Glasgow/Wetlab

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[[Glasgow/Wetlab/Protocols|PROTOCOLS]]
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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/References|REFERENCES]]
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----
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{|cellspacing="6px" cellpadding="16" border="0" width="100%"
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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>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 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 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 Protocol 3).
 
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#Tutorial in BioEdit and Primer3.
 
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#Started designing primers for amplification and site directed mutagenesis of DmpR and DmpR #24 (see Protocol 4).
 
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=== 09/07 ===
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{|cellspacing="6px" cellpadding="16" border="0" width="100%"
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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 Protocol 4, and to check Tm http://www.itt-biotech.de/itt-cgi/oligo-tm.pl
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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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!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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#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 Quiagen prepkit manual (see 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 Tutorials).
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#Christine made tetracycline stock – 250 mg tetracycline in 50ml 100% ethanol to make 5mg/ml stock.
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#Maija made thiamine stock (0.8g thiamine in 20ml dH2O and filter sterilized. Kept in freezer in foil (light sensitive).
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*'''First selection of biobricks transformed.'''
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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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*'''Preparation of stocks.'''
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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 (3.)  for DmpR and DmpR #24 did not grow, and restriction digests on 9/7/07 (2.) 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 (50ug/ml) with DmpR
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*'''Researched DmpR, XylR and DntR sensor systems.'''
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#*2x  5ml LB containing carb (50ug/ml) with DmpR #24
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*'''Designed primers for DmpR, DntR, XylR and associated promoters.'''
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#*2x  5ml LB containing Tc (50ug/ml) with DmpR
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#*2x  5ml LB containing Tc (50ug/ml) with DmpR #24
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|- align="center"
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#*2x  5ml LB containing Tc (10ug/ml) with DmpR
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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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#*2x  5ml LB containing Tc (10ug/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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!width="50%" |[https://2007.igem.org/Glasgow/Wetlab/Week4 <font face=georgia color=#3366CC size=7><b>Week 4</b></font>] <br>
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|- valign=top
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*'''BioBricks tested by digestion and PCR.'''
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*'''PCR with new primers associated with DntR and XylR.'''
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* '''phzM and phzS cloned into TOPO vectors.'''
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*'''Primer design for 7 gene operon.'''
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*'''DntR cloned into TOPO vector.'''
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*'''Primer design for XylR and associated genes.'''
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*'''Death gene cloned into TOPO vector.'''
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*'''Primer design for death gene.'''
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|- align="center"
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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>
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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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|- valign=top
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*'''Suspected amplification of 7 gene operon.'''
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* '''phzM and phzS confirmed as present in TOPO vectors.'''
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*'''Confirmed presence of death gene plasmid in some vectors.'''
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*'''Miller assay performed with DntR system.'''
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*'''Yeast cells.'''
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*'''Biobricks made of phzM, phzS, and DntR.'''
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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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!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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|- 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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|- 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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!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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|}
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----
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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"