Glasgow/Wetlab

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[[Glasgow|Glasgow Main Page]]
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!align=center|[[Image:Uog.jpg]] ||    ||  [[Glasgow|<font face=georgia color=#3366CC size=4>Back To <br> Glasgow's <br> Main Page</font>]] || [[Glasgow/Modeling|<font face=georgia color=#3366CC size=4>Go To <br> Glasgow's <br> Modelling Page</font>]]
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|[[Glasgow/Wetlab/Protocols|PROTOCOLS]]
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|[https://2007.igem.org/Glasgow/Wetlab/Protocols <font face=georgia color=#3366CC size=5><b>Protocols</b></font>]
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|[[Glasgow/Wetlab/References|REFERENCES]]
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|[https://2007.igem.org/Glasgow/Wetlab/References <font face=georgia color=#3366CC size=5><b>References</b></font>]
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|[[Glasgow/Wetlab/Orders|ORDERS]]
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|[https://2007.igem.org/Glasgow/Wetlab/Resources <font face=georgia color=#3366CC size=5><b>Resources</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Orders <font face=georgia color=#3366CC size=5><b>Sequences</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Biobricks <font face=georgia color=#3366CC size=5><b>Biobricks<br>Used</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Gels <font face=georgia color=#3366CC size=5><b>Gels</b></font>]
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== Week 1 ==
 
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=== 03/07 ===
 
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#Maija and Christine prepared LB broth and LB agar with [[Glasgow/Wetlab/Protocols|Protocol 1]].  Made 4 batches of plates, 2 with kanamycin and 2 with carbenicillin.
 
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=== 04/07 ===
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#All wetlab researched BioBricks.
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#Reporter constructs and mini-Tn5 stocks looked out.
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#Streaked the following:
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#*''P. putida'' PAW 340 pJAK14 (Carb. plate)
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#*Tn5 lux AB (Carb. plate)
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#*Mini-Tn5 lux AB (Carb. plate)
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#*''P. fluorescens'' NCIMB 9815 (Carb. plate)
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#*''P. putida'' KT2440 (LB)
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#*JM109 pBluescript 5k+ (Carb. plate)
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#*Mini-Tn5 Tc (Carb. Plate)
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#*pQF52 (Carb. plate)
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#*''P. fluorescens'' 9815 (LB)
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#*''E. coli'' pJAK14 (Km plate)
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#*Il DntR in pOF52 (Carb. plate)
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#*pUCINR in Ω strain C (Carb. plate)
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#*pGLTUR (Carb. plate)
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#*Mini-Tn5 Kan (Carb. plate)
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#*Mini-Tn5 Sm/Sp (Carb. plate)
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#*Mini-Tn5 1cc2 (Carb. plate)
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#*''E. coli'' Sa1 (LB)
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#*DmpR #24 (Carb. plate)
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#*Mini-Tn5 lac 32 in ''E. coli'' 517 (Carb. plate)
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#*Mini-Tn5 Tc (Carb. Plate)
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#*Mini-Tn5 Cm (Carb. plate)
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#*DmpR WT (Carb. plate)
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#*''E.coli'' sm 10 pESD15 Tn5 GFP (Carb. plate)
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#*pUJ8 (Carb. Plate)
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=== 05/07 ===
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''Click on a week number for more detailed lab book...''
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#BioBricks – Maija and Scott transformed using [[Glasgow/Wetlab/Protocols | Protocol 2]]
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#*BBa_p1010 (DB3.1) (death gene plasmid) Plate 4: 7A – p5B1A10, 11E – p5B1A10 and 11C – p5B3K3
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#*BBa_IS2001 (Top10) (high copy number plasmid) Plate 4: 5I – p5B4A5, 5D – p5B4K5, 6B – p5B3K5, 6D – p5B4K5 and 6E – p5B4A5 (Also 5K, 5M, 4J, 4L, 4N, 4P)
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#*BBa_J23119 (Top 10) (strong constitutive promoter) Plate 3: 19A – pB1A2 (V1013)
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#*BBa_R0062 (Top 10) (HSL and luxR inducible) Plate 1: 9G – pSB1A2 (V1004)
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#*BBa_J04500 (Top 10) (IPTG inducible prom + RBS) Plate 1: 16P – p5B1AK3 (V1009)
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#*BBa_E0040 (Top 10) (GFP mutant no promoter (3b)) Plate 1: 5H – p5B1A2 (V1001)
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#Maia and Christine researched restriction enzymes for the reporter constructs and Mini-Tn5s streaked on 4/7/07 and digested some. Had planned to make biobricks using Mini-Tn5s etc but discovered that a previous attempt to use transposable elements to make biobricks had been unsuccessful due to scarring at the restriction site. http://partsregistry.org/Part:BBa_J61206
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=== 06/07 ===
 
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#Maija and Christine made 10x stocks for M9 (see [[Glasgow/Wetlab/Protocols|Protocol 3.2]]).
 
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#Tutorial in [http://www.mbio.ncsu.edu/BioEdit/bioedit.html BioEdit] and [http://biotools.umassmed.edu/bioapps/primer3_www.cgi Primer3].
 
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#Started designing primers for amplification and site directed mutagenesis of DmpR and DmpR #24 (see [[Glasgow/Wetlab/Protocols|Protocol 4]]).
 
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== Week 2 ==
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{|cellspacing="6px" cellpadding="16" border="0" width="100%"
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=== 09/07 ===
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#Christine and Maija designed primers for site directed mutagenesis in DmpR, DmpR #24, ****, **** and ****, and amplification of DmpR and DmpR #24.  Used [[Glasgow/Wetlab/Protocols|Protocol 4]], and to check Tm http://www.itt-biotech.de/itt-cgi/oligo-tm.pl
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!width="50%" | [https://2007.igem.org/Glasgow/Wetlab/Week1 <font face=georgia color=#3366CC size=7><b>Week 1</b></font>] <br>
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#Mai carried out restriction digests of DmpR and DmpR #24.  Results were poor and gel gave poor visibility.
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#Scott retransformed any of the transformations that did not work from the transformations from 5/7/07.
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#*4/11C BBa_p1010 pSB3K3 death gene
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#*4/5I BBa_I522001 pSB4A5 hi-copy
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#*4/5D BBa_I522001 pSB4K5 hi-copy
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#*4/6B BBa_I522001 pSB3K3 hi-copy
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#*4/6D BBa_I522001 pSB4K hi-copy
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#*1/5H BBa_E0040 pSB1A2 GFP non promoter
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#Used transformations that did work and set them up tubes of LB for minipreps tomorrow.
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#*BBa_I52001 death gene plasmid (hi copy number)
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#*BBa_J23119 strong constitutive promoter
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#*BBa_R0062 HSL and luxR inducible
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#*BBa_306500 IPTG inducible and RBS
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Plan is to use DmpR and DmpR #24 to detect phenol and produce lacZ.  Grown on Xgal the better the bacteria detect phenol, the more blue they will be in a spectrophotometer.
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=== 10/07 ===
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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week2 <font face=georgia color=#3366CC size=7><b>Week 2</b></font>] <br>
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#Mai did minipreps, according to Qiagen prepkit manual (see [[Glasgow/Wetlab/Protocols|Protocol 5]]), of the transformations grown in LB last night (9/7/07).
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#Wetlab and Drylab gave presentations to the team to explain key terms used in the lab (see [[Wet to Dry|Tutorials]]).
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#Christine made tetracycline stock – 250 mg tetracycline in 50 ml 100% ethanol to make 5 mg/ml stock.
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#Maija made thiamine stock (0.8 g thiamine in 20 ml dH2O and filter sterilized).  Kept in freezer in foil (light sensitive).
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#Christine grew ''E. coli'' pJAK14 in LB overnight at 37°C following protocol from Wise et al, 2000).
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#Scott and Lynsey grew DmpR and DmpR #24 overnight in LB with Tc and Carb.  This is because the plates streaked on 4/7/07  for DmpR and DmpR #24 did not grow, and restriction digests on 9/7/07 used up most of the DNA.  What is grown will be mini-prepped and restriction digested tomorrow.
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#*2x  5ml LB containing carb (50 µg/ml) with DmpR
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#*2x  5ml LB containing carb (50 µg/ml) with DmpR #24
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#*2x  5ml LB containing Tc (50 µg/ml) with DmpR
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#*2x  5ml LB containing Tc (50 µg/ml) with DmpR #24
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#*2x  5ml LB containing Tc (10 µg/ml) with DmpR
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#*2x  5ml LB containing Tc (10 µg/ml) with DmpR #24
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#Scott's retransformations (9/7/07) all worked (esp Top 10s, not so much DB3.1).  To be mini-prepped tomorrow.
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=== 11/07 ===
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|- valign=top
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#Lynsey mini-prepped Scott's retransformed biobricks (9/7/07) according to Qiagen Prepkit Manual.
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#Maija prepared glycerol for freezing the transformations in LB.
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*'''First selection of biobricks transformed.'''
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#Can not mini-prep DmpR or DmpR #24 in Tc because it did not grow well overnight.  Time for Plan B.
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*'''Preparation of stocks.'''
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#*'''Plan B'''<br>DmpR is not working – not growing or digesting as we would expect it to.  Instead of DmpR we will try XylR which detects BETX compounds (benzene, toluene, and xylene) and DntR which detects salicylate and could be modified to detect TNT and DNT.  For this we will be using pGLTUR and pQF52.
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#We all began to design primers for site directed mutagenesis (SDM) and amplification of XylR, Pr, Pu and DntR.
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*'''Researched DmpR, XylR and DntR sensor systems.'''
 +
*'''Designed primers for DmpR, DntR, XylR and associated promoters.'''
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=== 12/07 ===
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|- align="center"
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<u>XylR, Pr, and Pu</u>
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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week3 <font face=georgia color=#3366CC size=7><b>Week 3</b></font>] <br>
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#Searched [http://www.ncbi.nlm.nih.gov/Genbank GenBank] for pWW0 which contains XylR, Pr and Pu.  Saved in BioEdit.
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#Using the XylR sequence (Inouye et al, 1988) we were able to design primers for the amplification of XylR.
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#Using previously designed primers (Willardson et al, 1998) we were able to locate the beginning of Pr and designed primers to amplify the sequence between the beginning of Pr and the beginning of XylR.
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#From previously designed primers (Willardson et al, 1998) we were able to locate a sequence we believed to be Pu and designed primers. To be sure we also searched pWW0 sequence with [http://biotools.umassmed.edu/bioapps/primer3_www.cgi Primer3] to locate on the plasmid where the Willardson Pu primers would attach. Using BioEdit we located another sequence we also suspect to be Pu. We now have primers designed for both suspected sequences.
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#Also designed primers for site directed mutagenesis of the PstI site in XylR.
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(We were unable to use the Willardson primers for our purposes because they were designed to contain restriction sites, instead we used them to locate the genes of interest).
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<u>DntR</u>
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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week4 <font face=georgia color=#3366CC size=7><b>Week 4</b></font>] <br>  
-
#Scott found an article containing the sequence for DntA and some of DntR, then we used BlastX to find the sequence of DntR.  From this we were able to design primers to amplify the sequence.
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#Also designed primers so we can sequence pQF52 because the lacZ gene is not complete in the plasmid and we need to know its sequence.
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=== 13/07 ===
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|- valign=top
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#Began typing up protocols for the Wiki.
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#Ordered primers, made changes to DntR_suffix_1 which will arrive later. (See [[Glasgow/Wetlab/Orders|Orders]])
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*'''BioBricks tested by digestion and PCR.'''
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#Wiki meeting. Maija, Christine H, Majeik, Toby, Christine M, Mai, Scott and Lynsey.
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*'''PCR with new primers associated with DntR and XylR.'''
 +
|
 +
* '''phzM and phzS cloned into TOPO vectors.'''
 +
*'''Primer design for 7 gene operon.'''
 +
*'''DntR cloned into TOPO vector.'''
 +
*'''Primer design for XylR and associated genes.'''
 +
*'''Death gene cloned into TOPO vector.'''
 +
*'''Primer design for death gene.'''
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== Week 3 ==
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|- align="center"
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=== 17/07 ===
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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week5 <font face=georgia color=#3366CC size=7><b>Week 5</b></font>] <br>
-
#Restriction Digests:
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#*BBa_J23119 (strong constitutive promoter) pSB1A2. 1 x NheI, 1 x PvuI. 680bp, 1430bp
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#*BBa_R0062 (HSL and luxR) pBB1A2.  1 x EcoRI, 1 x PvuI. 1460bp, 660bp
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#*BBa_J04500 (IPTG inducer and RBS) pSB1AK3.  1 x PvuII, 2 x PvuI. 2250bp, 1030bp, 730bp
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#*BBa_p1010 (death gene) pSB3K3. 1 x BamHI, 2 x XhoI. 190bp, 2390bp,840bp.
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#*BBa_E0040 (GFP no promoter) pSB1A2. 1 x Hime II, 1 x PvuI. 1630bp, 1170bp.
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#*BBa_I52001 (6D) p5B4K5. 2 x AvaI, 1 x PvuI. 920bp, 1470bp, 2120bp.
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#*See [[Glasgow/Wetlab/Protocols|Protocol 7]] for Restriction Digests.
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=== 18/07 ===
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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week6 <font face=georgia color=#3366CC size=7><b>Week 6</b></font>] <br>
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#Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
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#Maija ran a gel of the restriction digests to check the sizes of the biobricks.  These were the maps we made yesterday.
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#Mai is extracting DNA from 3 samples of ****, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see [[Glasgow/Wetlab/Protocols|Prorocol 8]]), only change was to shorten "shaking" time from 10 to 2 minutes.
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#PCR trial run using Reddymix and Touch 2 done on:
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#*pGLTUR: XylR_prefix / XylR_suffix, Pr_prefix / Pr_suffix, Pr_prefix / XylR_suffix, Pu_prefix_EM / Pu_suffix_EM.
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#*pQF52 plasmid: DntR_prefix / DntR_suffix
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#*DmpR WT/24: DntR_prefix / DntR_suffix
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#*Biobricks: R0062, E0040, J04500, J23119, p1010, I52001.
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#*See [[Glasgow/Wetlab/Protocols|Prorocol 9]] for PCR.
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=== 19/07 ===
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|- valign=top
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#Mai redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (See [[Glasgow/Wetlab/Protocols|Prorocol 9]] for PCR protocol) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
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#*XylR prefix and XylR suffix
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*'''Suspected amplification of 7 gene operon.'''
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#*Pr prefix and Pr suffix
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* '''phzM and phzS confirmed as present in TOPO vectors.'''
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#*Pr prefix and XylR suffix
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*'''Confirmed presence of death gene plasmid in some vectors.'''
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#*Pu prefix and Pu suffix
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*'''Miller assay performed with DntR system.'''
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#*DntR prefix and DntR suffix 2
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*'''Yeast cells.'''
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*'''Biobricks made of phzM, phzS, and DntR.'''
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=== 20/07 ===
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|- align="center"
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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week7 <font face=georgia color=#3366CC size=7><b>Week 7</b></font>] <br>
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== Week 4 ==
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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week8 <font face=georgia color=#3366CC size=7><b>Week 8</b></font>] <br>
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=== 23/07 ===
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#PCR Program "Touch 2" with Emma's primers (see [[Glasgow/Wetlab/Orders|Orders 1]]) using Reddy Mix (See [[Glasgow/Wetlab/Protocols|Protocol 9.1]]) and ''Pseudomonas as our template DNA. The primer pairs used:
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##Methyl_1 and Methyl_2
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##Oxy_1 and Oxy_2
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##Bbp_Methyl_1 and Bbp_Methyl_2
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##Bbp_Oxy_1 and Bbp_Oxy_2
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##(***)_S_for_1 and (***)_S_rev_1
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##Bbp_(***)_S_1 and Bbp_(***)_S_1
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##(***)_M_for_1 and (***)_M_rev_1
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##Bbp_(***)_M_for_1 and Bbp_(***)_M_rev_1
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#PCR Program "Touch 2" with extension time of 8 mins with Emma's primers (see [[Glasgow/Wetlab/Orders|Orders 1]]) and site directed mutagenesis primers (see [[Glasgow/Wetlab/Orders|Orders 2]]) using Reddy Mix (See [[Glasgow/Wetlab/Protocols|Protocol 9.1]]) and ''Pseudomonas as our template DNA. The primer pairs used:
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##(***)Up and (***)Low
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##bbp(***)Up and bbs(***)Low
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##bbp(***)Up and (***)_SDM_EcoRI_rev
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##bbs(***)Low and (***)_SDM_EcoRI_for
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#KOD polymerase reaction on functioning DntR primers.
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=== 24/07 ===
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|- valign=top
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*'''Utilised new biobricks.'''
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*'''Site directed mutagenesis of phzM completed.'''
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*'''Site directed mutagenesis of phzS completed.'''
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=== 25/07 ===
 
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=== 26/07 ===
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|- align="center"
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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week9 <font face=georgia color=#3366CC size=7><b>Week 9</b></font>] <br>
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=== 27/07 ===
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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week10 <font face=georgia color=#3366CC size=7><b>Week 10+</b></font>] <br>
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|- valign=top
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*'''phzD→phzG successfully cloned into TOPO vector.'''
 +
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*'''Contains further work achieved.'''
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*'''XylR and Pr cloned into construction vectors.'''
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*'''XylR and Pr put next to responsive promoter and luciferase.'''
 +
|}
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{|cellspacing="6px" cellpadding="16" border="0" width="100%"
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|- align=center
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|[https://2007.igem.org/Glasgow/Wetlab/Protocols <font face=georgia color=#3366CC size=5><b>Protocols</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/References <font face=georgia color=#3366CC size=5><b>References</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Resources <font face=georgia color=#3366CC size=5><b>Resources</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Orders <font face=georgia color=#3366CC size=5><b>Orders</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Biobricks <font face=georgia color=#3366CC size=5><b>Biobricks<br>Used</b></font>]
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|[https://2007.igem.org/Glasgow/Wetlab/Gels <font face=georgia color=#3366CC size=5><b>Gels</b></font>]
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|}

Latest revision as of 11:54, 26 October 2007

Uog.jpg Back To
Glasgow's
Main Page 		Go To
Glasgow's
Modelling Page
Protocols References Resources Sequences Biobricks
Used 		Gels

Click on a week number for more detailed lab book...


Week 1
Week 2
  • First selection of biobricks transformed.
  • Preparation of stocks.
  • Researched DmpR, XylR and DntR sensor systems.
  • Designed primers for DmpR, DntR, XylR and associated promoters.
Week 3
Week 4
  • BioBricks tested by digestion and PCR.
  • PCR with new primers associated with DntR and XylR.
  • phzM and phzS cloned into TOPO vectors.
  • Primer design for 7 gene operon.
  • DntR cloned into TOPO vector.
  • Primer design for XylR and associated genes.
  • Death gene cloned into TOPO vector.
  • Primer design for death gene.
Week 5
Week 6
  • Suspected amplification of 7 gene operon.
  • phzM and phzS confirmed as present in TOPO vectors.
  • Confirmed presence of death gene plasmid in some vectors.
  • Miller assay performed with DntR system.
  • Yeast cells.
  • Biobricks made of phzM, phzS, and DntR.
Week 7
Week 8
  • Utilised new biobricks.
  • Site directed mutagenesis of phzM completed.
  • Site directed mutagenesis of phzS completed.


Week 9
Week 10+
  • phzD→phzG successfully cloned into TOPO vector.
  • Contains further work achieved.
  • XylR and Pr cloned into construction vectors.
  • XylR and Pr put next to responsive promoter and luciferase.
Protocols References Resources Orders Biobricks
Used 		Gels
Retrieved from "http://2007.igem.org/wiki/index.php/Glasgow/Wetlab"