (P) <br />
:3. IPTG: this is an artificial Lactose equivalent that is un-metabolizable by the cell. This allows us to neglect the effects of the other genomic Lac Operon Genes.<br />
<br />
[[Image:StateIng.jpg|center]]<br />
<br />
<br />
These equations are sufficient to model the dynamics relevant to the functioning of the system and nor overly complex, to avoid [[Bologna University/Equations mess | biologist melancholy]].<br />
<br />
The inputs of this system are the extracellular concentrations of Glucose and IPTG, that are processed by the non linear functions described in the table below.<br />
<br />
{| border="1" cellpadding="5" cellspacing="0" align="center"<br />
|-<br />
! style="background:white;" | [[Image:Beta1.jpg]]<br />
| style="background:white;" | Glucose Dependent LacY Transcription Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot1 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta2.jpg]]<br />
| style="background:white;" | IPTG Dependent LacY Transcription Enhancer<br />
| style="background:white;" | [[Bologna University/Plot2 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta3.jpg]]<br />
| style="background:white;" | Glucose Dependent IPTG Uptake Inhibitor<br />
| style="background:white;" | [[Bologna University/Plot3 | Plot]]<br />
|-<br />
! style="background:white;" | [[Image:Beta4.jpg]]<br />
| style="background:white;" | IPTG Dependent IPTG Uptake Enhancer<br />
| style="background:white;" | [[Bologna University/Plot4 | Plot]]<br />
|-<br />
|}<br />
<br />
<br />
<br />
<br />
and have their own [[Bologna University/formulas | formulas]] and [[Bologna University/trends | trends]].<br />
<br />
Since our output is the reporter protein GFP we need another equation to link the protein LacY (P) to GFP: in first approximation, we simply assume:<br />
<br />
[[Image:outputEqn.jpg|center]]<br />
<br />
All the genomic parameters (from Santillàn et al, 2007) are defined [[Bologna_University/Literature Parameters | here]], while the parameters adopted to model the synthetic parts are defined in the table of [[Bologna University/SynthPar| Synthetic Parameters]].<br />
<br />
===Numerical Simulation===<br />
To analyse the circuit’s behaviour under different stimulations we implemented model equations in [[Bologna University/simulink | simulink]] (Mathworks). As model input we impose a periodic change in the extracellular IPTG levels (slow enough to assume that the system is near to the equilibrium for each IPTG value) while Glucose is kept constant. <br />
Several simulations were run by assigning a different value to Glucose. The results are shown in Figure 7: the system shows hysteresis, distinct thresholds depending on the Glucose and, once induced, constant level of expression. Notably, the circuit shows bistability (two stable equilibria) for IPTG values between the thresholds. <br />
<br />
<br />
<br />
[[Image:Iptg_Gfp3.jpg|center]]<br />
<br />
Figure 7. ''Shows the typical hysteresis profile, different curves are due to different values of the external Glucose.''<br />
<br />
<br />
Dependence of IPTG thresholds on Glucose are shown in Figure 8: the points that lie between the thresholds are values (of the inputs) at which the system exhibit bistability; that is, we are not able of predict the final state with no information on the initial state. Outside these values, system reaches without any doubts, steady induced or uninduced state.<br />
<br />
<br />
[[Image:Stability_plane_def.jpg|center]]<br />
<br />
Figure 8. ''Shows how the 2 thresholds value changes for varying levels of external glucose. All the points that lie between the two curves represent states for which the trascription can occur or not, the points below the blue curve correspond to pLac full repression (trascription off) while points over the red curve correpond to a condition where Plac is partially repressed (trascription on).''<br />
<br />
<br />
When the higher threshold is crossed transcripion takes place with an expression level unsensitive to the ITPG stimulus (see Figure 7). In this condition (i.e. IPTG 900 microM), the saturation level depends on the Glucose (see figure 8), and the circuit acts like a glucose sensor.<br />
<br />
<br />
<br />
<br />
[[Image:grafico3.jpg|center]]<br />
<br />
Figure 9.''Shows that the final level of GFP concentration (output of the system) in the fully induced steady state depends on the External Glucose.''<br />
<br />
<br />
In conclusion, numerical simulation predicts a circuit functionality which is coherent with our initial speculation, i.e. the circuit presented in Figure 6 can operate like a [[#Introduction | Schmitt Trigger]]. <br />
The values of few parameters were assigned with some uncertainty. In particular, we used a value of LacI that is about 30 times the genomic one for a circuit copy number of 10. Since these values can affect the model prediction we will perform specific experiment to identify this parameters. However, the LacI values was incresed in order to trigger off the system for low values of IPTG. To realize this increase we decided to build a sub-circuit where the LacI gene is under the control of pTet promoter. <br />
<br />
At the same way, the GFP steady state expression decreases with extracellular Glucose (see Figure 9), while we want, for an intuitive use of the sensor a fluorescent signal that increases with Glucose. To obtain this behavior we plan to build a sub-circuit that acts like an inverter, with an RFP under the control of pλ, and to put in the main circuit the cI gene which encode for the protein that regulates pλ.<br />
<br />
==Image Acquisition and Analysis==<br />
<br />
===Image Acquisition System===<br />
<br />
[[Image:Microscopy.jpg|center]]<br />
<br />
Figure 10. Image acquisition system based on the fluorescence microscope.<br />
<br />
<br />
<br />
For complete description of Acquisition System, see [https://2007.igem.org/Bologna_University/Microscopy:_Experimental_Set_Up here].<br />
<br />
<br />
<br />
===Image Acquisition===<br />
<br />
Using the experimental set up, we obtain short movies of fluorescent bacteria; then we extract several frames from each of them, using a dedicated software. Example images are shown below.<br />
<br />
<br />
<br />
[[Image:batteri1.jpg|center]]<br />
<br />
Figure 11.''Fluorescent bacteria''<br />
<br />
<br />
<br />
===Image Elaboration===<br />
<br />
Since we need to know how much of the total image area is occupied by bacteria, we process the images with a segmentation algorithm implemented in Matlab.<br />
<br />
Here are the processing phases:<br />
<br />
*the RGB image is read and the green channel is extracted;<br />
*a morphological top hat filtering is performed on the grayscale image;<br />
*by means of an adaptive threshold, the image is tranformed in a binary one;<br />
*the distance between every point and the nearest black pixel is calculated;<br />
*on this last image the [http://en.wikipedia.org/wiki/Watershed_%28algorithm%29 watershed] algorithm is applied: every pixel is assigned a label, depending on the segmentated region it belongs; then, every labeled region is represented with a different colour, as in the images shown below;<br />
<br />
<br />
[[Image:batteri2.jpg|center]]<br />
<br />
<br />
Figure 12.''Algorithm output.''<br />
<br />
<br />
<br />
*the area of every segmented region is calculated, checking that the summation of these areas (including the background) balances the image total area;<br />
*two complementary matrices are created: <br />
1) ''ImageWithOutBackground'', containing the intensity positive values corresponding only to pixels recognised as bacteria, with zeros elsewhere;<br />
<br />
2) ''ImageBackground'', which contains the intensity positive values of pixels recognised as background, with zeros as other entries;<br />
*with a summation over all the ''ImageWithOutBackground'' matrix' entries, the total intensity is obtained;<br />
*dividing this value by the total bacteria area, the output is the normalized intensity we use to compare the fluorescence of different kinds of bacteria.<br />
<br />
This image acquisition and elaboration '''protocol''' has been '''validated''' with a series of measures. For the '''results''', see [[Bologna_University/Protocol validation measures | here]].<br />
<br />
''Thanks to Camilo Melani for his competence and kindness during the algorithm implementation.''<br />
<br />
<br />
<br />
==Biodevice==<br />
<br />
===Components===<br />
<br />
The Genetic Schmitt Trigger [http://partsregistry.org/Part:BBa_I763029 (I763029)], built up with iGEM 2007 Biobricks, consists of 3 main parts combined in the same plasmid: pTeTR-LacI [http://partsregistry.org/Part:BBa_I763026 (I763026)], pLac-cI-LacY-GFP [http://partsregistry.org/Part:BBa_I763019 (I763019)], pλ-RFP [http://partsregistry.org/Part:BBa_I763007 (I763007)].<br \><br />
Each part displays a specific function depending on the promoter and the coded gene(s).<br />
<br />
[[Image:Tutto3.jpg]]<br />
<br />
''Figure 13: The Genetic Schmitt Trigger biodevice ([http://partsregistry.org/Part:BBa_I763029 I763029]).''<br />
<br />
<br />
<br />
:1.<u>pTetR-LacI</u> [http://partsregistry.org/Part:BBa_I763026 (I763026)] codes for ''LacI'' gene [http://partsregistry.org/Part:BBa_C0012 (C0012)] regulated by pTetR [http://partsregistry.org/Part:BBa_R0040 (R0040)] inverting promoter. pTetR can be considered a constitutive promoter in absence of tetracycline (or its analog aTc). Its action is inhibited by the addition of this antibiotic. This promoter regulates the expression of ''LacI'' gene whose protein inhibits the activation of pLac promoter. This part is important to make up for endogenous LacI and to prevent pLac activation in absence of induction with lactose or IPTG.<br />
<br />
:2.<u>pLac-cI-LacY-GFP</u> [http://partsregistry.org/Part:BBa_I763019 (I763019)]: LacY permease [http://partsregistry.org/Part:BBa_J22101 (J22101)] controlled by pLac promoter [http://partsregistry.org/Part:BBa_R0010 (R0010)] introduces a positive feedback necessary for hysteresis. LacY is a membrane transporter allowing the uptake of lactose (or IPTG) in the cell. Lactose (or IPTG) on his own, causes the LacI repressor release from pLac operators. At the same time GFP proteins [http://partsregistry.org/Part:BBa_J04031 (J04031)] are produced as reporters of the induction. We also introduced the gene for cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)] that binds to the cI regulator [http://partsregistry.org/Part:BBa_R0051 (R0051)] and inhibits its action.<br />
<br />
:3.<u>pλ-RFP</u> [http://partsregistry.org/Part:BBa_I763007 (I763007)] acts as reporter of standard condition. Pλ promoter [http://partsregistry.org/Part:BBa_R0051 (R0051)] is a constitutive promoter from phage-λ. Here it regulates RFP protein [http://partsregistry.org/Part:BBa_E1010 (E1010)] expression and it is inhibited by cI repressor [http://partsregistry.org/Part:BBa_C0051 (C0051)]. So, when external glucose is high, pLac transcription level, cI and GFP production are high, while RFP production is low. When external glucose is low pLac transcription level, cI and GFP production are low while RFP production is high. As a consequence, we can consider RFP fluorescence levels as an indicator of glucose concentration in culture medium.<br />
<br />
===How it works===<br />
In standard conditions, without IPTG induction pLac promoter is repressed by the LacI repressor binding on the operator sites and cI, LacY and GFP proteins are not expressed. In this condition pλ promoter is active and RFP is expressed. The cells show red fluorescence.<br />
<br />
[[Image:no_IPTG.jpg]]<br />
<br />
''Figure 14: Project in action: no IPTG in the medium.''<br />
<br />
<br />
<br />
For induction IPTG is added in the culture medium. Due to inductor binding to LacI repressor, Plac promoter is activated and ''cI'', ''LacY'' and ''GFP'' genes are expressed. In this condition pλ promoter is repressed by cI inhibitor and so RFP is not expressed. The cells show green fluorescence.<br />
<br />
[[Image:con_IPTG1.jpg]]<br />
<br />
''Figure 15: Project in action: IPTG in the medium.''<br />
<br />
<br />
<br />
===Intermediates===<br />
<br />
The table below shows all the intermediates that we have obtained to build up our final device.<br />
For each part you can find: <br />
<br />
-in the first column the link to the Registry;<br />
<br />
-in the second column the components for each part;<br />
<br />
-in the third column the plasmid the part has been cloned in;<br />
<br />
-in the fourth the insert length after double digestion with Xba1/Pst1 enzymes linked to the electrophoresis gel run;<br />
<br />
-in the fifth column cell vitality after transformation;<br />
<br />
-in the sixth column fluo data for each part.<br />
<br />
<br />
{| align="center" style="color:black;" border="0" cellspacing="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Plasmid''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
:26<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I763020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763027 I763027]<br />
<br />
<br />
| align="left" width="560pt" valign="top" bgcolor="#B0FF80"| <br />
:RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T <br />
:pλ-RBS-RFP-T <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T<br />
:pLac-RBS-cI-RBS-GFP-T <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T <br />
:pLac-RBS-LacY-RBS-GFP-T <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:Genetic Schmitt Trigger<br />
:Genetic Schmitt Trigger<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
| align="left" width="160pt" valign="top" bgcolor="#B0FF80"|<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB4A3 pSB4A3 (LC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
:[http://partsregistry.org/Part:pSB1AK3 pSB1AK3 (HC)]<br />
:[http://partsregistry.org/Part:pSB1A2 pSB1A2 (HC)]<br />
<br />
| align="left" width="130" valign="top" bgcolor="#B0FF80"|<br />
:[[Bologna University/Gel/RBS-GFP-T | 914]]<br />
:[[Bologna University/Gel/pLac-RBS-GFP-T | 1122]]<br />
:[[Bologna University/Gel/pλ-RBS-GFP-T | 971]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (low copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pλ-RBS-RFP-T (high copy plasmid) | 918]]<br />
:[[Bologna University/Gel/pTetR-RBS-RFP-T | 923]]<br />
:[[Bologna University/Gel/RBS-LacY | 1306]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY | 1514]]<br />
:[[Bologna University/Gel/pLac-RBS-cI | 1001]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | 1923]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (low copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY (high copy plasmid) | 2315]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-T | 2452]]<br />
:[[Bologna University/Gel/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | 3237]]<br />
:[[Bologna University/Gel/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid)| 2436]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (high copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI (low copy plasmid) | 1233]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T | 1370]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-RBS-GFP-T | 2155]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T| 4615]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (high copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/Genetic Schmitt Trigger (low copy plasmid) | 5525]]<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pλ-RBS-RFP | 2296]]<br />
:2053<br />
:[[Bologna University/Gel/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | 2500]]<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:No<br />
:Yes<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:-<br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:- <br />
:- <br />
:- <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/Genetic Schmitt Trigger (high copy plasmid) | >>]]<br />
:-<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]]<br />
:[[Bologna University/TetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
Clicking on the length of the fragment you will be shown the electrophoresis gel of the digestion of the fragment excised from the plasmid by cutting it with a double digestion using Xba and Pst1 enzymes.<br />
<br />
<br />
<br />
==Components Characterization==<br />
<br />
===Fluorescence intensity dependence on Promoter type===<br />
<br />
The three promoters used in the biodevice ([http://partsregistry.org/Part:BBa_I763004 I763004], [http://partsregistry.org/Part:BBa_I763035 I763035] and [http://partsregistry.org/Part:BBa_I763011 I763011]) have been separately characterized in order to gain useful information for the model. We chose to characterize the behavior of promoters escluding the LacY positive feedback.<br />
<br />
First, the three different intermediate parts have been tested in LB medium to stabilish differences in transcription rates. All of the three intermediates have GFP protein as a reporter to avoid difference in protein synthetis and degradation.<br />
<br />
Mesuarements have been performed at OD value of 1.2. We have taken pictures of two fields from each of the five slides we prepared; then, we calculated the mean value for each slide and finally the mean and the standard deviation on these values. <br />
Results are in figure below:<br />
<br />
[[Image:Istogramma1.jpg|center]]<br />
<br />
<br />
<br />
Fluorescence intensity levels for [http://partsregistry.org/Part:BBa_I763004 I763004] and [http://partsregistry.org/Part:BBa_I763035 I763035] are similar, while [http://partsregistry.org/Part:BBa_I763011 I763011] beams a significantely lower fluorescence (30%).<br />
<br />
<br />
Experiments have been repeated with bacteria grown into a different culture medium (M9 medium), that is the one chosen for testing the Genetic Schmitt Trigger. <br />
Results are in figure below:<br />
<br />
<br />
[[Image:Istogramma21.jpg|center]]<br />
<br />
<br />
In this case, the three promoters produce levels of fluorescence significantely different being pTet the one with the highest transcription activity.<br />
<br />
===Comparison between fluorescence elicited by the two media===<br />
<br />
To highlight the differences between the two media, we have directely compared for each intermediate fluorescence intensity in the two media.<br />
The comparison is shown in figure below:<br />
<br />
[[Image:Istogramma3333.jpg|center]]<br />
<br />
pλ promoter seems to not been influenced by the growth in different media.As expected, pLac promoter seems to have a higher transcription rate in a medium not added with carbon sources (M9 minimal medium). This result strectly agree with the prediction obteined by our model (20% experimental vs 23% simulation). Surprisingly, pTet promoter increases its activity changing the medium. We are not able to justify this result. Anyway, our model does not include such dependence and this aspect deserve to be investigated to be clarify.<br />
<br />
<br />
Complete data set, summarized in the table below, is [[Bologna University/Fluo results (Promoter - Medium) | here]].<br />
<br />
<br />
<br />
{| border="0" cellpadding="5" cellspacing="1" align="center"<br />
|-<br />
! style="background:white;" | <br />
! style="background:white;" |<br />
! style="background:#ffdead;" | LB<br />
! style="background:#ffdead;" | M9<br />
|-<br />
| style="background:#ffdead;" | I763004<br />
| style="background:#ffdead;" | pLac-GFP<br />
| style="background:#ffefbe;" | 102.9 ± 13.4<br />
| style="background:#ffefbe;" | 123.2 ± 13.1<br />
|-<br />
| style="background:#ffdead;" | I763035<br />
| style="background:#ffdead;" | pTetR-LacI-GFP<br />
| style="background:#ffefbe;" | 106.6 ± 6.0<br />
| style="background:#ffefbe;" | 138.1 ± 7.7<br />
|-<br />
| style="background:#ffdead;" | I763011<br />
| style="background:#ffdead;" | pλ-GFP<br />
| style="background:#ffefbe;" | 71.6 ± 12.3<br />
| style="background:#ffefbe;" | 81.3 ± 13.9<br />
|-<br />
|}<br />
<br />
<br />
===Switch Time Course===<br />
<br />
We also tested the intermediate device pTet-LacI-pLac-GFP [http://partsregistry.org/Part:BBa_I763027 (I763027)] in a high copy plasmid [http://partsregistry.org/Part:pSB1A2 pSB1A2] to verify if it really works as a toggle switch.In this intermediate device only LacY is not included since we would study transcriptional activity without the positive feedback due to the lactose permease.<br />
<br />
Following the [[Bologna University/Fluorescence Test | Fluorescence Test protocol]], bacteria grew in LB medium O/N. The day after we tested output fluorescence intensity in M9 with 1M glucose. We induced them with two different IPTG concentrations (2mM and 8mM) to activate pLac promoter. We measured output fluorescence intensity at regular time intervals and we verified that it incresed esponentially. After 90 minutes we eliminated IPTG induction resuspendig the cells in M9 medium with 1M glucose and we verified that fluorescence output intensity decreased quickly to the initial level.<br />
We could not measure fluorescence output intensity of cells in 8mM IPTG at 140 minutes because cells died.<br />
<br />
Our device seems to work as expected like a switch as the mathematical model predicted.<br />
<br />
<br />
<br />
[[Image:curvetempi2.jpg|center]]<br />
<br />
=Concluding Remarks=<br />
<br />
<br />
=Acknowledgements=<br />
Our Team was funded by: <br />
<br />
*'''European Union SYNBIOCOMM project''' <br />
<br />
<br />
[[Image:Synbiocomm.jpg|left]] [[Image:Bandiera_comunita_europea.jpg|absright]]<br />
<br />
<br />
*'''Ser.In.Ar. Cesena''' <br />
<br />
<br />
[[Image:serinar.jpg]]<br />
<br />
<br />
*'''University of Bologna - Cesena Campus'''<br />
<br />
[[Image:logounibo2.jpg|center]]<br />
<br />
=Overview Table=<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="340pt" |<br />
:'''Undergraduate Students'''<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/IB| Iros Barozzi]]<br />
:'''PhD student'''<br />
::[[Bologna_University/AP | Alice Pasini]]<br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisors'''<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Emanuele Giordano]<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Marco Tartagni]<br />
<br />
| align="left" width="310pt"| <br />
:[[#Biodevice | '''Biodevice''']]<br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna#Experimental SetUp | Microscopy: Experimental SetUp]]<br />
::4. [[Bologna#Image acquisition and analysis | Image Acquisition and Analysis]]<br />
<br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
| align="left" width="300"|<br />
:[[#Mathematical Model | '''Mathematical Model''']]<br />
:'''Results'''<br />
::1. [[Bologna#Intermediates | Intermediates]]<br />
::2. [[Bologna_University#Fluorescence intensity dependence on Promoter type | Fluorescence reliance on Promoter and Medium]]<br />
::3. [[#Components Characterization | Components Characterization]]<br />
:[[#Concluding Remarks | '''Concluding Remarks''']]<br />
|-<br />
|}<br />
<br />
=Diary=<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center>'''[[Week 13]]'''</center><br />
|<center>'''[[Week 14]]'''</center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|}</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/ResultsBologna University/Results2007-10-22T17:57:15Z<p>Alice.pasini: </p>
<hr />
<div>{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I7630020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763040 I763040]<br />
<br />
<br />
| align="left" width="700pt" valign="top" bgcolor="#B0FF80"| <br />
: RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T (low copy plasmid) <br />
:pλ-RBS-RFP-T (high copy plasmid) <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T (low copy plasmid)<br />
:pLac-RBS-cI-RBS-GFP-T (high copy plasmid) <br />
:pLac-RBS-cI-RBS-LacY (low copy plasmid) <br />
:pLac-RBS-cI-RBS-LacY (high copy plasmid) <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) <br />
:pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) <br />
:pTetR-RBS-LacI (high copy plasmid) <br />
:pTetR-RBS-LacI (low copy plasmid) <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
<br />
| align="left" width="150" valign="top" bgcolor="#B0FF80"|<br />
:914<br />
:1122<br />
:971<br />
:918<br />
:918<br />
:923<br />
:1306<br />
:1514<br />
:1001<br />
:1923<br />
:1923<br />
:2315<br />
:2315<br />
:2452<br />
:3237<br />
:2436<br />
:1233<br />
:1233<br />
:1370<br />
:2155<br />
:4615<br />
:5525<br />
:2296<br />
:2053<br />
:2500<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:Yes<br />
:-<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:-<br />
:-<br />
:-<br />
:Yes<br />
:Yes<br />
:-<br />
:-<br />
:-<br />
:Yes<br />
:Yes<br />
:-<br />
:-<br />
:-<br />
:Yes<br />
:Yes<br />
:<br />
:<br />
:<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T | >>]] <br />
:[[Bologna University/RBS-LacY | >>]] <br />
:[[Bologna University/pLac-RBS-LacY | >>]]<br />
:[[Bologna University/pLac-RBS-cI | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY (low copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-T | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI (high copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI (low copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T | >>]] <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]]<br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/ResultsBologna University/Results2007-10-22T17:53:57Z<p>Alice.pasini: </p>
<hr />
<div>{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I7630020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763039 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763034 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763040 I763040]<br />
<br />
<br />
| align="left" width="700pt" valign="top" bgcolor="#B0FF80"| <br />
: RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T (low copy plasmid) <br />
:pλ-RBS-RFP-T (high copy plasmid) <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T (low copy plasmid)<br />
:pLac-RBS-cI-RBS-GFP-T (high copy plasmid) <br />
:pLac-RBS-cI-RBS-LacY (low copy plasmid) <br />
:pLac-RBS-cI-RBS-LacY (high copy plasmid) <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) <br />
:pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) <br />
:pTetR-RBS-LacI (high copy plasmid) <br />
:pTetR-RBS-LacI (low copy plasmid) <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
<br />
| align="left" width="150" valign="top" bgcolor="#B0FF80"|<br />
:914<br />
:1122<br />
:971<br />
:918<br />
:918<br />
:923<br />
:1306<br />
:1514<br />
:1001<br />
:1923<br />
:1923<br />
:2315<br />
:2315<br />
:2452<br />
:3237<br />
:2436<br />
:1233<br />
:1233<br />
:1370<br />
:2155<br />
:4615<br />
:5525<br />
:2288<br />
:2045<br />
:2492<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:Yes<br />
:-<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:-<br />
:-<br />
:-<br />
:Yes<br />
:Yes<br />
:-<br />
:-<br />
:-<br />
:Yes<br />
:Yes<br />
:-<br />
:-<br />
:-<br />
:Yes<br />
:Yes<br />
:<br />
:<br />
:<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T | >>]] <br />
:[[Bologna University/RBS-LacY | >>]] <br />
:[[Bologna University/pLac-RBS-LacY | >>]]<br />
:[[Bologna University/pLac-RBS-cI | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY (low copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-T | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI (high copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI (low copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T | >>]] <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]]<br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/ResultsBologna University/Results2007-10-22T17:52:47Z<p>Alice.pasini: </p>
<hr />
<div>{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="white" color="white" height="30pt" align="center" | ''' '''<br />
| bgcolor="#00E040" color="white" align="center" | '''Igem Code''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fragment''' <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Length (bp)''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Vitality''' <br />
| bgcolor="#00E040" color="white" align="center" | '''Fluo Data'''<br />
|- style="color:black;"<br />
<br />
| align="left" width="50pt" valign="top" bgcolor="#B0FF80"|<br />
:1<br />
:2<br />
:3<br />
:4<br />
:5<br />
:6<br />
:7<br />
:8<br />
:9<br />
:10<br />
:11<br />
:12<br />
:13<br />
:14<br />
:15<br />
:16<br />
:17<br />
:18<br />
:19<br />
:20<br />
:21<br />
:22<br />
:23<br />
:24<br />
:25<br />
<br />
| align="left" width="100pt" valign="top" bgcolor="#B0FF80"| <br />
:[http://partsregistry.org/Part:BBa_I7630020 I763020]<br />
:[http://partsregistry.org/Part:BBa_I763004 I763004]<br />
:[http://partsregistry.org/Part:BBa_I763011 I763011]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763007 I763007]<br />
:[http://partsregistry.org/Part:BBa_I763033 I763033]<br />
:[http://partsregistry.org/Part:BBa_I763015 I763015]<br />
:[http://partsregistry.org/Part:BBa_I763012 I763012]<br />
:[http://partsregistry.org/Part:BBa_I763005 I763005]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763031 I763031]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763016 I763016]<br />
:[http://partsregistry.org/Part:BBa_I763036 I763036]<br />
:[http://partsregistry.org/Part:BBa_I763019 I763019]<br />
:[http://partsregistry.org/Part:BBa_I763032 I763032]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763025 I763025]<br />
:[http://partsregistry.org/Part:BBa_I763026 I763026]<br />
:[http://partsregistry.org/Part:BBa_I763035 I763035]<br />
:[http://partsregistry.org/Part:BBa_I763028 I763028]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763029]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763039]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763034]<br />
:[http://partsregistry.org/Part:BBa_I763029 I763040]<br />
<br />
<br />
| align="left" width="700pt" valign="top" bgcolor="#B0FF80"| <br />
: RBS-GFP-T <br />
:pLac-RBS-GFP-T <br />
:pλ-RBS-GFP-T <br />
:pλ-RBS-RFP-T (low copy plasmid) <br />
:pλ-RBS-RFP-T (high copy plasmid) <br />
:pTetR-RBS-RFP-T <br />
:RBS-LacY <br />
:pLac-RBS-LacY<br />
:pLac-RBS-cI <br />
:pLac-RBS-cI-RBS-GFP-T (low copy plasmid)<br />
:pLac-RBS-cI-RBS-GFP-T (high copy plasmid) <br />
:pLac-RBS-cI-RBS-LacY (low copy plasmid) <br />
:pLac-RBS-cI-RBS-LacY (high copy plasmid) <br />
:pLac-RBS-cI-RBS-LacY-T <br />
:pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) <br />
:pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) <br />
:pTetR-RBS-LacI (high copy plasmid) <br />
:pTetR-RBS-LacI (low copy plasmid) <br />
:pTetR-RBS-LacI-T <br />
:pTetR-RBS-LacI-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP-pLac-RBS-cI-RBS-LacY-RBS-GFP-T<br />
:pTetR-RBS-LacI-T-pλ-RBS-RFP<br />
:pTetR-RBS-RFP-T-pLac-RBS-GFP-T <br />
:pTetR-RBS-LacI-T-pLac-RBS-GFP-T <br />
<br />
<br />
| align="left" width="150" valign="top" bgcolor="#B0FF80"|<br />
:914<br />
:1122<br />
:971<br />
:918<br />
:918<br />
:923<br />
:1306<br />
:1514<br />
:1001<br />
:1923<br />
:1923<br />
:2315<br />
:2315<br />
:2452<br />
:3237<br />
:2436<br />
:1233<br />
:1233<br />
:1370<br />
:2155<br />
:4615<br />
:5525<br />
:2288<br />
:2045<br />
:2492<br />
<br />
| align="left" width="100" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:Yes<br />
:-<br />
:Yes<br />
:Yes<br />
:N.D.<br />
:-<br />
:-<br />
:-<br />
:Yes<br />
:Yes<br />
:-<br />
:-<br />
:-<br />
:Yes<br />
:Yes<br />
:-<br />
:-<br />
:-<br />
:Yes<br />
:Yes<br />
:<br />
:<br />
:<br />
<br />
| align="left" width="50" valign="top" bgcolor="#B0FF80"|<br />
:-<br />
:[[Bologna University/pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-GFP-T | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (low copy plasmid) | >>]] <br />
:[[Bologna University/pλ-RBS-RFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-RFP-T | >>]] <br />
:[[Bologna University/RBS-LacY | >>]] <br />
:[[Bologna University/pLac-RBS-LacY | >>]]<br />
:[[Bologna University/pLac-RBS-cI | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (low copy plasmid) | >>]]<br />
:[[Bologna University/pLac-RBS-cI-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY (low copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-T | >>]] <br />
:[[Bologna University/pLac-RBS-cI-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pLac-RBS-LacY-RBS-GFP-T (high copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI (high copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI (low copy plasmid) | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T | >>]] <br />
:[[Bologna university/pTetR-RBS-LacI-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP-pLac-RBS-cI-RBS-LacY-RBS-GFP-T | >>]]<br />
:[[Bologna University/pTetR-RBS-LacI-T-pλ-RBS-RFP | >>]]<br />
:[[Bologna University/pTetR-RBS-RFP-T-pLac-RBS-GFP-T | >>]] <br />
:[[Bologna University/pTetR-RBS-LacI-T-pLac-RBS-GFP-T | >>]]<br />
<br />
|-<br />
|}<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/ProtocolsBologna University/Protocols2007-10-04T15:48:22Z<p>Alice.pasini: </p>
<hr />
<div>::1. [[Bologna_University/Chemiocompetent cells | Chemiocompetent cells]]<br />
::2. [[Bologna_University/Mediums and buffers | Mediums and buffers]]<br />
::3. [[Bologna_University/Antibiotics stocks preparation | Antibiotics stocks preparation]]<br />
::4. [[Bologna_University/Plates preparation | Plates preparation]]<br />
::5. [[Bologna_University/Transformation | Transformation]]<br />
::6. [[Bologna_University/Digestion reaction | Digestion reaction]]<br />
::7. [[Bologna_University/Ligation reaction | Ligation reaction]]<br />
::8. [[Bologna_University/IPTG stocks preparation | IPTG stocks preparation]]<br />
::9. [[Bologna_University/Fluorescence issue | Fluorescence Test ]]<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/ProtocolsBologna University/Protocols2007-10-04T15:47:42Z<p>Alice.pasini: </p>
<hr />
<div>::1. [[Bologna_University/Chemiocompetent cells | Chemiocompetent cells]]<br />
::2. [[Bologna_University/Mediums and buffers | Mediums and buffers]]<br />
::3. [[Bologna_University/Antibiotics stocks preparation | Antibiotics stocks preparation]]<br />
::4. [[Bologna_University/Plates preparation | Plates preparation]]<br />
::5. [[Bologna_University/Transformation | Transformation]]<br />
::6. [[Bologna_University/Digestion reaction | Digestion reaction]]<br />
::7. [[Bologna_University/Ligation reaction | Ligation reaction]]<br />
::8. [[Bologna_University/IPTG stocks preparation | IPTG stocks preparation]]<br />
::9. [[Bologna_University/Fluorescence Test | Fluorescence Test ]]<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/Fluorescence_issueBologna University/Fluorescence issue2007-10-04T15:46:45Z<p>Alice.pasini: </p>
<hr />
<div>::1. Growth O/N for 15h in LB (5ml) of:<br />
<br />
::::-transformed E. Coli with appropriate antibiotic;<br />
<br />
::::-E. Coli.<br />
<br />
::2. '''All measures have to be done in M9 with OD=1.'''<br />
::3. The day after in the morning measure OD.<br />
::4. To obtain OD=1:<br />
::::-centrifugate 4ml of bacteria culture at 4400rpm for 3min at 25°C;<br />
::::-discard the supernatant;<br />
::::-resuspende cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x).<br />
::5. Measure OD: adjust OD to 1 through further diluition or growth.<br />
::6. Adjust PMT offset with untransformed E. Coli.<br />
::7. Test culture fluorescence before IPTG induction.<br />
::8. IPTG induction:<br />
::::-centrifugate bacteria culture at 4400rpm for 3min at 25°C;<br />
::::-discard the supernatant;<br />
::::-resuspende cell pellet in M9 medium and 2mM IPTG with appropriate antibiotic (1000X);<br />
::9. Incubate at 37°C.<br />
::10. Test fluorescence after 10min, 20min, 30min, 1h, 2h.<br />
<br />
[[Bologna University/Protocols | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/Fluorescence_issueBologna University/Fluorescence issue2007-10-04T15:46:23Z<p>Alice.pasini: </p>
<hr />
<div>::1. Growth O/N for 15h in LB (5ml) of:<br />
<br />
::::-transformed E. Coli with appropriate antibiotic;<br />
<br />
::::-E. Coli.<br />
<br />
::2. '''All measures have to be done in M9 with OD=1.'''<br />
::3. The day after in the morning measure OD.<br />
::4. To obtain OD=1:<br />
::::-centrifugate 4ml of bacteria culture at 4400rpm for 3min at 25°C;<br />
::::-discard the supernatant;<br />
::::-resuspende cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x).<br />
::5. measure OD: adjust OD to 1 through further diluition or growth.<br />
::6. Adjust PMT offset with untransformed E. Coli.<br />
::7. Test culture fluorescence before IPTG induction.<br />
::8. IPTG induction:<br />
::::-centrifugate bacteria culture at 4400rpm for 3min at 25°C;<br />
::::-discard the supernatant;<br />
::::-resuspende cell pellet in M9 medium and 2mM IPTG with appropriate antibiotic (1000X);<br />
::9. Incubate at 37°C.<br />
::10. Test fluorescence after 10min, 20min, 30min, 1h, 2h.<br />
<br />
[[Bologna University/Protocols | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/Fluorescence_issueBologna University/Fluorescence issue2007-10-04T15:45:04Z<p>Alice.pasini: </p>
<hr />
<div>::1. Growth O/N for 15h in LB (5ml) of:<br />
<br />
::::-transformed E. Coli with appropriate antibiotic;<br />
<br />
::::-E. Coli.<br />
<br />
::2. '''All measures have to be done in M9 with OD=1.'''<br />
::3. The day after in the morning measure OD.<br />
::4. To obtain OD=1:<br />
::::-centrifugate 4ml of bacteria culture at 4400rpm for 3min at 25°C;<br />
::::-discard the supernatant;<br />
::::-resuspende cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x).<br />
::5. measure OD: adjust OD to 1 through further diluition or growth.<br />
::6. Adjust PMT offset with untransformed E. Coli.<br />
::7. Test culture fluorescence before IPTG induction.<br />
::8. IPTG induction:<br />
::::-centrifugate bacteria culture at 4400rpm for 3min at 25°C;<br />
::::-discard the supernatant;<br />
::::-resuspende cell pellet in M9 medium and 2mM IPTG with appropriate antibiotic (1000X);<br />
::9. Incubate at 37°C.<br />
::10. Test fluorescence after 10min, 20min, 30min, 1h, 2h.<br />
<br />
[[Bologna University/Protocols | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/ProtocolsBologna University/Protocols2007-10-04T15:36:55Z<p>Alice.pasini: </p>
<hr />
<div>::1. [[Bologna_University/Chemiocompetent cells | Chemiocompetent cells]]<br />
::2. [[Bologna_University/Mediums and buffers | Mediums and buffers]]<br />
::3. [[Bologna_University/Antibiotics stocks preparation | Antibiotics stocks preparation]]<br />
::4. [[Bologna_University/Plates preparation | Plates preparation]]<br />
::5. [[Bologna_University/Transformation | Transformation]]<br />
::6. [[Bologna_University/Digestion reaction | Digestion reaction]]<br />
::7. [[Bologna_University/Ligation reaction | Ligation reaction]]<br />
::8. [[Bologna_University/IPTG stocks preparation | IPTG stocks preparation]]<br />
::9. [[Bologna_University/Fluorescence issue | Fluorescence Test ]]<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/FCBologna University/FC2007-10-02T08:46:41Z<p>Alice.pasini: </p>
<hr />
<div>[[Image: Francesca_Ceroni.jpg|center]]<br />
<br />
I got my Laurea Degree in Pharmaceutical Biotechnologies at the University of Bologna and now here I'm in Igem 2007!<br />
In my thesis I worked on the regulation of the ABC genes by the Myc oncoproteins. Now I have the chance to be in the synthetic biology and I really love it.<br />
<br />
I have one great passion... jazz! That's why I play the saxophone!<br />
<br />
<br />
'''E-mail:''' ''francesca.ceroni2@unibo.it''<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Bologna_University/FCBologna University/FC2007-10-02T08:45:54Z<p>Alice.pasini: </p>
<hr />
<div>[[Image: Francesca_Ceroni.jpg|center]]<br />
<br />
I got my Laurea Degree in Pharmaceutical Biotechnologies at the University of Bologna and now here I'm in Igem 2007!<br />
In my thesis I worked on the regulation of the ABC genes by the Myc oncoprotein. Now I have the chance to be in the synthetic biology and I really love it.<br />
<br />
I have one great passion... jazz! That's why I play the saxophone!<br />
<br />
<br />
'''E-mail:''' ''francesca.ceroni2@unibo.it''<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_9Week 92007-09-03T15:37:03Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/27/07'''<br />
*New [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation with a new protocol (3h 15°C and 1.2 ul Ligase).<br />
*Transformation of [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation and of new biobricks [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103]. <br />
<br />
<br />
<br />
<br />
::'''08/28/07'''<br />
*Lab meeting on: <br />
-model<br />
-LacI cloning under the pTetR [http://partsregistry.org/Part:BBa_R0040 R0040])promoter control; <br />
<br />
*We transform new biobricks:[http://partsregistry.org/Part:BBa_E0020 E0020] and [http://partsregistry.org/Part:BBa_R0040 R0040]<br />
<br />
* We inoculate a [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103] colony in 5 ml;<br />
<br />
* There are no colonies for [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412]([http://partsregistry.org/Part:BBa_I763010 (I763010)] ligation.<br />
<br />
::'''08/29/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103].<br />
*[http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103] digestions with Xba/Pst1.<br />
*[http://partsregistry.org/Part:BBa_B0034 B0034] digestion with Spe/Pst1.<br />
*Run on electroforesi gel and [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034] band extraction. We get some problems with [http://partsregistry.org/Part:BBa_J06550 J06550] plasmid vector molecular wheigh (too heavy) and with [http://partsregistry.org/Part:BBa_S0103 S0103] insert molecular weight (too heavy). So, we decide to use another biobrick.<br />
*Ligation for [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_J06550 J06550] ([http://partsregistry.org/Part:BBa_I763016 I763016]), [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_S0103 S0103] (missed in the sendbox),[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J22101 J22101] (missed in the sendbox), [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763020 I763020]).<br />
*We transform the ligations. <br />
<br />
<br />
<br />
<br />
::'''08/30/07'''<br />
*There are colonies in the 07/30/07 plates.<br />
*We inoculate a [http://partsregistry.org/Part:BBa_E0020 E0020] and [http://partsregistry.org/Part:BBa_R0040 R0040] colony in 5 ml.<br />
*We perform some tests to set up the fluorescence acquisition with the PMT (photomultiplier tube).<br />
<br />
<br />
<br />
::'''08/31/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_J06550 J06550] [http://partsregistry.org/Part:BBa_I763016 (I763016)], [http://partsregistry.org/Part:BBa_I763012 I763012]+ [http://partsregistry.org/Part:BBa_S0103 S0103] (missed in the sandbox), [http://partsregistry.org/Part:BBa_B0034 B0034] + <br />
[http://partsregistry.org/Part:BBa_J22101 J22101] (missed in the sandbox), [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] [http://partsregistry.org/Part:BBa_I763020 (I763020)], [http://partsregistry.org/Part:BBa_E0020 E0020 ] and of [http://partsregistry.org/Part:BBa_R0040 R0040].<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_9Week 92007-09-03T15:35:44Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/27/07'''<br />
*New [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation with a new protocol (3h 15°C and 1.2 ul Ligase).<br />
*Transformation of [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation and of new biobricks [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103]. <br />
<br />
<br />
<br />
<br />
::'''08/28/07'''<br />
*Lab meeting on: <br />
-model<br />
-LacI cloning under the pTetR [http://partsregistry.org/Part:BBa_R0040 R0040])promoter control; <br />
<br />
*We transform new biobricks:[http://partsregistry.org/Part:BBa_E0020 E0020] and [http://partsregistry.org/Part:BBa_R0040 R0040]<br />
<br />
* We inoculate a [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103] colony in 5 ml;<br />
<br />
* There are no colonies for [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412]([http://partsregistry.org/Part:BBa_I763010 (I763010)] ligation.<br />
<br />
::'''08/29/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103].<br />
*[http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103] digestions with Xba/Pst1.<br />
*[http://partsregistry.org/Part:BBa_B0034 B0034] digestion with Spe/Pst1.<br />
*Run on electroforesi gel and [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034] band extraction. We get some problems with [http://partsregistry.org/Part:BBa_J06550 J06550] plasmid vector molecular wheigh (too heavy) and with [http://partsregistry.org/Part:BBa_S0103 S0103] insert molecular weight (too heavy). So, we decide to use another biobrick.<br />
*Ligation for [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_J06550 J06550] ([http://partsregistry.org/Part:BBa_I763016 I763016]), [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_S0103 S0103] (missed in the sendbox),[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J22101 J22101] (missed in the sendbox), [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763020 I763020]).<br />
*We transform the ligations. <br />
<br />
<br />
<br />
<br />
::'''08/30/07'''<br />
*There are colonies in the ligations.<br />
*We inoculate ligations, [http://partsregistry.org/Part:BBa_E0020 E0020] and [http://partsregistry.org/Part:BBa_R0040 R0040] colony in 5 ml.<br />
*We perform some tests to set up the fluorescence acquisition with the PMT (photomultiplier tube).<br />
<br />
<br />
<br />
::'''08/31/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_J06550 J06550] [http://partsregistry.org/Part:BBa_I763016 (I763016)], [http://partsregistry.org/Part:BBa_I763012 I763012]+ [http://partsregistry.org/Part:BBa_S0103 S0103] (missed in the sandbox), [http://partsregistry.org/Part:BBa_B0034 B0034] + <br />
[http://partsregistry.org/Part:BBa_J22101 J22101] (missed in the sandbox), [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] [http://partsregistry.org/Part:BBa_I763020 (I763020)], [http://partsregistry.org/Part:BBa_E0020 E0020 ] and of [http://partsregistry.org/Part:BBa_R0040 R0040].<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_8Week 82007-09-03T15:32:03Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/20/07'''<br />
*[http://partsregistry.org/Part:BBa_I763010 I763010] and [http://partsregistry.org/Part:BBa_C0012 C0012] transformation. <br />
<br />
<br />
<br />
::'''08/21/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_I763010 I763010] and [http://partsregistry.org/Part:BBa_C0012 C0012]. We can't amplify these biobricks.<br />
*[http://partsregistry.org/Part:BBa_J04631 J04631] digestion. <br />
*Band extraction. <br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation.<br />
<br />
<br />
<br />
<br />
::'''08/22/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010])<br />
<br />
<br />
<br />
::'''08/23/07'''<br />
*A new [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation with a new protocol (3h 15°C and 1.2 ul T4 Ligase).<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051], [http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe/Pst1.<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_J04631 J04631] digestion with Xba/Pst1.<br />
*Run and extraction from gel.<br />
*Transformation of [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/24/07'''<br />
*We plan new amplifications.<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_5Week 52007-09-03T15:30:58Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/30/07'''<br />
*We check bacterial growth curve (link).<br />
*Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012], [http://partsregistry.org/Part:BBa_J52034 J52034] and [http://partsregistry.org/Part:BBa_B0015 B0015].<br />
*Ligation [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 2 ul, insert 6 ul) and the ligation [http://partsregistry.org/Part:BBa_J04500 J04500]+ [http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)](vector 2 ul, insert 10 ul).<br />
<br />
<br />
<br />
<br />
::'''07/31/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012] , so we inoculate [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015] colony in 5 ml.<br />
*We strake on plates 30/07 ligations.<br />
*We get along with the [http://partsregistry.org/Part:BBa_J04431 J04431] fluorescence test:<br />
-From the glycerol stock we sprout 1 ml at 37°C for 1 hour in agitation.<br />
<br />
-Every 15 minutes we check bacterial OD and fluorescence. Cells are fluorescent at t= 210 min and OD=0.5.<br />
* We decide to repeat the test to understand if we can switch off the fluorescent cells with a higher amount of glucose.<br />
We will perform this test on 07/08.<br />
<br />
<br />
<br />
<br />
::'''08/01/07'''<br />
<br />
-Miniprep of [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015];<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_B0015 B0015] with Xba/Pst1;<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_I52034 I52034] with Spe/Pst1;<br />
<br />
- Gel extraction (we can’t see [http://partsregistry.org/Part:BBa_B0015 B0015]);<br />
<br />
-[http://partsregistry.org/Part:BBa_R0015 R0015]+[http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)] ligation which doesn’t give colonies so we transform with the other 10 ul of ligation; <br />
<br />
-[http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051][http://partsregistry.org/Part:BBa_I763005 (I763005)]ligation which gives colonies so we inoculate in 5ml;<br />
<br />
-Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012] (4ul) and for [http://partsregistry.org/Part:BBa_J22101 J22101] (2ul);<br />
<br />
-A new [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 4ul, insert 8ul) ligation. <br />
<br />
<br />
<br />
<br />
::'''08/02/07'''<br />
<br />
-Miniprep of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)];<br />
<br />
-Control digestion of 5 ul with Eco/Pst1;<br />
<br />
-A new [http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Xba/Pst1;<br />
<br />
-Run on electrophoresis gel. We have problems with gel because we don’t see the band;<br />
<br />
-Band extraction for [http://partsregistry.org/Part:BBa_B0015 B0015] e [http://partsregistry.org/Part:BBa_I763005 I763005];<br />
<br />
-There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012], but there are colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)]. So, we identify the correct ligation protocol;<br />
<br />
- [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_C0012 C0012] transformation;<br />
<br />
-Inoculation in 5 ml for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] and for [http://partsregistry.org/Part:BBa_J22101 J22101];<br />
<br />
- [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] fluorescence test to check if the RFP filter works properly.<br />
<br />
<br />
<br />
::'''08/03/07'''<br />
<br />
-Miniprep for [http://partsregistry.org/Part:BBa_J22101 J22101] and for [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J22101 J22101] digestion with Xba/Pst1 ;<br />
<br />
- Control digestion of [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_B0015 B0015]and [http://partsregistry.org/Part:BBa_J22101 J22101] and [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] run on electrophoresis gel and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_J22101 J22101] extraction;<br />
<br />
-Glycerol stocks preparation;<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T15:29:51Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*On the plates we observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
* Transformation again for [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test ligation protocol. We try with vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C 1 h and at 65°C for 10 minutes. <br />
*Then we transform 4 ul from the ligation reaction. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. We inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051],[http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we inoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. After 30 minutes the cells are already green.<br />
*We harvest and put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We measure OD every 20 minutes.<br />
<br />
*We get along with another GFP induction: after 10 minutes the cells are already green.<br />
*We transform bacteria with 4 ul of the [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul from [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*Midiprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Midiprep Quantification: conc. 65 ng/ul;<br />
<br />
*We observe colonies for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We inoculate in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform again with [http://partsregistry.org/Part:BBa_R0051 R0051] and with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We let colonies from [http://partsregistry.org/Part:BBa_R0051 R0051] and from[http://partsregistry.org/Part:BBa_R1051 R1051] grow at 37°C during the day. We inoculate in 5 ml in the evening and we leave them O/N.<br />
*We perform a fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if it is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_3Week 32007-09-03T15:28:12Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/17/07'''<br />
<br />
*We amplify some bio- bricks of interest for our project from the IGEM plates. We resuspend and transform: <br />
**[http://partsregistry.org/Part:BBa_J04430 J04430] to test the GFP fluorescence detection with our experimental set up;<br />
**[http://partsregistry.org/Part:BBa_J04431 J04431] to test the GFP (+LVA) half-life since we need a GFP with a short one for our project;<br />
**[http://partsregistry.org/Part:BBa_J04500 J04500], the PLac promoter inducible by IPTG;<br />
**[http://partsregistry.org/Part:BBa_J04631 J04631], the GFP (+LVA) protein. <br />
*We strake on plates with the right antibiotic.<br />
<br />
<br />
::'''07/18/07'''<br />
<br />
*We perform a test for GFP induction with IPTG. We pick a colony from the [http://partsregistry.org/Part:BBa_J04430 J04430] and [http://partsregistry.org/Part:BBa_J04431 J04431] plates and grow it in 5 ml of LB medium during the day. In the afternoon we diluite the 5 ml cultures in 50 ml and let them growing overnight.<br />
<br />
*We pick a colony from 07/17 plates, inoculate each in 5 ml of LB medium and incubate overnight. <br />
<br />
<br />
::'''07/19/07'''<br />
<br />
*In the morning we grow the [http://partsregistry.org/Part:BBa_J04430 J04430] and [http://partsregistry.org/Part:BBa_J04431 J04431] cultures to an OD = 0.4 – 0.6. We add 1 mM IPTG to induce GFP expression to test fluorescence after 2 h. ([[photos]]).<br />
*Althought [http://partsregistry.org/Part:BBa_J04431 J04431] works rigth in presence of IPTG, [http://partsregistry.org/Part:BBa_J04430 J04430] doesn't. So, we perform a run on electrophoresis gel for the [http://partsregistry.org/Part:BBa_J04430 J04430] plasmid. We find that it doesn't match the rigth molecular weight. We think maybe there's something wrong with the plasmid.<br />
<br />
<br />
::'''07/20/07'''<br />
<br />
*''Plasmid digestion (link):''we digest [http://partsregistry.org/Part:BBa_J04500 J04500] with Spe1 and Pst1 and [http://partsregistry.org/Part:BBa_J04631 J04631] with Xba1 and Pst1.<br />
*We perform the gel extraction procedure and store at -20°C.<br />
*We amplify [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051]from the IGEM plates. <br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_6Week 62007-09-03T15:25:53Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/06/07'''<br />
* [http://partsregistry.org/Part:BBa_I763005 I763005] digestion with Spe1/Pst1;<br />
*Ligation for [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]); <br />
*We inoculate a [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010]colony in 5 ml.<br />
<br />
<br />
<br />
<br />
::'''08/07/07'''<br />
*Miniprep for [http://partsregistry.org/Part:BBa_P0412 P0412] and [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
*We inoculate a colony of [http://partsregistry.org/Part:BBa_J04431 J04431] in 5 ml to perform the fluorescence test with glucose (see the following protocol).<br />
<br />
'''[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:'''<br />
<br />
''-growth of 10 aliquots (1 ml/each) at 37°C for 1h;''<br />
<br />
''-collection of 5 ml in 2 tubes;''<br />
<br />
''-fluorescence control: bacterial cells from one tube are fluorescent and cells from the other not;''<br />
<br />
''-add glucose 2 mM in each falcon;''<br />
<br />
''-incubation at 37°C for 1 h;''<br />
<br />
''-there are no fluorescence bacteria in the two cultures;''<br />
<br />
''-after 2 hours the fluorescence is the same.''<br />
<br />
We check bacteria from the stock and we see that some of them are fluorescent, even if we don’t expect this result.<br />
*We strake on plate from falcon with glucose, we discard the supernatant, we clean in LB for 2 times, we resuspend in 5 ml of LB. Bacteria become fluorescent. When we add 2 mM of glucose the bacteria are no fluorescent.<br />
<br />
So, it is possible to control Plac activation with glucose. As it's known in literature, endogenous LacI is not enough to repress Plac promoter from a high copy number plasmid since a small amount of glucose arises the cinetic rateo of Plac trascription.<br />
We are going to insert LacI in our construct to solve this problem.<br />
This test enables us to verify GFP emivita which is about 40 minutes (link literature).<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412] digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_P0412 P0412] band extraction;<br />
*Transformation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligation. <br />
<br />
<br />
<br />
<br />
<br />
::'''08/08/07'''<br />
*Model analysis.<br />
<br />
<br />
<br />
::'''08/09/07'''<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] (I763010) ligation;<br />
*We inoculate [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/10/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] and of [http://partsregistry.org/Part:BBa_I763016 I763016]; <br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe1/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] control digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] extraction from gel. <br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_3Week 32007-09-03T15:11:26Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/17/07'''<br />
<br />
*We amplify some bio- bricks of interest for our project from the IGEM plates. We resuspend and transform: <br />
**[http://partsregistry.org/Part:BBa_J04430 J04430] to test the GFP fluorescence detection with our experimental set up;<br />
**[http://partsregistry.org/Part:BBa_J04431 J04431] to test the GFP (+LVA) half-life since we need a GFP with a short one for our project;<br />
**[http://partsregistry.org/Part:BBa_J04500 J04500], the PLac promoter inducible by IPTG;<br />
**[http://partsregistry.org/Part:BBa_J04631 J04631], the GFP (+LVA) protein. <br />
*We strake on plates with the right antibiotic.<br />
<br />
<br />
::'''07/18/07'''<br />
<br />
*We perform a test for GFP induction with IPTG. We pick a colony from the [http://partsregistry.org/Part:BBa_J04430 J04430] and [http://partsregistry.org/Part:BBa_J04431 J04431] plates and grow it in 5 ml of LB medium during the day. In the afternoon we diluite the 5 ml cultures in 50 ml and let them growing overnight.<br />
<br />
*We pick a colony from 07/17 plates, inoculate each in 5 ml of LB medium and incubate overnight. <br />
<br />
<br />
::'''07/19/07'''<br />
<br />
*In the morning we grow the [http://partsregistry.org/Part:BBa_J04430 J04430] and [http://partsregistry.org/Part:BBa_J04431 J04431] cultures to an OD = 0.4 – 0.6. We add 1 mM IPTG to induce GFP expression to test fluorescence after 2 h. ([[photos]]).<br />
*Althought [http://partsregistry.org/Part:BBa_J04431 J04431] works rigth in presence of IPTG, [http://partsregistry.org/Part:BBa_J04430 J04430] doesn't. So, we perform a run on electroforesis gel for the [http://partsregistry.org/Part:BBa_J04430 J04430] plasmid. We find that it doesn't match the rigth molecular weight. We think maybe there's something wrong with the plasmid.<br />
<br />
<br />
::'''07/20/07'''<br />
<br />
*''Plasmid digestion (link):''we digest [http://partsregistry.org/Part:BBa_J04500 J04500] with Spe1 and Pst1 and [http://partsregistry.org/Part:BBa_J04631 J04631] with Xba1 and Pst1.<br />
*We perform the gel extraction procedure and store at -20°C.<br />
*We amplify [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051]from the IGEM plates. <br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_3Week 32007-09-03T15:10:46Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/17/07'''<br />
<br />
*We amplify some bio- bricks of interest for our project from the IGEM plates. We resuspend and transform: <br />
**[http://partsregistry.org/Part:BBa_J04430 J04430] to test the GFP fluorescence detection with our experimental set up;<br />
**[http://partsregistry.org/Part:BBa_J04431 J04431] to test the GFP (+LVA) half-life since we need a GFP with a short one for our project;<br />
**[http://partsregistry.org/Part:BBa_J04500 J04500], the PLac promoter inducible by IPTG;<br />
**[http://partsregistry.org/Part:BBa_J04631 J04631], the GFP (+LVA) protein. <br />
*We strake on plates with the right antibiotic.<br />
<br />
<br />
::'''07/18/07'''<br />
<br />
*We perform a test for GFP induction with IPTG. We pick a colony from the [http://partsregistry.org/Part:BBa_J04430 J04430] and [http://partsregistry.org/Part:BBa_J04431 J04431] plates and grow it in 5 ml of LB medium during the day. In the afternoon we diluite the 5 ml cultures in 50 ml and let them growing overnight.<br />
<br />
*We pick a colony from 07/17 plates, inoculate each in 5 ml of LB medium and incubate overnight. <br />
<br />
<br />
::'''07/19/07'''<br />
<br />
*In the morning we grow the [http://partsregistry.org/Part:BBa_J04430 J04430] and [http://partsregistry.org/Part:BBa_J04431 J04431] cultures to an OD = 0.4 – 0.6. We add 1 mM IPTG to induce GFP expression to test fluorescence after 2 h. ([[photos]]).<br />
*Althought [http://partsregistry.org/Part:BBa_J04431 J04431] works rigth in presence of IPTG, [http://partsregistry.org/Part:BBa_J04430 J04430] doesn't. So, we perform a run on electroforesis gel for the [http://partsregistry.org/Part:BBa_J04430 J04430] plasmid. We find that it doesn't match the rigth molecular weight. We think maybe there's something wrong with the plasmid.<br />
<br />
<br />
::'''07/20/07'''<br />
<br />
*''Plasmid digestion (link):''we digest [http://partsregistry.org/Part:BBa_J04500 J04500] with Spe1 and Pst1 and [http://partsregistry.org/Part:BBa_J04631 J04631] with Xba1 and Pst1.<br />
*We perform the gel extraction procedure and store at -20°C.<br />
*We amplify amd transform [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051]from the IGEM plate. <br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_5Week 52007-09-03T15:09:02Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/30/07'''<br />
*We check bacterial growth curve (link).<br />
*Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012], [http://partsregistry.org/Part:BBa_J52034 J52034] and [http://partsregistry.org/Part:BBa_B0015 B0015].<br />
*Ligation [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 2 ul, insert 6 ul) and the ligation [http://partsregistry.org/Part:BBa_J04500 J04500]+ [http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)](vector 2 ul, insert 10 ul).<br />
<br />
<br />
<br />
<br />
::'''07/31/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012] , so we inoculate [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015] colony in 5 ml.<br />
*We strake on plates 30/07 ligations.<br />
*We get along with the [http://partsregistry.org/Part:BBa_J04431 J04431] fluorescence test:<br />
-From the glycerol stock we sprout 1 ml at 37°C for 1 hour in agitation.<br />
<br />
-Every 15 minutes we check bacterial OD and fluorescence. Cells are fluorescent at t= 210 min and OD=0.5.<br />
* We decide to repeat the test to understand if we can switch off the fluorescent cells with a higher amount of glucose.<br />
We will perform this test on 07/08.<br />
<br />
<br />
<br />
<br />
::'''08/01/07'''<br />
<br />
-Miniprep of [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015];<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_B0015 B0015] with Xba/Pst1;<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_I52034 I52034] with Spe/Pst1;<br />
<br />
- Gel extraction (we can’t see [http://partsregistry.org/Part:BBa_B0015 B0015]);<br />
<br />
-[http://partsregistry.org/Part:BBa_R0015 R0015]+[http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)] ligation which doesn’t give colonies so we transform with the other 10 ul of ligation; <br />
<br />
-[http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051][http://partsregistry.org/Part:BBa_I763005 (I763005)]ligation which gives colonies so we inoculate in 5ml;<br />
<br />
-Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012] (4ul) and for [http://partsregistry.org/Part:BBa_J22101 J22101] (2ul);<br />
<br />
-A new [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 4ul, insert 8ul) ligation. <br />
<br />
<br />
<br />
<br />
::'''08/02/07'''<br />
<br />
-Miniprep of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)];<br />
<br />
-Control digestion of 5 ul with Eco/Pst1;<br />
<br />
-A new [http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Xba/Pst1;<br />
<br />
-Run on electroforesis gel. We have problems with gel because we don’t see the band;<br />
<br />
-Band extraction for [http://partsregistry.org/Part:BBa_B0015 B0015] e [http://partsregistry.org/Part:BBa_I763005 I763005];<br />
<br />
-There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012], but there are colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)]. So, we identify the correct ligation protocol;<br />
<br />
- [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_C0012 C0012] transformation;<br />
<br />
-Inoculation in 5 ml for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] and for [http://partsregistry.org/Part:BBa_J22101 J22101];<br />
<br />
- [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] fluorescence test to check if the RFP filter works properly.<br />
<br />
<br />
<br />
::'''08/03/07'''<br />
<br />
-Miniprep for [http://partsregistry.org/Part:BBa_J22101 J22101] and for [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J22101 J22101] digestion with Xba/Pst1 ;<br />
<br />
- Control digestion of [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_B0015 B0015]and [http://partsregistry.org/Part:BBa_J22101 J22101] and [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] run on electroforesis gel and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_J22101 J22101] extraction;<br />
<br />
-Glycerol stocks preparation;<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T15:07:58Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
* Transformation again for [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test ligation protocol. We try with vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C 1 h and at 65°C for 10 minutes. <br />
*Then we transform 4 ul from the ligation reaction. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. We inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051],[http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we inoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. After 30 minutes the cells are already green.<br />
*We harvest and put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We measure OD every 20 minutes.<br />
<br />
*We get along with another GFP induction: after 10 minutes the cells are already green.<br />
*We transform bacteria with 4 ul of the [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul from [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*Midiprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Midiprep Quantification: conc. 65 ng/ul;<br />
<br />
*We observe colonies for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We inoculate in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform again with [http://partsregistry.org/Part:BBa_R0051 R0051] and with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We let colonies from [http://partsregistry.org/Part:BBa_R0051 R0051] and from[http://partsregistry.org/Part:BBa_R1051 R1051] grow at 37°C during the day. We inoculate in 5 ml in the evening and we leave them O/N.<br />
*We perform a fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if it is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T15:02:19Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We get along with:<br />
- another transformation for [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes. <br />
*Then we transform 4 ul from the ligation reaction. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. We inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051],[http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we inoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. After 30 minutes the cells are already green.<br />
*We harvest and put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We measure OD every 20 minutes.<br />
<br />
*We get along with another GFP induction: yet after 10 minutes the cells are green.<br />
*We transform bacteria with 4 ul from [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul from [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Quantification: conc. 65 ng/ul;<br />
<br />
-Cleanness n.:1.8.<br />
*We observe colonies for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We inoculate in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform again with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We let colonies from [http://partsregistry.org/Part:BBa_R0051 R0051] and from[http://partsregistry.org/Part:BBa_R1051 R1051] grow at 37°C during the day. We inoculate in 5 ml in the evening and we leave them O/N.<br />
*We perform a fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if it is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_9Week 92007-09-03T14:04:12Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/27/07'''<br />
*New [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation with a new protocol (3h 15°C and 1.2 ul Ligase).<br />
*Transformation of [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation and of new biobricks [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103]. <br />
<br />
<br />
<br />
<br />
::'''08/28/07'''<br />
*Lab meeting on: <br />
-model<br />
-LacI cloning under the pTetR [http://partsregistry.org/Part:BBa_R0040 R0040])promoter control; <br />
<br />
*We transform new biobricks:[http://partsregistry.org/Part:BBa_E0020 E0020] and [http://partsregistry.org/Part:BBa_R0040 R0040]<br />
<br />
* We inoculate a [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103] colony in 5 ml;<br />
<br />
* We observe that there are no colonies to [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412]([http://partsregistry.org/Part:BBa_I763010 (I763010)] ligation.<br />
<br />
::'''08/29/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103].<br />
*[http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103] digestions with Xba/Pst1.<br />
*[http://partsregistry.org/Part:BBa_B0034 B0034] digestion with Spe/Pst1.<br />
*Run on electroforesi gel and [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034] band extraction. We notice problems with [http://partsregistry.org/Part:BBa_J06550 J06550] band height (the insert is too heavy) and with [http://partsregistry.org/Part:BBa_S0103 S0103] band because the plasmid too heavy, however we extract insert bands for both.<br />
*Ligation with a new protocol of [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_J06550 J06550] ([http://partsregistry.org/Part:BBa_I763016 I763016]), [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_S0103 S0103] (missed in the sendbox),[http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J22101 J22101] (missed in the sendbox), [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763020 I763020]).<br />
*We transform the ligations. <br />
<br />
<br />
<br />
<br />
::'''08/30/07'''<br />
*There are colonies in the ligations.<br />
*We inoculate ligations, [http://partsregistry.org/Part:BBa_E0020 E0020] and [http://partsregistry.org/Part:BBa_R0040 R0040] colony in 5 ml.<br />
*We perform some tests to set up the fluorescence acquisition with the PMT (photomultiplier tube).<br />
<br />
<br />
<br />
::'''08/31/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] + [http://partsregistry.org/Part:BBa_J06550 J06550] [http://partsregistry.org/Part:BBa_I763016 (I763016)], [http://partsregistry.org/Part:BBa_I763012 I763012]+ [http://partsregistry.org/Part:BBa_S0103 S0103] (missed in the sandbox), [http://partsregistry.org/Part:BBa_B0034 B0034] + <br />
[http://partsregistry.org/Part:BBa_J22101 J22101] (missed in the sandbox), [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_J04631 J04631] [http://partsregistry.org/Part:BBa_I763020 (I763020)], [http://partsregistry.org/Part:BBa_E0020 E0020 ] and of [http://partsregistry.org/Part:BBa_R0040 R0040].<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_8Week 82007-09-03T13:53:58Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/20/07'''<br />
*[http://partsregistry.org/Part:BBa_I763010 I763010] and [http://partsregistry.org/Part:BBa_C0012 C0012] transformation. <br />
<br />
<br />
<br />
::'''08/21/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_I763010 I763010] and [http://partsregistry.org/Part:BBa_C0012 C0012]. We can't amplify these biobricks.<br />
*[http://partsregistry.org/Part:BBa_J04631 J04631] digestion. <br />
*Band extraction. <br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010])ligation.<br />
<br />
<br />
<br />
<br />
::'''08/22/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010])<br />
<br />
<br />
<br />
::'''08/23/07'''<br />
*A new [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation with a new protocol (3h 15°C and 1.2 ul T4 Ligase).<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051], [http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe/Pst1.<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_J04631 J04631] digestion with Xba/Pst1.<br />
*Run and extraction from gel.<br />
*Transformation of [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/24/07'''<br />
*We plan new amplifications.<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_6Week 62007-09-03T13:52:48Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/06/07'''<br />
* [http://partsregistry.org/Part:BBa_I763005 I763005] digestion with Spe1/Pst1;<br />
*Ligation for [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]); <br />
*We inoculate in 5 ml for [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
<br />
<br />
<br />
<br />
::'''08/07/07'''<br />
*Miniprep for [http://partsregistry.org/Part:BBa_P0412 P0412] and [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
*We inoculate a colony of [http://partsregistry.org/Part:BBa_J04431 J04431] in 5 ml to perform the fluorescence test with glucose (see the following protocol).<br />
<br />
'''[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:'''<br />
<br />
''-growth of 1 ml 10 aliquot at 37°C for 1h;''<br />
<br />
''-collection of 5 ml in 2 falcon;''<br />
<br />
''-fluorescence control (1 green, 1 not);''<br />
<br />
''-add glucose 2 mM in each falcon;''<br />
<br />
''-incubation at 37°C for 1 h;''<br />
<br />
''-in each falcon there aren't fluorescence bacteria;''<br />
<br />
''-idem after 2 hours.''<br />
<br />
We check bacteria from the stock and we see that some of them are fluorescent, even if we don’t expect this result.<br />
*We strake on plate from falcon with glucose, we discard the supernatant, we clean in LB for 2 times, we resuspend in 5 ml of LB. Bacteria become fluorescent. When we add 2 mM of glucose the bacteria are no fluorescent.<br />
<br />
So, it is possible to control Plac activation with glucose. As it's known in literature, endogenous LacI is not enough to repress Plac promoter from a high copy number plasmid and a small amount of glucose arises the cinetic rateo of Plac trascription.<br />
We are going to insert LacI in our construct to solve this problem.<br />
This test enables us to verify GFP emivita which is about 40 minutes (link literature).<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412] digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_P0412 P0412] band extraction;<br />
*Transformation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligation. <br />
<br />
<br />
<br />
<br />
<br />
::'''08/08/07'''<br />
*Model analysis.<br />
<br />
<br />
<br />
::'''08/09/07'''<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] (I763010) ligation;<br />
*We inoculate [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/10/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] and of [http://partsregistry.org/Part:BBa_I763016 I763016]; <br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe1/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] control digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] extraction from gel. <br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_5Week 52007-09-03T13:47:06Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/30/07'''<br />
*We check bacterial growth curve (link).<br />
*Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012], [http://partsregistry.org/Part:BBa_J52034 J52034] and [http://partsregistry.org/Part:BBa_B0015 B0015].<br />
*We get along with the ligation [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 2 ul, insert 6 ul) and the ligation [http://partsregistry.org/Part:BBa_J04500 J04500]+ [http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)](vector 2 ul, insert 10 ul).<br />
<br />
<br />
<br />
<br />
::'''07/31/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012] , so we inoculate [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015] colony in 5 ml.<br />
*We strake on plates 30/07 ligations.<br />
*We get along with the [http://partsregistry.org/Part:BBa_J04431 J04431] fluorescence test:<br />
-From the glycerol stock we sprout 1 ml at 37°C for 1 hour in agitation.<br />
<br />
-Every 15 minutes we check bacterial OD and fluorescence. Cells are fluorescent at t= 210 min and OD=0.5.<br />
* We decide to repeat the test to understand if we can switch off the fluorescent cells with a higher amount of glucose.<br />
We will perform this test on 07/08.<br />
<br />
<br />
<br />
<br />
::'''08/01/07'''<br />
<br />
-Miniprep of [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015];<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_B0015 B0015] with Xba/Pst1;<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_I52034 I52034] with Spe/Pst1;<br />
<br />
- Gel extraction (we can’t see [http://partsregistry.org/Part:BBa_B0015 B0015]);<br />
<br />
-[http://partsregistry.org/Part:BBa_R0015 R0015]+[http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)] ligation which doesn’t give colonies so we transform with the other 10 ul of ligation; <br />
<br />
-[http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051][http://partsregistry.org/Part:BBa_I763005 (I763005)]ligation which gives colonies so we inoculate in 5ml;<br />
<br />
-Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012] (4ul) and for [http://partsregistry.org/Part:BBa_J22101 J22101] (2ul);<br />
<br />
-A new [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 4ul, insert 8ul) ligation. <br />
<br />
<br />
<br />
<br />
::'''08/02/07'''<br />
<br />
-Miniprep of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)];<br />
<br />
-Control digestion of 5 ul with Eco/Pst1;<br />
<br />
-A new [http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Xba/Pst1;<br />
<br />
-Run on electroforesis gel. We have problems with gel because we don’t see the band;<br />
<br />
-Band extraction for [http://partsregistry.org/Part:BBa_B0015 B0015] e [http://partsregistry.org/Part:BBa_I763005 I763005];<br />
<br />
-There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012], but there are colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)]. So, we identify the correct ligation protocol;<br />
<br />
- [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_C0012 C0012] transformation;<br />
<br />
-Inoculation in 5 ml for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] and for [http://partsregistry.org/Part:BBa_J22101 J22101];<br />
<br />
- [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] fluorescence test to check if the RFP filter works properly.<br />
<br />
<br />
<br />
::'''08/03/07'''<br />
*We get along with:<br />
-Miniprep for [http://partsregistry.org/Part:BBa_J22101 J22101] and for [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J22101 J22101] digestion with Xba/Pst1 ;<br />
<br />
- Control digestion of [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_B0015 B0015]and [http://partsregistry.org/Part:BBa_J22101 J22101] and [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] run on electroforesis gel and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_J22101 J22101] extraction;<br />
<br />
-Glycerol stocks preparation;<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_5Week 52007-09-03T13:46:49Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/30/07'''<br />
*We check bacterial growth curve (link).<br />
*Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012], [http://partsregistry.org/Part:BBa_J52034 J52034] and [http://partsregistry.org/Part:BBa_B0015 B0015].<br />
*We get along with the ligation [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 2 ul, insert 6 ul) and the ligation [http://partsregistry.org/Part:BBa_J04500 J04500]+ [http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)](vector 2 ul, insert 10 ul).<br />
<br />
<br />
<br />
<br />
::'''07/31/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012] , so we inoculate [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015] colony in 5 ml.<br />
*We strake on plates 30/07 ligations.<br />
*We get along with the [http://partsregistry.org/Part:BBa_J04431 J04431] fluorescence test:<br />
-From the glycerol stock we sprout 1 ml at 37°C for 1 hour in agitation.<br />
<br />
-Every 15 minutes we check bacterial OD and fluorescence. Cells are fluorescent at t= 210 min and OD=0.5.<br />
* We decide to repeat the test to understand if we can switch off the fluorescent cells with a higher amount of glucose.<br />
We will perform this test on 07/08.<br />
<br />
<br />
<br />
<br />
::'''08/01/07'''<br />
*<br />
-Miniprep of [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015];<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_B0015 B0015] with Xba/Pst1;<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_I52034 I52034] with Spe/Pst1;<br />
<br />
- Gel extraction (we can’t see [http://partsregistry.org/Part:BBa_B0015 B0015]);<br />
<br />
-[http://partsregistry.org/Part:BBa_R0015 R0015]+[http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)] ligation which doesn’t give colonies so we transform with the other 10 ul of ligation; <br />
<br />
-[http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051][http://partsregistry.org/Part:BBa_I763005 (I763005)]ligation which gives colonies so we inoculate in 5ml;<br />
<br />
-Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012] (4ul) and for [http://partsregistry.org/Part:BBa_J22101 J22101] (2ul);<br />
<br />
-A new [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 4ul, insert 8ul) ligation. <br />
<br />
<br />
<br />
<br />
::'''08/02/07'''<br />
*<br />
-Miniprep of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)];<br />
<br />
-Control digestion of 5 ul with Eco/Pst1;<br />
<br />
-A new [http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Xba/Pst1;<br />
<br />
-Run on electroforesis gel. We have problems with gel because we don’t see the band;<br />
<br />
-Band extraction for [http://partsregistry.org/Part:BBa_B0015 B0015] e [http://partsregistry.org/Part:BBa_I763005 I763005];<br />
<br />
-There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012], but there are colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)]. So, we identify the correct ligation protocol;<br />
<br />
- [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_C0012 C0012] transformation;<br />
<br />
-Inoculation in 5 ml for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] and for [http://partsregistry.org/Part:BBa_J22101 J22101];<br />
<br />
- [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] fluorescence test to check if the RFP filter works properly.<br />
<br />
<br />
<br />
::'''08/03/07'''<br />
*We get along with:<br />
-Miniprep for [http://partsregistry.org/Part:BBa_J22101 J22101] and for [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J22101 J22101] digestion with Xba/Pst1 ;<br />
<br />
- Control digestion of [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_B0015 B0015]and [http://partsregistry.org/Part:BBa_J22101 J22101] and [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] run on electroforesis gel and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_J22101 J22101] extraction;<br />
<br />
-Glycerol stocks preparation;<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_5Week 52007-09-03T13:45:48Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/30/07'''<br />
*We check bacterial growth curve (link).<br />
*Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012], [http://partsregistry.org/Part:BBa_J52034 J52034] and [http://partsregistry.org/Part:BBa_B0015 B0015].<br />
*We get along with the ligation [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 2 ul, insert 6 ul) and the ligation [http://partsregistry.org/Part:BBa_J04500 J04500]+ [http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)](vector 2 ul, insert 10 ul).<br />
<br />
<br />
<br />
<br />
::'''07/31/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012] , so we inoculate [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015] colony in 5 ml.<br />
*We strake on plates 30/07 ligations.<br />
*We get along with the [http://partsregistry.org/Part:BBa_J04431 J04431] fluorescence test:<br />
-From the glycerol stock we sprout 1 ml at 37°C for 1 hour in agitation.<br />
<br />
-Every 15 minutes we check bacterial OD and fluorescence. Cells are fluorescent at t= 210 min and OD=0.5.<br />
* We decide to repeat the test to understand if we can switch off the fluorescent cells with a higher amount of glucose.<br />
We will perform this test on 07/08.<br />
<br />
<br />
<br />
<br />
::'''08/01/07'''<br />
*We get along with:<br />
-Miniprep of [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015];<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_B0015 B0015] with Xba/Pst1;<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_I52034 I52034] with Spe/Pst1;<br />
<br />
- Gel extraction (we can’t see [http://partsregistry.org/Part:BBa_B0015 B0015]);<br />
<br />
-[http://partsregistry.org/Part:BBa_R0015 R0015]+[http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)] ligation which doesn’t give colonies so we transform with the other 10 ul of ligation; <br />
<br />
-[http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051][http://partsregistry.org/Part:BBa_I763005 (I763005)]ligation which gives colonies so we inoculate in 5ml;<br />
<br />
-Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012] (4ul) and for [http://partsregistry.org/Part:BBa_J22101 J22101] (2ul);<br />
<br />
-A new [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 4ul, insert 8ul) ligation. <br />
<br />
<br />
<br />
<br />
::'''08/02/07'''<br />
*Today we do:<br />
-Miniprep of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)];<br />
<br />
-Control digestion of 5 ul with Eco/Pst1;<br />
<br />
-A new [http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Xba/Pst1;<br />
<br />
-Run on electroforesis gel. We have problems with gel because we don’t see the band;<br />
<br />
-Band extraction for [http://partsregistry.org/Part:BBa_B0015 B0015] e [http://partsregistry.org/Part:BBa_I763005 I763005];<br />
<br />
-There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012], but there are colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)]. So, we identify the correct ligation protocol;<br />
<br />
- [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_C0012 C0012] transformation;<br />
<br />
-Inoculation in 5 ml for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] and for [http://partsregistry.org/Part:BBa_J22101 J22101];<br />
<br />
- [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] fluorescence test to check if the RFP filter works properly.<br />
<br />
<br />
<br />
::'''08/03/07'''<br />
*We get along with:<br />
-Miniprep for [http://partsregistry.org/Part:BBa_J22101 J22101] and for [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J22101 J22101] digestion with Xba/Pst1 ;<br />
<br />
- Control digestion of [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_B0015 B0015]and [http://partsregistry.org/Part:BBa_J22101 J22101] and [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] run on electroforesis gel and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_J22101 J22101] extraction;<br />
<br />
-Glycerol stocks preparation;<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_5Week 52007-09-03T13:45:32Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/30/07'''<br />
*We check bacterial growth curve (link).<br />
*Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012] and [http://partsregistry.org/Part:BBa_J52034 J52034] and [http://partsregistry.org/Part:BBa_B0015 B0015].<br />
*We get along with the ligation [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 2 ul, insert 6 ul) and the ligation [http://partsregistry.org/Part:BBa_J04500 J04500]+ [http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)](vector 2 ul, insert 10 ul).<br />
<br />
<br />
<br />
<br />
::'''07/31/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012] , so we inoculate [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015] colony in 5 ml.<br />
*We strake on plates 30/07 ligations.<br />
*We get along with the [http://partsregistry.org/Part:BBa_J04431 J04431] fluorescence test:<br />
-From the glycerol stock we sprout 1 ml at 37°C for 1 hour in agitation.<br />
<br />
-Every 15 minutes we check bacterial OD and fluorescence. Cells are fluorescent at t= 210 min and OD=0.5.<br />
* We decide to repeat the test to understand if we can switch off the fluorescent cells with a higher amount of glucose.<br />
We will perform this test on 07/08.<br />
<br />
<br />
<br />
<br />
::'''08/01/07'''<br />
*We get along with:<br />
-Miniprep of [http://partsregistry.org/Part:BBa_I52034 I52034] and [http://partsregistry.org/Part:BBa_B0015 B0015];<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_B0015 B0015] with Xba/Pst1;<br />
<br />
-Digestion of [http://partsregistry.org/Part:BBa_I52034 I52034] with Spe/Pst1;<br />
<br />
- Gel extraction (we can’t see [http://partsregistry.org/Part:BBa_B0015 B0015]);<br />
<br />
-[http://partsregistry.org/Part:BBa_R0015 R0015]+[http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)] ligation which doesn’t give colonies so we transform with the other 10 ul of ligation; <br />
<br />
-[http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051][http://partsregistry.org/Part:BBa_I763005 (I763005)]ligation which gives colonies so we inoculate in 5ml;<br />
<br />
-Transformation for [http://partsregistry.org/Part:BBa_C0012 C0012] (4ul) and for [http://partsregistry.org/Part:BBa_J22101 J22101] (2ul);<br />
<br />
-A new [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] (vector 4ul, insert 8ul) ligation. <br />
<br />
<br />
<br />
<br />
::'''08/02/07'''<br />
*Today we do:<br />
-Miniprep of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)];<br />
<br />
-Control digestion of 5 ul with Eco/Pst1;<br />
<br />
-A new [http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Xba/Pst1;<br />
<br />
-Run on electroforesis gel. We have problems with gel because we don’t see the band;<br />
<br />
-Band extraction for [http://partsregistry.org/Part:BBa_B0015 B0015] e [http://partsregistry.org/Part:BBa_I763005 I763005];<br />
<br />
-There are no colonies for [http://partsregistry.org/Part:BBa_C0012 C0012], but there are colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507][http://partsregistry.org/Part:BBa_I763007 (I763007)]. So, we identify the correct ligation protocol;<br />
<br />
- [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_C0012 C0012] transformation;<br />
<br />
-Inoculation in 5 ml for [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] and for [http://partsregistry.org/Part:BBa_J22101 J22101];<br />
<br />
- [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] fluorescence test to check if the RFP filter works properly.<br />
<br />
<br />
<br />
::'''08/03/07'''<br />
*We get along with:<br />
-Miniprep for [http://partsregistry.org/Part:BBa_J22101 J22101] and for [http://partsregistry.org/Part:BBa_R0051 R0051]+ [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J22101 J22101] digestion with Xba/Pst1 ;<br />
<br />
- Control digestion of [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)];<br />
<br />
- [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_B0015 B0015]and [http://partsregistry.org/Part:BBa_J22101 J22101] and [http://partsregistry.org/Part:BBa_R0051 R0051] + [http://partsregistry.org/Part:BBa_I13507 I13507] [http://partsregistry.org/Part:BBa_I763007 (I763007)] run on electroforesis gel and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_C0051 C0051] [http://partsregistry.org/Part:BBa_I763005 (I763005)] and [http://partsregistry.org/Part:BBa_J22101 J22101] extraction;<br />
<br />
-Glycerol stocks preparation;<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T13:33:25Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We get along with:<br />
- another transformation for [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes. <br />
*Then we transform 4 ul from the ligation reaction. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. We inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051],[http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we inoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. After 30 minutes the cells are already green.<br />
*We harvest and put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We measure OD every 20 minutes.<br />
<br />
*We get along with another GFP induction: yet after 10 minutes the cells are green.<br />
*We transform bacteria with 4 ul from [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul from [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Quantification: conc. 65 ng/ul;<br />
<br />
-Cleanness n.:1.8.<br />
*We observe colonies for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We inoculate in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform again with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We leave colonies from [http://partsregistry.org/Part:BBa_R0051 R0051] and from[http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml in the evening and we leave them O/N.<br />
*We get along with fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if it is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T13:29:54Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We get along with:<br />
- another transformation for [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes. <br />
*Then we transform 4 ul from the ligation reaction. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. We inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051],[http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we inoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. Yet after 30 minutes the cells are green.<br />
*We harvest and put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We measure OD every 20 minutes.<br />
<br />
*We get along with another GFP induction: yet after 10 minutes the cells are green.<br />
*We transform bacteria with 4 ul of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul of [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Quantification: conc. 65 ng/ul;<br />
<br />
-Cleanness n.:1.8.<br />
*We observe colony for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We preinoculate in 5 ml and at evening in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and alternatively with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We leave colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] and for [http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml at evening and we leave them O/N.<br />
*We get along with fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if fluorescence is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T13:29:05Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We get along with:<br />
- another transformation for [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes. <br />
*Then we transform 4 ul from the ligation reaction. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plates. We inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051],[http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we preinoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. Yet after 30 minutes the cells are green.<br />
*We harvest and put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We measure OD every 20 minutes.<br />
<br />
*We get along with another GFP induction: yet after 10 minutes the cells are green.<br />
*We transform bacteria with 4 ul of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul of [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Quantification: conc. 65 ng/ul;<br />
<br />
-Cleanness n.:1.8.<br />
*We observe colony for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We preinoculate in 5 ml and at evening in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and alternatively with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We leave colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] and for [http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml at evening and we leave them O/N.<br />
*We get along with fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if fluorescence is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T10:38:23Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We get along with:<br />
- another transformation of [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes. <br />
*Then we transforme 4 ul of ligate. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colony for [http://partsregistry.org/Part:BBa_C0051 C0051] on the plate. Then we preinoculate in 5 ml all the day and at evening we inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we preinoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. Yet after 30 minutes the cells are green.<br />
*We harvest and put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We measure OD every 20 minutes.<br />
<br />
*We get along with another GFP induction: yet after 10 minutes the cells are green.<br />
*We transform bacteria with 4 ul of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul of [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Quantification: conc. 65 ng/ul;<br />
<br />
-Cleanness n.:1.8.<br />
*We observe colony for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We preinoculate in 5 ml and at evening in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and alternatively with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We leave colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] and for [http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml at evening and we leave them O/N.<br />
*We get along with fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if fluorescence is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T10:37:23Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We get along with:<br />
- another transformation of [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes. <br />
*Then we transforme 4 ul of ligate. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colony for [http://partsregistry.org/Part:BBa_C0051 C0051] on the plate. Then we preinoculate in 5 ml all the day and at evening we inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we preinoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. Yet after 30 minutes the cells are green.<br />
*We harvest them and we put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We read every 20 minutes.<br />
<br />
*We get along with another GFP induction: yet after 10 minutes the cells are green.<br />
*We transform bacteria with 4 ul of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul of [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Quantification: conc. 65 ng/ul;<br />
<br />
-Cleanness n.:1.8.<br />
*We observe colony for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We preinoculate in 5 ml and at evening in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and alternatively with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We leave colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] and for [http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml at evening and we leave them O/N.<br />
*We get along with fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if fluorescence is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T10:36:14Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We get along with:<br />
- another transformation of [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes. <br />
*Then we transforme 4 ul of ligate. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colony for [http://partsregistry.org/Part:BBa_C0051 C0051] on the plate. Then we preinoculate in 5 ml all the day and at evening we inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we preinoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. Yet after 30 minutes the cells are green.<br />
*We harvest them and we put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We read every 20 minutes.<br />
<br />
*We get along with another GFP induction: yet after 10 minutes the cells are green.<br />
*We transform bacteria with 4 ul of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul of [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Quantification: conc. 65 ng/ul;<br />
<br />
-Cleanness n.:1.8.<br />
*We observe colony for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We preinoculate in 5 ml and at evening in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and alternatively with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We leave colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] and for [http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml at evening and we leave them O/N.<br />
*We get along with fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) and [http://partsregistry.org/Part:BBa_J04431 J04431] cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if fluorescence is caused by glucose depletion in the culture medium and/or by a deficient LacI quantity inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T10:34:41Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We get along with:<br />
- another transformation of [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes. <br />
*Then we transforme 4 ul of ligate. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colony for [http://partsregistry.org/Part:BBa_C0051 C0051] on the plate. Then we preinoculate in 5 ml all the day and at evening we inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we preinoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. Yet after 30 minutes the cells are green.<br />
*We harvest them and we put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We read every 20 minutes.<br />
<br />
*We get along with another GFP induction: yet after 10 minutes the cells are green.<br />
*We transform bacteria with 4 ul of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul of [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Quantification: conc. 65 ng/ul;<br />
<br />
-Cleanness n.:1.8.<br />
*We observe colony for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We preinoculate in 5 ml and at evening in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and alternatively with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We leave colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] and for [http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml at evening and we leave them O/N.<br />
*We get along with fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) cells and in IPTG not yet inducted [http://partsregistry.org/Part:BBa_J04431 J04431] cells.So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if fluorescence is caused by glucose depletion in the culture medium and/or by a deficient LacI quantity inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_4Week 42007-09-03T10:23:21Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/23/07'''<br />
*We observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] on the plate and none of [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We get along with:<br />
- another transformation of [http://partsregistry.org/Part:BBa_R0051 R0051] (2 ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];<br />
<br />
- the ligation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes. <br />
*Then we transforme 4 ul of ligate. <br />
<br />
<br />
<br />
::'''07/24/07''' <br />
*We observe colony for [http://partsregistry.org/Part:BBa_C0051 C0051] on the plate. Then we preinoculate in 5 ml all the day and at evening we inoculate in 50 ml and we leave at 37°C O/N.<br />
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.<br />
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we preinoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. Yet after 30 minutes the cells are green.<br />
*We harvest them and we put them in 5 ml without IPTG to see the extinction time of the protein.<br />
*We read every 20 minutes.<br />
<br />
*We get along with another GFP induction: yet after 10 minutes the cells are green.<br />
*We transform bacteria with 4 ul of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10 ul of [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) ligations.<br />
*We strake on plates.<br />
<br />
<br />
<br />
::'''07/25/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:<br />
-Quantification: conc. 65 ng/ul;<br />
<br />
-Cleanness n.:1.8.<br />
*We observe colony for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We preinoculate in 5 ml and at evening in 50 ml.<br />
*We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051]. <br />
*We transform with all the [http://partsregistry.org/Part:BBa_R0051 R0051] we have and alternatively with [http://partsregistry.org/Part:BBa_R1051 R1051].<br />
<br />
<br />
<br />
<br />
::'''07/26/07'''<br />
*We leave colonies for [http://partsregistry.org/Part:BBa_R0051 R0051] and for [http://partsregistry.org/Part:BBa_R1051 R1051] at 37°C during the day. We inoculate in 5 ml at evening and we leave them O/N.<br />
*We get along with fluorescence test of J04431 and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocoll.<br />
*Then we inoculate in 5 ml O/N.<br />
<br />
<br />
<br />
<br />
::'''07/27/07'''<br />
*We get along with Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:<br />
-Quantification: conc.:56 ng/ul.<br />
*Digestion for : <br />
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;<br />
<br />
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;<br />
<br />
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.<br />
*Then we extract from gel.<br />
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] (I763004) cells and in IPTG not yet inducted [http://partsregistry.org/Part:BBa_J04431 J04431] cells. To explain this unespected observation, we decide to identify the istant during the bacterial growth when the fluorescence appears to understand if fluorescence appearance is determinated by glucose depletion in the culture medium and/or by a deficient LacI quantity inside the cell. Infact the indogenous LacI in coli is sufficient for an only Plac and we suspect it can’t repress the high copy number plasmid Plac we have used. (We will perform this test on 31/07)<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_6Week 62007-09-03T10:20:58Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/06/07'''<br />
* [http://partsregistry.org/Part:BBa_I763005 I763005] digestion with Spe1/Pst1;<br />
*Ligation for [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]); <br />
*We inoculate in 5 ml for [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
<br />
<br />
<br />
<br />
::'''08/07/07'''<br />
*Miniprep for [http://partsregistry.org/Part:BBa_P0412 P0412] and [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
*We inoculate a colony of [http://partsregistry.org/Part:BBa_J04431 J04431] in 5 ml to perform the fluorescence test with glucose (see the following protocol).<br />
<br />
'''[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:'''<br />
<br />
''-growth of 1 ml 10 aliquot at 37°C for 1h;''<br />
<br />
''-collection of 5 ml in 2 falcon;''<br />
<br />
''-fluorescence control (1 green, 1 not);''<br />
<br />
''-add glucose 2 mM in each falcon;''<br />
<br />
''-incubation at 37°C for 1 h;''<br />
<br />
''-in each falcon there aren't fluorescence bacteria;''<br />
<br />
''-idem after 2 hours.''<br />
<br />
Perhaps we have problem with the stock, infact we see some fluorescent bacteria in 10 ul of the stock on the micro slide, even if we don’t expect this result.<br />
*We strake on plate from falcon with glucose, we discard the supernatant, we clean in LB for 2 times, we risuspend in 5 ml of LB. We observe the bacteria become fluorescent. When we add 2 mM of glucose the bacteria don’t become fluorescent.<br />
At the end of this test we conclude it is possible to control the Plac attivation with glucose. As it's known in literature, endogenous LacI is not sufficient to repress Plac promoter from a high copy number plasmid and a small amount of glucose arises the cinetic rateo of Plac trascription.<br />
We are going to insert in our construct esogenous LacI to solve this problem.<br />
This test enables us to verify GFP emivita which is about 40 minutes (link literature).<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412] digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_P0412 P0412] band extraction;<br />
*Transformation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligation. <br />
<br />
<br />
<br />
<br />
<br />
::'''08/08/07'''<br />
*Model analysis.<br />
<br />
<br />
<br />
::'''08/09/07'''<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] (I763010) ligation;<br />
*We inoculate [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/10/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] and of [http://partsregistry.org/Part:BBa_I763016 I763016]; <br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe1/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] control digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] extraction from gel. <br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_6Week 62007-09-03T10:18:11Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/06/07'''<br />
* [http://partsregistry.org/Part:BBa_I763005 I763005] digestion with Spe1/Pst1;<br />
*Ligation for [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]); <br />
*We inoculate in 5 ml for [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
<br />
<br />
<br />
<br />
::'''08/07/07'''<br />
*Miniprep for [http://partsregistry.org/Part:BBa_P0412 P0412] and [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
*We inoculate a colony of [http://partsregistry.org/Part:BBa_J04431 J04431] in 5 ml to perform the fluorescence test with glucosio (see the following protocol).<br />
<br />
'''[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:'''<br />
<br />
''-growth of 1 ml 10 aliquot at 37°C for 1h;''<br />
<br />
''-collection of 5 ml in 2 falcon;''<br />
<br />
''-fluorescence control (1 green, 1 not);''<br />
<br />
''-add glucose 2 mM in each falcon;''<br />
<br />
''-incubation at 37°C for 1 h;''<br />
<br />
''-in each falcon there aren't fluorescence bacteria;''<br />
<br />
''-idem after 2 hours.''<br />
<br />
Perhaps we have problem with the stock, infact we see some fluorescent bacteria in 10 ul of the stock on the micro slide, even if we don’t expect this result.<br />
*We strake on plate from falcon with glucose, we discard the supernatant, we clean in LB for 2 times, we risuspend in 5 ml of LB. We observe the bacteria become fluorescent. When we add 2 mM of glucose the bacteria don’t become fluorescent.<br />
At the end of this test we conclude it is possible to control the Plac attivation with glucose. As it's known in literature, endogenous LacI is not sufficient to repress Plac promoter from a high copy number plasmid and a small amount of glucose arises the cinetic rateo of Plac trascription.<br />
We are going to insert in our construct esogenous LacI to solve this problem.<br />
This test enables us to verify GFP emivita which is about 40 minutes (link literature).<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412] digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_P0412 P0412] band extraction;<br />
*Transformation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligation. <br />
<br />
<br />
<br />
<br />
<br />
::'''08/08/07'''<br />
*Model analysis.<br />
<br />
<br />
<br />
::'''08/09/07'''<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] (I763010) ligation;<br />
*We inoculate [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/10/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] and of [http://partsregistry.org/Part:BBa_I763016 I763016]; <br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe1/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] control digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] extraction from gel. <br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_6Week 62007-09-03T10:16:37Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/06/07'''<br />
* [http://partsregistry.org/Part:BBa_I763005 I763005] digestion with Spe1/Pst1;<br />
*Ligation for [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]); <br />
*We inoculate in 5 ml for [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
<br />
<br />
<br />
<br />
::'''08/07/07'''<br />
*Miniprep for [http://partsregistry.org/Part:BBa_P0412 P0412] and [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
*We inoculate a colony of [http://partsregistry.org/Part:BBa_J04431 J04431] in 5 ml to perform the fluorescence test with glucosio (see the following protocol).<br />
<br />
'''[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:'''<br />
<br />
''-growth of 1 ml 10 aliquot at 37°C for 1h;''<br />
<br />
''-collection of 5 ml in 2 falcon;''<br />
<br />
''-fluorescence control (1 green, 1 not);''<br />
<br />
''-add glucose 2 mM in each falcon;''<br />
<br />
''-incubation at 37°C for 1 h;''<br />
<br />
''-in each falcon there aren't fluorescence bacteria;''<br />
<br />
''-idem after 2 hours.''<br />
<br />
Perhaps we have problem with the stock, infact we see some fluorescent bacteria in 10 ul of the stock on the micro slide, even if we don’t expect this result.<br />
*We strake on plate from falcon with glucose, we discard the supernatant, we clean in LB for 2 times, we risuspend in 5 ml of LB. We observe the bacteria become fluorescent. When we add 2 mM of glucose the bacteria don’t become fluorescent.<br />
At the end of this test we conclude it is possible to control the Plac attivation with glucose. As it's known in literature, endogenous LacI is not sufficient to repress it and a little quantity of glucose arises the cinetic rateo of Plac trascription.<br />
We are going to insert in our construct esogenous LacI to solve this problem.<br />
This test enables us to verify GFP emivita which is about 40 minutes (link literature).<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412] digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_P0412 P0412] band extraction;<br />
*Transformation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligation. <br />
<br />
<br />
<br />
<br />
<br />
::'''08/08/07'''<br />
*Model analysis.<br />
<br />
<br />
<br />
::'''08/09/07'''<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] (I763010) ligation;<br />
*We inoculate [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/10/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] and of [http://partsregistry.org/Part:BBa_I763016 I763016]; <br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe1/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] control digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] extraction from gel. <br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_6Week 62007-09-03T10:15:17Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/06/07'''<br />
* [http://partsregistry.org/Part:BBa_I763005 I763005] digestion with Spe1/Pst1;<br />
*Ligation for [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]); <br />
*We inoculate in 5 ml for [http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
<br />
<br />
<br />
<br />
::'''08/07/07'''<br />
*Miniprep for [http://partsregistry.org/Part:BBa_P0412 P0412] and [http://partsregistry.org/Part:BBa_R0010 R0010].<br />
*We inoculate a colony of [http://partsregistry.org/Part:BBa_J04431 J04431] in 5 ml to perform the fluorescence test with glucosio (see the following protocol).<br />
<br />
'''[http://partsregistry.org/Part:BBa_J04431 J04431] bacteria from glycerol stock:'''<br />
<br />
''-growth of 1 ml 10 aliquot at 37°C for 1h;''<br />
<br />
''-collection of 5 ml in 2 falcon;''<br />
<br />
''-fluorescence control (1 green, 1 not);''<br />
<br />
''-add glucose 2 mM in each falcon;''<br />
<br />
''-incubation at 37°C for 1 h;''<br />
<br />
''-in each falcon there aren't fluorescence bacteria;''<br />
<br />
''-idem after 2 hours.''<br />
<br />
Perhaps we have problem with the stock, infact we see some fluorescent bacteria in 10 ul of the stock on the micro slide, even if we don’t expect this result.<br />
*We strake on plate from falcon with glucose, we discard the supernatant, we clean in LB for 2 times, we risuspend in 5 ml of LB. We observe the bacteria become fluorescent. When we add 2 mM of glucose the bacteria don’t become fluorescent.<br />
At the end of this test we conclude it is possible to control the Plac attivation with glucose. As it's known in literature, endogenous LacI is not sufficient to repress it and a little quantity of glucose arises the cinetic rateo of Plac trascription.<br />
We are going to insert in our construct esogenous LacI to solve this problem.<br />
This test enable us to verify GFP emivita which is about 40 minutes as is know in literature.<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_B0015 B0015] digestion with Eco/Xba;<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412] digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_J04431 J04431], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_P0412 P0412] band extraction;<br />
*Transformation of [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligation. <br />
<br />
<br />
<br />
<br />
<br />
::'''08/08/07'''<br />
*Model analysis.<br />
<br />
<br />
<br />
::'''08/09/07'''<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] (I763010) ligation;<br />
*We inoculate [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J22101 J22101] ([http://partsregistry.org/Part:BBa_I763012 I763012]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/10/07'''<br />
*Miniprep of [http://partsregistry.org/Part:BBa_I763012 I763012] and of [http://partsregistry.org/Part:BBa_I763016 I763016]; <br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe1/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] control digestion with Xba/Pst1;<br />
*[http://partsregistry.org/Part:BBa_I763012 I763012] extraction from gel. <br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_3Week 32007-09-03T10:12:10Z<p>Alice.pasini: </p>
<hr />
<div>::'''07/17/07'''<br />
<br />
*We amplify some bio- bricks of interest for our project from the IGEM plates. We resuspend and transform: <br />
**[http://partsregistry.org/Part:BBa_J04430 J04430] to test the GFP fluorescence detection with our experimental set up;<br />
**[http://partsregistry.org/Part:BBa_J04431 J04431] to test the GFP (+LVA) half-life since we need a GFP with a short one for our project;<br />
**[http://partsregistry.org/Part:BBa_J04500 J04500], the PLac promoter inducible by IPTG;<br />
**[http://partsregistry.org/Part:BBa_J04631 J04631], the GFP (+LVA) protein. <br />
*We strake on plates with the right antibiotic.<br />
<br />
<br />
::'''07/18/07'''<br />
<br />
*We perform a test for GFP induction with IPTG. We pick a colony from the [http://partsregistry.org/Part:BBa_J04430 J04430] and [http://partsregistry.org/Part:BBa_J04431 J04431] plates and grow it in 5 ml of LB medium during the day. In the afternoon we diluite the 5 ml cultures in 50 ml and let them growing overnight.<br />
<br />
*We pick a colony from 07/17 plates, inoculate each in 5 ml of LB medium and incubate overnight. <br />
<br />
<br />
::'''07/19/07'''<br />
<br />
*In the morning we diluite the [http://partsregistry.org/Part:BBa_J04430 J04430] and [http://partsregistry.org/Part:BBa_J04431 J04431] cultures to an OD=0.05. We grow the cultures till an OD = 0.4 – 0.6. We add 1 mM IPTG to induce GFP expression and we test fluorescence after 2 h. ([[photos]]).<br />
*Althought [http://partsregistry.org/Part:BBa_J04431 J04431] works rigth in presence of IPTG, [http://partsregistry.org/Part:BBa_J04430 J04430] doesn't. So, we perform a run on electroforesis gel for the [http://partsregistry.org/Part:BBa_J04430 J04430] plasmid. We find that it doesn't match the rigth molecular weight. We think maybe there's something wrong with the plasmid.<br />
<br />
<br />
::'''07/20/07'''<br />
<br />
*''Plasmid digestion (link):''we digest [http://partsregistry.org/Part:BBa_J04500 J04500] with Spe1 and Pst1 and [http://partsregistry.org/Part:BBa_J04631 J04631] with Xba1 and Pst1.<br />
*We perform the gel extraction procedure and store at -20°C.<br />
*We amplify amd transform [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051]from the IGEM plate. <br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_8Week 82007-09-03T10:07:57Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/20/07'''<br />
*[http://partsregistry.org/Part:BBa_I763010 I763010] and [http://partsregistry.org/Part:BBa_C0012 C0012] transformation. <br />
<br />
<br />
<br />
::'''08/21/07'''<br />
*There aren't colonies for [http://partsregistry.org/Part:BBa_I763010 I763010] and [http://partsregistry.org/Part:BBa_C0012 C0012]. We can't amplify these biobricks.<br />
*[http://partsregistry.org/Part:BBa_J04631 J04631] digestion. <br />
*Band extraction. <br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010])ligation.<br />
<br />
<br />
<br />
<br />
::'''08/22/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010])<br />
<br />
<br />
<br />
::'''08/23/07'''<br />
*A new [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation with a new protocol (3h 15°C and 1.2 ul T4 Ligase).<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051], [http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe/Pst1.<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_J04631 J04631] digestion with Xba/Pst1.<br />
*Run and extraction from gel.<br />
*Transformation of [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/24/07'''<br />
*We plan new amplifications.<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_8Week 82007-09-03T10:07:16Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/20/07'''<br />
*[http://partsregistry.org/Part:BBa_I763010 I763010] and [http://partsregistry.org/Part:BBa_C0012 C0012] transformation. <br />
<br />
<br />
<br />
::'''08/21/07'''<br />
*There aren't colonies for [http://partsregistry.org/Part:BBa_I763010 I763010] and [http://partsregistry.org/Part:BBa_C0012 C0012]. We can't amplify this biobrick.<br />
*[http://partsregistry.org/Part:BBa_J04631 J04631] digestion. <br />
*Band extraction. <br />
*[http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010])ligation.<br />
<br />
<br />
<br />
<br />
::'''08/22/07'''<br />
*There are no colonies for [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010])<br />
<br />
<br />
<br />
::'''08/23/07'''<br />
*A new [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation with a new protocol (3h 15°C and 1.2 ul T4 Ligase).<br />
*[http://partsregistry.org/Part:BBa_R0051 R0051], [http://partsregistry.org/Part:BBa_I763012 I763012] digestion with Spe/Pst1.<br />
*[http://partsregistry.org/Part:BBa_P0412 P0412], [http://partsregistry.org/Part:BBa_J04631 J04631] digestion with Xba/Pst1.<br />
*Run and extraction from gel.<br />
*Transformation of [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligations.<br />
<br />
<br />
<br />
<br />
::'''08/24/07'''<br />
*We plan new amplifications.<br />
<br />
<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/Week_9Week 92007-08-30T15:48:34Z<p>Alice.pasini: </p>
<hr />
<div>::'''08/27/07'''<br />
*New [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation with a new protocol (3h 15°C and 1.2 ul Ligasi).<br />
*Transformation of [http://partsregistry.org/Part:BBa_R0051 R0051]+[http://partsregistry.org/Part:BBa_P0412 P0412] ([http://partsregistry.org/Part:BBa_I763010 I763010]) ligation and of new biobricks [http://partsregistry.org/Part:BBa_S03520 S03520], [http://partsregistry.org/Part:BBa_S0100 S0100], [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_J06550 J06550], [http://partsregistry.org/Part:BBa_S0103 S0103].<br />
<br />
<br />
<br />
<br />
::'''08/28/07'''<br />
<br />
<br />
<br />
<br />
::'''08/29/07'''<br />
<br />
<br />
<br />
<br />
::'''08/30/07'''<br />
<br />
<br />
<br />
<br />
::'''08/31/07'''<br />
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<br />
<br />
[[Bologna | Back]]</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/BolognaBologna2007-08-27T15:06:46Z<p>Alice.pasini: </p>
<hr />
<div> <br />
[[Image:Bollo_UNIBO.png]]<br />
<br />
"Ὁ βίος βραχὺς, ἡ δὲ τέχνη μακρὴ, ὁ δὲ καιρὸς ὀξὺς, ἡ δὲ πεῖρα σφαλερὴ, ἡ δὲ κρίσις χαλεπή" <br />
<br />
"Vita brevis, ars longa, occasio praeceps, experimentum periculosum, iudicium difficile"<br />
<br />
"Art is long, life is short, opportunity is fleeting, experience is deceitful, judgement is difficult"<br />
<br />
"La vita è breve, l'arte è lunga, l'occasione fuggevole, l'esperimento pericoloso, il giudizio difficile"<br />
<br />
<br />
<br />
''(Aforisma di Ippocrate di Coo)''<br />
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<br />
<br />
<br />
== About Us ==<br />
<br />
Welcome to Bologna’s IGEM Wiki!<br />
<br />
<br />
Our [[Bologna_University/team | team]] consists of [[Bologna_University/FP | Francesco Pasqualini]], [[Bologna_University/GC | Guido Costa]], [[Bologna_University/ST | Silvia Tamarri]], [[Bologna_University/FB | Francesca Buganè]], and [[Bologna_University/MM| Michela Mirri]],<br />
all soon-to-be juniors majoring in Biomedical Engineering at Bologna University; and [[Bologna_University/AP | Alice Pasini]], a just graduated student.<br />
<br />
<br />
We are advised by [http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni], Professor of Engineering and [http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr. Emanuele Giordano], Professor of Biomedical Engineering. We are grateful to our advisors for their time and support!<br />
<br />
Our instructors are [http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti], Professor of Biomedical Engineering, [[Bologna_University/FC | Francesca Ceroni]], Graduated student in Pharmaceutical Biotechnologies, [[Bologna_University/SM | Sara Montagna]], Graduated student in Biomedical Engineering and [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini], PhD in Bioengineering.<br />
<br />
== Our Project==<br />
Visit our [[University of Bologna Cesena Campus iGEM2007 Team/Project_Description]]<br />
<br />
<br />
<br />
{| align="center" style="color:black;" border="1"<br />
|- <br />
| bgcolor="#00E040" color="white" height="30pt" align="center" | '''Our Team''' <br />
| bgcolor="white" color="white" align="center" | '''Project Design''' <br />
| bgcolor="#D00010" color="white" align="center" | '''Project Results''' <br />
|- style="color:#990000;"<br />
| align="left" width="375pt" |<br />
:'''Undergraduate Students''' <br />
::[[Bologna_University/FP | Francesco Pasqualini]] • [[Bologna_University/GC | Guido Costa]]<br />
::[[Bologna_University/ST| Silvia Tamarri]] • [[Bologna_University/MM| Michela Mirri]]<br />
::[[Bologna_University/FB | Francesca Buganè]] <br />
:'''Graduate Student''' <br />
::[[Bologna_University/AP | Alice Pasini]] <br />
:'''Instructors'''<br />
::[http://www.ing2.unibo.it/Ingegneria%20Cesena/Strumenti%20del%20Portale/Cerca/paginaWebDocente?UPN=silvio.cavalcanti@unibo.it Prof. Silvio Cavalcanti] • [[Bologna_University/FC | Francesca Ceroni]]<br />
::[[Bologna_University/SM | Sara Montagna]] • [http://www-micrel.deis.unibo.it/~christine/ Christine Nardini]<br />
:'''Advisor'''<br />
::[http://www-micro.deis.unibo.it/cgi-bin/user?tartagni Prof. Marco Tartagni]<br />
::[http://www.ing2.unibo.it/Ingegneria+Cesena/Strumenti+del+Portale/Cerca/paginaWebDocente.htm?NRMODE=Published&TabControl1=TabContatti&UPN=emanuele.giordano%40unibo.it Dr. Emanuele Giordano]<br />
| align="left" width="350pt"| <br />
:'''[[Bologna_University/Literature | Literature]]'''<br />
<br />
:'''Materials and Methods'''<br />
::1. [[Bologna_University/Procedure | Procedure]]<br />
::2. [[Bologna_University/Protocols | Protocols]]<br />
::3. [[Bologna_University/Microscopy: Sperimental Set Up| Microscopy: Sperimental Set Up]]<br />
<br />
| align="left" width="250"|<br />
:'''Results''' <br />
|-<br />
|}<br />
<br />
==Diary==<br />
<br />
{| cellspacing="2px" cellpadding="20" border="0" width="100%"<br />
|-<br />
|<center>'''[[Week 1]]''' </center><br />
|<center>'''[[Week 2]]''' </center><br />
|<center>'''[[Week 3]]''' </center><br />
|<center>'''[[Week 4]]'''</center><br />
|<center>'''[[Week 5]]''' </center><br />
|-<br />
|<center>'''[[Week 6]]''' </center><br />
|<center> '''[[Week 7]]''' </center><br />
|<center> '''[[Week 8]]'''</center><br />
|<center> '''[[Week 9]]''' </center><br />
|<center>'''[[Week 10]]'''</center><br />
|-<br />
|<center>'''[[Week 11]]''' </center><br />
|<center> '''[[Week 12]]''' </center><br />
|<center> '''[[Week 13]]'''</center><br />
|<center> '''[[Week 14]]''' </center><br />
|<center>'''[[Week 15]]'''</center><br />
|-<br />
|<center>'''[[Week 16]]''' </center><br />
|<center> '''[[Week 17]]''' </center><br />
|<center> '''[[Week 18]]'''</center><br />
|}</div>Alice.pasinihttp://2007.igem.org/wiki/index.php/File:Bologna.jpgFile:Bologna.jpg2007-08-27T14:53:26Z<p>Alice.pasini: </p>
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<div></div>Alice.pasini