ETHZ/Internal

From 2007.igem.org

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<center><font size = '+2'><b> .:: ETH Zurich - EducatETH ''E. coli'' ::. </b></font></center><br>
<center><font size = '+2'><b> .:: ETH Zurich - EducatETH ''E. coli'' ::. </b></font></center><br>
 +
 +
=='''.:: Synthetic Biology Boot camp ::.'''==
 +
To make everybody familiar with synthetic biology concepts and to assist coming up with ideas for an interesting project, we will read and present publications on important topics. The list to be covered is the following:
 +
 +
* Introduction to synthetic biology (Markus, Martin)
 +
* DNA de novo design (Rico, Raphael)
 +
* DNA circuits (Christian, Nan))
 +
* Hysteresis (Tim, Sylke)
 +
* Oscillators (Christos, Joe, Katerina and Markus, Martin)
 +
* Zinc fingers (Sylke)
 +
* Noise in single cell measurements
 +
* Distance communication (Rico, Raphael)
 +
* Parameter manipulations (Christos, Joe, Katerina)
 +
* Orthogonal systems (Christos, Joe, Katerina)
 +
* Minimal genomes (Markus, Martin)
 +
* RNA regulators (Rico, Raphael)
 +
 +
All presentations have been uploaded in the known web space. You may also contact the people who did it if you need additional information.
 +
 +
=='''.:: Brainstorming ::.'''==
 +
 +
==='''.:: Brainstorming sessions::.'''===
 +
During the first two weeks of July, the team has to come up with a project. The team is divided into three groups, which will brainstorm individually. Sven and Joerg are available on the 4th and 5th of July in case groups needs to consult them. The objective is that each group comes up with many fancy ideas. To facilitate this, keep in mind the following "brainstorming rules":
 +
 +
# Defer judgment - the rules of nature don't apply
 +
# Encourage wild ideas
 +
# Build on the ideas of others
 +
# Be visual
 +
# Go for quantity
 +
# Stay focused on topic
 +
 +
==='''.:: Preliminary ideas ::.'''===
 +
 +
* '''PID Controller''': Design a PID controller out of biological elements. The P component can be a simple output to a regulatory protein, and the I component can be the overall protein production at a time period. What can the D component be?
 +
* '''Motion Detector''': Cells are grown on a petri dish. Below the dish, moving images are displayed. A 3-state automaton is proposed. Output A is created when light is present. Output B is created when light is absent. Moving patterns will cause some cells to create both outputs over time. This will result in some “inspector” cells producing output C, by collecting outputs A and B.
 +
* '''Analog-to-Digital Converter''': Compare the level of protein concentration with thresholds, and digitize the output.
 +
* '''Neural Network''': Create a sort of biological neural network with bacteria. We should address the issue of learning, and find a way to incorporate the feedback in the cell decision making process. Directed evolution can be a sort of feedback, but we want to avoid this. (This idea was the basis for the “learning project”)
 +
* '''Paramedic Cells''': Some cells are able to detect signals coming from other cells, and create food for them, or create proteins in order to save them and make them function better.
 +
* '''Cell Batteries''': Cells are able to create and store large quantities of ATP, during a “storing process”. Afterwards, they can detect a signal and give back all the energy they stored, in a short burst, like a capacitor. Other ideas are that the cells can “blow up” and emit large amounts of GFP, based on the ATP that they have accumulated.
 +
* '''Flashing Bacteria''': Cells are grown on a light pattern. The cells that are on the bright parts of the image are oscillating in phase, while the others are remaining dark. This results in the observation of a flashing pattern.
 +
* '''Biocam''': Visible to Fluorescent light converter.
 +
* '''BioCD''': “Print” cells on a film, then read them out and “reconstruct” the original data. Basically, it is an analog to digital converter, followed by a system that can interpret the digitized data. (This idea was the basis for the “Music of life project”, where cells would produce fluorescent proteins based on an analog input. Then, the amount and type of fluorescence would code some music).
 +
* '''Clock''': A follow-the-leader system. We have to groups of cells. The first group creates something that repels the second group. The second group creates a protein that attracts the first group. This way, they first group wants to “catch” the second group, whereas the second group wants to “avoid” the first group. This results in them moving around. We can say that the second group is the leader, and the first group exhibits a "follow-the-leader” behavior.
 +
* '''Sensors''': Various systems that can sense PH, pressure, temperature, meat quality, moisture e.t.c. have been proposed.
 +
 +
==='''.:: Preferred projects::.'''===
 +
Three ideas of the above are chosen for further examination. The team will be again split up in three groups (different than before, to make sure that new ideas come up with mxing of people). Each group has to come up with an initial system, with remarks on its feasibility and coolness. Our results
 +
will be presented to all team members, so that potential projects may be limited down to two and subsequently to
 +
one. The preferred projects are:
 +
 +
# '''Music of Life''': The basic idea is that instead of having an analog-to-digital converter with four outputs (three fluorescent proteins, and no output), we can have two switches. When switch A is on, RFP is produced. When switch B is on, GFP is produced. When both switches A and B are on, a yellowish output is observed. By recording these outputs, we can later create music, by assigning each fluorescent protein to a chord. For example, RFP would correspond to a G chord. The strength of the fluorescence can signify the strength of the chord. If the cells are placed on a spinning disk, we can have something like a vinyl player. A camera is observing the cells, and music is created on the fly.
 +
# '''Learning''': Based on the idea of the neural network, we want to create a biological system, where the cells can learn a specific behavior. In order to simplify the system, we decided that the cells can learn to recognize a specific type of other cells. We divide the process in a learning phase, and a recognition phase. First, cells A are put together with cells B. Then, cells A are “learning” to recognize cells B. If afterwards they are put in a petri dish with cells B, they will emit GPF. Otherwise, they will stay dark.
 +
 +
==='''.:: Final project::.'''===
 +
 +
The chosen project is a modified version of Learning which was presented at the last meeting. The system proposed is modified as in its current state may be implemented with two switches only, something which is not exciting enough. Keeping the idea of learning, and of training and testing phases, we have come up with EducatETH <i>E.coli</i>.
=='''.:: Task List ::.'''==
=='''.:: Task List ::.'''==
Line 20: Line 76:
# '''Wiki (Project and part documentation due on 26.10)''' <br> ''Martin'': See the points above, from next Monday I will give everything, now I'm doing my best... <br> ''Rico'': I can help. I guess this will have to contain the materials that we will also use for poster and presenttion anyway. <br> ''Nan'': Shouldn't it be updated with every going on process? <br> ''Christos'': I am trying to put stuff in, as it comes along. I will update the bio pages with the presentation material, this weekend (I hope). <br> ''Katerina'': You guys have done a great job so far, will help with whatever needed.
# '''Wiki (Project and part documentation due on 26.10)''' <br> ''Martin'': See the points above, from next Monday I will give everything, now I'm doing my best... <br> ''Rico'': I can help. I guess this will have to contain the materials that we will also use for poster and presenttion anyway. <br> ''Nan'': Shouldn't it be updated with every going on process? <br> ''Christos'': I am trying to put stuff in, as it comes along. I will update the bio pages with the presentation material, this weekend (I hope). <br> ''Katerina'': You guys have done a great job so far, will help with whatever needed.
# '''T-Shirts''' <br> ''Sylke'': in print (2007-09-24)
# '''T-Shirts''' <br> ''Sylke'': in print (2007-09-24)
-
 
-
==='''.:: Wiki Task List ::.'''===
 
-
 
-
'''''Katerina'':Please write here your remarks on the wiki. Thank you.'''
 
=='''.:: In the Lab ::.'''==
=='''.:: In the Lab ::.'''==

Revision as of 12:45, 15 October 2007

Eth zh logo 2.png
Main Page      Biology Pespective      Engineering Perspective      Meet the Team      Team Notes      Pictures!

.:: ETH Zurich - EducatETH E. coli ::.

Contents

.:: Synthetic Biology Boot camp ::.

To make everybody familiar with synthetic biology concepts and to assist coming up with ideas for an interesting project, we will read and present publications on important topics. The list to be covered is the following:

  • Introduction to synthetic biology (Markus, Martin)
  • DNA de novo design (Rico, Raphael)
  • DNA circuits (Christian, Nan))
  • Hysteresis (Tim, Sylke)
  • Oscillators (Christos, Joe, Katerina and Markus, Martin)
  • Zinc fingers (Sylke)
  • Noise in single cell measurements
  • Distance communication (Rico, Raphael)
  • Parameter manipulations (Christos, Joe, Katerina)
  • Orthogonal systems (Christos, Joe, Katerina)
  • Minimal genomes (Markus, Martin)
  • RNA regulators (Rico, Raphael)

All presentations have been uploaded in the known web space. You may also contact the people who did it if you need additional information.

.:: Brainstorming ::.

.:: Brainstorming sessions::.

During the first two weeks of July, the team has to come up with a project. The team is divided into three groups, which will brainstorm individually. Sven and Joerg are available on the 4th and 5th of July in case groups needs to consult them. The objective is that each group comes up with many fancy ideas. To facilitate this, keep in mind the following "brainstorming rules":

  1. Defer judgment - the rules of nature don't apply
  2. Encourage wild ideas
  3. Build on the ideas of others
  4. Be visual
  5. Go for quantity
  6. Stay focused on topic

.:: Preliminary ideas ::.

  • PID Controller: Design a PID controller out of biological elements. The P component can be a simple output to a regulatory protein, and the I component can be the overall protein production at a time period. What can the D component be?
  • Motion Detector: Cells are grown on a petri dish. Below the dish, moving images are displayed. A 3-state automaton is proposed. Output A is created when light is present. Output B is created when light is absent. Moving patterns will cause some cells to create both outputs over time. This will result in some “inspector” cells producing output C, by collecting outputs A and B.
  • Analog-to-Digital Converter: Compare the level of protein concentration with thresholds, and digitize the output.
  • Neural Network: Create a sort of biological neural network with bacteria. We should address the issue of learning, and find a way to incorporate the feedback in the cell decision making process. Directed evolution can be a sort of feedback, but we want to avoid this. (This idea was the basis for the “learning project”)
  • Paramedic Cells: Some cells are able to detect signals coming from other cells, and create food for them, or create proteins in order to save them and make them function better.
  • Cell Batteries: Cells are able to create and store large quantities of ATP, during a “storing process”. Afterwards, they can detect a signal and give back all the energy they stored, in a short burst, like a capacitor. Other ideas are that the cells can “blow up” and emit large amounts of GFP, based on the ATP that they have accumulated.
  • Flashing Bacteria: Cells are grown on a light pattern. The cells that are on the bright parts of the image are oscillating in phase, while the others are remaining dark. This results in the observation of a flashing pattern.
  • Biocam: Visible to Fluorescent light converter.
  • BioCD: “Print” cells on a film, then read them out and “reconstruct” the original data. Basically, it is an analog to digital converter, followed by a system that can interpret the digitized data. (This idea was the basis for the “Music of life project”, where cells would produce fluorescent proteins based on an analog input. Then, the amount and type of fluorescence would code some music).
  • Clock: A follow-the-leader system. We have to groups of cells. The first group creates something that repels the second group. The second group creates a protein that attracts the first group. This way, they first group wants to “catch” the second group, whereas the second group wants to “avoid” the first group. This results in them moving around. We can say that the second group is the leader, and the first group exhibits a "follow-the-leader” behavior.
  • Sensors: Various systems that can sense PH, pressure, temperature, meat quality, moisture e.t.c. have been proposed.

.:: Preferred projects::.

Three ideas of the above are chosen for further examination. The team will be again split up in three groups (different than before, to make sure that new ideas come up with mxing of people). Each group has to come up with an initial system, with remarks on its feasibility and coolness. Our results will be presented to all team members, so that potential projects may be limited down to two and subsequently to one. The preferred projects are:

  1. Music of Life: The basic idea is that instead of having an analog-to-digital converter with four outputs (three fluorescent proteins, and no output), we can have two switches. When switch A is on, RFP is produced. When switch B is on, GFP is produced. When both switches A and B are on, a yellowish output is observed. By recording these outputs, we can later create music, by assigning each fluorescent protein to a chord. For example, RFP would correspond to a G chord. The strength of the fluorescence can signify the strength of the chord. If the cells are placed on a spinning disk, we can have something like a vinyl player. A camera is observing the cells, and music is created on the fly.
  2. Learning: Based on the idea of the neural network, we want to create a biological system, where the cells can learn a specific behavior. In order to simplify the system, we decided that the cells can learn to recognize a specific type of other cells. We divide the process in a learning phase, and a recognition phase. First, cells A are put together with cells B. Then, cells A are “learning” to recognize cells B. If afterwards they are put in a petri dish with cells B, they will emit GPF. Otherwise, they will stay dark.

.:: Final project::.

The chosen project is a modified version of Learning which was presented at the last meeting. The system proposed is modified as in its current state may be implemented with two switches only, something which is not exciting enough. Keeping the idea of learning, and of training and testing phases, we have come up with EducatETH E.coli.

.:: Task List ::.

.:: Project Task List ::.

The things to do, from the most pressing (timewise), to the least pressing (timewise) is below. Please put your name next to the task that you believe that you can undertake.

  1. Team descriptions (overdue) and team photograph
    Christian: I guess I am the only semiprofessional photographer of the group. I can do some group pictures etc. but for this we need some ideas... - I would like to do something special. I also made the group-pics of the Synth. Biology 3.0 conference. Some references  ;-)  : [http://www.fotocommunity.de/pc/pc/mypics/461397]
    Raphael: What about a short movie of us?
  2. Team rosters due (1.9.)
    Martin: Does somebody know, what exactly should be done here?
    Nan: A list of team members, including some basic personal info.? (e.g. nationality, background, pet peeves...? )
    Katerina: Guys, to make this easier, either write things about you on your personal page on the wiki or link to a page about you. This way it'll be easy to put it all together afterwards.
  3. Labwork (parts have to be at the registry in Boston on 26.10.)
    Joe: I can be in the lab at least 2 evenings a week and some times through the weekdays.
    Martin: From Monday I can work every day for the whole day. At the moment I only work for several hours...
    Rico: I have my exam on Tuesday. Afterwards I can assist.
    Christian: I can do the introduction of the polylinker into the vectors beside my normal labwork on the Hoenggerberg. I could also do the whole biobrick assembly if you want this (I will go on holiday from the 5.10-24.10.07)
    Raphael: That's the part where I will mainly contribute, from 14.09. on I can work several days/week
    Christos: I can assist at the afternoons, if needed.
    Katerina: 7.09 - 30.09 generally plenty of time, apart from when I do my semester project presentation, will keep you posted when that is.
    Sylke: I'll not be available until 14th of October but from then on I can work several days per week fulltime.
  4. Testing/ Analysis/ Detection (due to 02.11.)
    Sylke: Meeting with Alfredo Franco-Obregon (FACS guy from center) on 18.09. concerning devices available for detection of GFP/ derivatives.
  5. Simulation and sensitivity analysis
    Martin: From 10. Sept. I've got plenty time to work on it. I think Markus would join here too ;-)
    Tim: I can contribute in running stuff and help identify parameters from literature
    Rico: I can do simulations, sensitivity analysis.
    Nan: I would like to do simulations and sensitivity analysis.
    Christos: Yeap, I guess I can be here too. I will check some toolboxes to automate things, this weekend.
    Katerina: Want to help (parameter identification, programming, sensitivity analysis). Discuss in upcoming meeting tasks, versioning and ask Christian about parameters.
  6. Presentation
    Joe: I'm American... I can sale anything.
    Martin: I'm bad in Layout stuff, but maybe I can help as an idea supplier or so.
    Rico: I like giving presentations. For preparation we will need a mixture of different background and excellent pictures!!!
    Christian: I can provide the molecular biology knowledge and part.
    Nan: I can work on the slides.
    Christos: I like this part, I guess everyone will contribute anyway...
    Katerina: I believe I can be of help in structure, layout and fancy stuff. Could help train a bit the people we decide to do the presentation (question answering, style). I also think that Joe and Christos could be good for doing the presentation (structured, pleasant voices and lively), can discuss this in a meeting.
    Sylke: I can help with the bio-part and I'm good at doing graphics and stuff. Can be a help when it needs to look good :-)
  7. Poster
    Joe: See 5. above
    Rico: I can help.
    Christian: I can provide the molecular biology knowledge and part.
    Nan: Partly art. I will help.
    Christos: You can sell, but can you trick? lol :)
    Katerina: Not my strong point, but have an eye for typos, fonts, layout etc, so can help in final checking.
    Sylke: See above. Good at layout.
  8. Wiki (Project and part documentation due on 26.10)
    Martin: See the points above, from next Monday I will give everything, now I'm doing my best...
    Rico: I can help. I guess this will have to contain the materials that we will also use for poster and presenttion anyway.
    Nan: Shouldn't it be updated with every going on process?
    Christos: I am trying to put stuff in, as it comes along. I will update the bio pages with the presentation material, this weekend (I hope).
    Katerina: You guys have done a great job so far, will help with whatever needed.
  9. T-Shirts
    Sylke: in print (2007-09-24)

.:: In the Lab ::.

.:: List of system building blocks ::.

Here is a list of all the registry parts we used as bulding blocks for our system parts. This list has to be updated and extended for the new, concatenated parts that the ETH Zurich has submitted to the registry.

  1. [http://partsregistry.org/Part:BBa_B0034 B0034]
  2. [http://partsregistry.org/Part:BBa_R0062 R0062]
  3. [http://partsregistry.org/Part:BBa_R0053 R0053]
  4. [http://partsregistry.org/Part:BBa_J23100 J23100]
  5. [http://partsregistry.org/Part:BBa_J37033 J37033]
  6. [http://partsregistry.org/Part:BBa_E0434 E0434]
  7. [http://partsregistry.org/Part:BBa_B0015 B0015]
  8. [http://partsregistry.org/Part:BBa_Q04400 Q04400]
  9. [http://partsregistry.org/Part:BBa_R0010 R0010]
  10. [http://partsregistry.org/Part:BBa_E0422 E0422]
  11. [http://partsregistry.org/Part:BBa_R0040 R0040]
  12. [http://partsregistry.org/Part:BBa_R0051 R0051]
  13. [http://partsregistry.org/Part:BBa_Q04121 Q04121]
  14. [http://partsregistry.org/Part:BBa_C0053 C0053]
  15. [http://partsregistry.org/Part:BBa_Q04510 Q4510]

.:: Lab book ::.

.:: Week 1 ::.

Date TODO's Completed People
Mon, 06. Aug. 2007
  • Preparing the Solutions
Sylke
Raphael
Stefan
Markus
Martin
Christos
Joe
Tue, 07. Aug. 2007
  • Prepare competent cells for all parts
  • Transformation of all the parts
Sylke
Raphael
Stefan
Markus
Martin
Christos
Joe
Wed, 08. Aug. 2007
  • Preparing the grown cultures (12) for the MINIPREP
    (o/n cultures)
Raphael
Stefan
Thu, 09. Aug. 2007
  • MINIPREP of the grown (10) o/n cultures
  • Gelelectrophoresis of the grown cultures (step: 0.8% Agarose)
Raphael
Stefan
Martin
Christos
Joe
Fri, 10. Aug. 2007
  • 7 working parts/plasmids (step after "DIGESTS"):
    (B0034, R0062, R0053, E0434, B0015, R0010, E0422)
  • 4 parts/plasmids minipreped:
    (R0040, R0051, Q04121, C0053)

Christos
Markus
Stefan

Sat, 11. Aug. 2007 no labwork
Sun, 12. Aug. 2007 labwork cancelled

.:: Week 2 ::.

Date TODO's Completed People
Mon, 13. Aug. 2007
start at 3 pm
  • Prepare competent cells
  • Transformations of J23100, J37033, Q04400, Q04510
  • Control Restrictions (step after "MINIPREP")
    R0040, R0051, Q04121, C0053
  • o/n culture (E.Coli Top10)
  • Control Restrictions (didn't work)
Martin
Markus
Christos
Tim
Tue, 14. Aug. 2007 Morning Shift:
  • Start Preparing competent cells (for J23100, J37033, Q04400, Q04510)

Evening Shift:

  • Transformations of J23100, J37033, Q04400, Q04510
Morning Shift:
  • Prepared competent cells (stored in the -80°C freezer in the basement)

Evening Shift:

  • Transformation of J23100, J37033, Q04400, Q04510 and R0040, R0051, Q04121, C0053 (in the 37°C incubator until Wednesday)
  • Prepared new Liquid LB, LB Agar (both in the autoclave), Agarose Gel with concentrations of 0.8% and 2.4%
Morning Shift (9am-1pm?):
Markus, Tim

Evening Shift (5pm-...):
Martin, Christos
Wed, 15. Aug. 2007
  • Ligation (step: "LINK ASSEMBLY"):
    R0053 + E0422
    R0010 + E0422
    R0010 + E0434
    S/P: R0053, R0010
    X/P: E0422, E0434
  • Ligation didn't work due to bad quality of enzymes (probably)
From 12:
Martin
Markus

Thu, 16. Aug. 2007
  • Miniprep (J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053)
  • Transformation of #13 and #14
  • Miniprep of #4 (J23100), #5 (J37033), #8 (Q04400), #11 (R0040), #12 (R0051), #15 (Q04510)
    One batch is miniprepped (after step 19 in the miniprep procedure) and a second batch is frozen as a backup (which is to be miniprepped from step 3 on)
  • Transformation of #13 (Q04121) and #14 (C0053)
    Numbers #13 and #14 are now growing in the 37°C incubator (step 13 in the transformation procedure)

Markus
Christos
(Martin)

Fri, 17. Aug. 2007
  • o/n of #13 and #14
  • Check whether miniprep of parts #4 #5 #8 #11 #12 (#13 #14) #15 was successful
  • #13 and #14 didn't grow
  • # 4, 8 and 11 had the plasmid, they were streaked out new on plates, that we have them now on plates
  • New white pipette tips prepared (autoclave)
  • New bottles of Liquid LB and LB Agar prepared (autoclave)
Martin
Sat, 18. Aug. 2007
Sun, 19. Aug. 2007

.:: Week 3 ::.

Little rearrangements of the parts. Planning of the sequences to order them.


.:: Week 4 ::.

Date TODO's Completed People
Mon, 27. Aug. 2007
Tue, 28. Aug. 2007
Wed, 29. Aug. 2007
Thu, 30. Aug. 2007
Fri, 31. Aug. 2007
Sat, 01. Sept. 2007
  • Transform pbr322, pcyc177 and pck01
  • Transform pbr322, pcyc177 and pck01 and plated them
Stefan
Sun, 02. Sept. 2007
  • Prepare o/n of pbr322, pcyc177, pck01
  • o/n of pcyc177, pck01
  • the plates of pcyc177 and pck01 are in the fridge
  • transformed pbr322 because the culture didn't grow on the plate
Stefan

.:: Week 5 ::.

Date TODO's Completed People
Mon, 03. Sept. 2007
  • Prepare new competent cells
  • Miniprep pcyc177 and pcK01
  • prepare new o/n culture of pbr322
  • Run agarose gel of Minipreped plasmids
  • New competent cells prepared, they are now in the -80° Frezzer in the basement, column #17, dark orange box (we have now 30-35 EDTs of competent cells...)
  • Minipreped pcyc177 and pck01 (in the -18° freezer, where the antibiotics are)
  • pbr322 didn't grow again, so no o/n could be prepared, but we get a culture from Andy on tuesday
  • new o/n of pcyc177 and pck01 prepared (3 Falcons each), because we need to have more plasmids
  • 2 boxes of blue pipette tips are in the autoclave
  • Stefan ran the agarose gel (?)
Martin
Stefan
Tue, 04. Sept. 2007
  • Miniprep pcyc177 and pck01
  • cut the prepped plasmids to test if we've got the right ones
  • run agarose gel to test the cut and uncut ones
  • prepare new o/n of pbr322 (from Andy)
  • Miniprep of pcyc177 and pck01 (but not yet tested)
  • Prepared 3 o/ns of pbr322 (finally ;-) and each 1 o/n of pcyc177 and pck01, just in case there are problems with the miniprep
Martin
Christian
Wed, 05. Sept. 2007
  • Miniprep of pbr322
  • Test-Digest of pcyc177 and pck01 and agarose gel...
  • Streak out all three plasmids on new plates, so we have them in reserve
  • New Plate of pbr322.
  • Minipreps and Agarose Gels will be done tomorrow
Martin
Thu, 06. Sept. 2007
  • Miniprep of pbr322, pacyc177, pck01
  • Test with agarose gel
  • Gel of the older plasmids -> plasmid present
Christian
Fri, 07. Sept. 2007
  • Miniprep of pbr322, pacyc177, pck01
  • Plasmids miniprepped
Martin
Sat, 08. Sept. 2007
Sun, 09. Sept. 2007

.:: Week 6 ::.

Date TODO's Completed People
Mon, 10. Sept. 2007
  • Miniprep pBR322
  • annealing of different MCSs
  • Digest of pCK01 with BamHI+AseI
  • digest of pACYC177 with BamHI+PstI
  • digest of pBR322 with EcoRI+PstI 
 all digests o/n

Christian

Tue, 11. Sept. 2007
  • Gelextraction of backbones pBR322, pCK01, pACYC digest did NOT work
  • 1x ligation of MCS inside backbones o/d, Trafo
  • 1x ligation of MCS inside backbones o/n
  • plate all 3 plasmids for new minipreps

Christian

Wed, 12. Sept. 2007
  • Trafo of o/n ligations
  • o/n cultures of putative clones

Christian

Thu, 13. Sept. 2007
  • Minipreps of putative clones pCK01-MCS and pBR322-MCS
  • control digests of putative clones
  • new o/n cultures of the putative clones of o/n ligations

Christian

Fri, 14. Sept. 2007
  • separation of control digests of putative clones

*pBR322-MCS (Tet-selection) clone2 positive

Christian

Sat, 15. Sept. 2007
Sun, 16. Sept. 2007

.:: Week 7 ::.

Date TODO's Completed People
Mon, 17. Sept. 2007
  • new digest of pACYC177 with BamHI+PstI o/n
  • digest of pACYC177, pBR322 AP
  • ligation of 177 and 322AP
  • digest of pBR322 AP (the concentration of DNA was too low for pacyc177...)
  • ligation of pBR322 o/n
  • 100 ml o/n culture to MAXIprep pacyc177
  • Transformation of pBR322 AP to have it on plates (because andy only miniprepped them)

Christian
Martin, Raphael

Tue, 18. Sept. 2007
  • different control digests of pBR322-MCS (Tet) (see last week)
  • separation of pACYC177 digest
  • Test Digests of pck01 with XbaI, SpeI, PstI, Xba/Pst, Xba/Spe (because all of them should be in the plasmid due to the sequence, and if they are it would be crap!!!)
  • Transformation of the ligated pbr322 AP (MCS)
  • Prep pacyc177
  • Digest prepped pacyc177

> no DNA on pACYC177 digest-gel, only degradation smear

  • Plates of pbr322 AP grew
  • No Digest of pck01 worked due to too low DNA concentration... (che cazzo di low copy plasmids !!!!)
  • Miniprepped only 20 ml of the pacyc o/n culture with Quiagen Kit, the results were great! We have loads of DNA! (thank god! )
  • Digest of pacyc177 with BamHI (45 µl), then precipitated, in the gel was still very much DNA, but there were still 3 bands, so we guess, that it hasn't cut, maybe because the BamHI in the center is very old, perhaps we should Digest it in Höngg again.
  • Digest of pacyc177 with PstI o/n (pray that it will work!)
  • New o/n cultures of pck01 (to prep it like pacyc177), pbr322 AP (to prep it too, to have something on stock again, if the ligation didn't work), top10 (to make new competent cells)
  • test digest of pck01 with notI, but due to the low DNA concentration I don't think it will work. I took glooves, if it now work, then we have caught some DNases in the earlier test digests

Christian
Martin
Raphael

Wed, 19. Sept. 2007
  • o/n culture of pbr322 AP (MCS), then test digest and see if it is ligated
  • Prep of pck01 and test digests (xba, pst, spe, pvuI, notI)
  • check the digests of pacyc177 (pst) and pck01 (notI)
  • design new linkers for pck01, design primers for PCR for the extraction of SpeI from pck01
Thu, 20. Sept. 2007
Fri, 21. Sept. 2007
Sat, 22. Sept. 2007
Sun, 23. Sept. 2007

.:: Week 8 ::.

Date TODO's Completed People
Mon, 24. Sept. 2007
Tue, 25. Sept. 2007
Wed, 26. Sept. 2007
Thu, 27. Sept. 2007
Fri, 28. Sept. 2007
Sat, 29. Sept. 2007
Sun, 30. Sept. 2007

.:: Cloning plan::.

.::Plasmid 1 (pbr322ap)::.

  1. Put parts 1,2,3 in pbr322ap plasmids.
  2. Merge plasmid containing part 2 (donor) with plasmid containing part 3 (acceptor). You should get a plasmid containing a 2.3 composite part.
  3. Merge plasmid containing part 1 (donor) with plasmid containing composite part 2.3 (acceptor). You should get a plasmid containing a 1.2.3 composite part.

.::Plasmid 2 (pck01cm)::.

  1. Put parts 4,5,8,9 in pck01cm plasmids.
  2. Merge plasmid containing part 4 (donor) with plasmid containing part 5 (acceptor). You should get a plasmid containing a 4.5 composite part.
  3. Merge plasmid containing part 8 (donor) with plasmid containing part 9 (acceptor). You should get a plasmid containing a 8.9 composite part. Note: this step can be done simultaneously with the above.
  4. Merge plasmid containing composite part 4.5 (donor) with plasmid containing composite part 8.9 (acceptor). You should get a plasmid containing a 4.5.8.9 composite part.

.::Plasmid 3 (pacyc177km)::.

  1. Put parts 6,7,10,11 in pacyc177km plasmids.
  2. Merge plasmid containing part 6 (donor) with plasmid containing part 7 (acceptor). You should get a plasmid containing a 6.7 composite part.
  3. Merge plasmid containing part 10 (donor) with plasmid containing part 11 (acceptor). You should get a plasmid containing a 10.11 composite part. Note: this step can be done simultaneously with the above.
  4. Merge plasmid containing composite part 6.7 (donor) with plasmid containing composite part 10.11 (acceptor). You should get a plasmid containing a 6.7.10.11 composite part.

.::Linkers::.

  1. pck01-1
  2. pck01-2
  3. pacyc177-1
  4. pacyc177-2
  5. pbr322-1
  6. pbr322-2
  7. pbr322-3
  8. pbr322-4

Four linkers are tested for pbr322, as two are used for the tetracycline-resistance version of pbr322 and two are used for the ampicillin-resistnace version.

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