Edinburgh/Lab

From 2007.igem.org

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[[Edinburgh]] > '''Lab Work'''
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[https://2007.igem.org/Edinburgh https://static.igem.org/mediawiki/2007/f/f5/800px-Edinburgh_City_15_mod.JPG]
[https://2007.igem.org/Edinburgh https://static.igem.org/mediawiki/2007/f/f5/800px-Edinburgh_City_15_mod.JPG]
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! Brick || Description || Well || Used for
! Brick || Description || Well || Used for
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|-
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| [http://partsregistry.org/Part:BBa_J04430 BBa_J04430] || IPTG induced GFP || Plate 2, 1L || Test biobrick revival process
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| [http://partsregistry.org/Part:BBa_E0241 BBa_E0241] || PoPS --> GFP || Plate 2, 15L || For use in [[Edinburgh/DivisionPopper/Design#Exp_1| dif experiment 1]]
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|-
|-
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| [http://partsregistry.org/Part:BBa_E0241 BBa_E0241] || PoPS --> GFP || Plate 2, 15L || For use in [[Edinburgh/DivisionPopper/Design#Exp_1| dif experement 1]]
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|[http://partsregistry.org/Part:BBa_E0422 BBa_E0422] || fast degrading ECFP || Plate 1, 11G || For use in [[Edinburgh/DivisionPopper/Design#Exp_1| dif experiment 1]]
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|-
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|[http://partsregistry.org/Part:BBa_J45700 BBa_J45700] || Wintergreen Scent || Plate 3, 7B || Place into lacto bacillus
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|[http://partsregistry.org/Part:BBa_I0500 BBa_I0500] || arabinose promoter || Plate 2, 9I || For use in [[Edinburgh/DivisionPopper/Design#Exp_3| dif experiment 3]]
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|-
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|[http://partsregistry.org/Part:BBa_E0422 BBa_E0422] || fast degrading ECFP || Plate 1, 11G || For use in [[Edinburgh/DivisionPopper/Design#Exp_1| dif experement 1]]
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|[http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510] || CI (Lambda) Inverter || Plate 2, 13K || For use in [[Edinburgh/DivisionPopper/Design#Exp_3| dif experiment 3]]
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|-
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|[http://partsregistry.org/Part:BBa_I0500 BBa_I0500] || arabinose promoter || Plate 2, 9I || For use in [[Edinburgh/DivisionPopper/Design#Exp_3| dif experement 3]]
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-
|-
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|[http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510] || CI (Lambda) Inverter || Plate 2, 13K || For use in [[Edinburgh/DivisionPopper/Design#Exp_3| dif experement 3]]
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|}
|}
==Lab Diary==
==Lab Diary==
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====Thu 12th July====
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===Condensed LabJournal for the Division PoPper===
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* IPTG induced GFP revived ([http://partsregistry.org/Part:BBa_J04430 BBa_J04430])
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::Test biobrick revival process
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====Fri 13th July====
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* Alfalfa Seeds Planted (in Agar)
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::Growing alfalfa plants, to get leaves from which to extract DNA for the caffeic acid-3-o-methyltransferase gene (part of the vanillin synthesis pathway)
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* Biobricks  [http://partsregistry.org/Part:BBa_E0241 BBa_E0241] and [http://partsregistry.org/Part:BBa_J45700 BBa_J45700] revived, using modified protocal from open wetware site
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====Mon 16th July====
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* Begin DNA extraction from Bamboo
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::Test DNA extraction process from plants
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* Prepared mini preps for GFP and mint genes
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====Tue 17th July====
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* Alfalfa Transferred to compost
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* DNA containing GFP and mint genes extracted from E.Coli
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====Wed 18th July====
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* Double digest GFP and mint mini preps, using restriction enzymes SpeI and EcoRI.  These were then run on a polyacramide gel
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* Biobrick ECFP [http://partsregistry.org/Part:BBa_E0422 BBa_E0422] revived and transformed into E.coli JM109 competent cells
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====Thu 19th July====
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* Repeat of double digest (using SpeI & EcoRI) of  GFP and mint mini preps with increased concentration of DNA
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* Replate colonies of ECFP, 4 overnight cultures prepared for making DNA mini preps
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* designed primers for COMT gene (part of vanillin biosynthesis pathway)
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====Fri 20th July====
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For the Proof-of-Concept:
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* Prepared GFP and winter green biobricks for sequencing
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Ordered oligonucleotides for reverse and forward dif-sites (difF and difR) were amplified, combined and biobricked.
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* Revived ''Pseudamonas fluorescens'' from freeze dried culture (''P. fluorescens'' contains two genes, ''ech'' and ''fcs'', which are part of the synthetic vanillin biosynthesis pathway)
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Primers for a reverse lac promoter (PlacRev) were ordered and the sequence amplified and biobricked. This reverse lac promoter was combined with the dif forward sequence to give the biobrick difF-PlacRev.
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* Made 4 mini preps of ECFP
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Yellow fluorescent protein (YFP) was revived from the Registry and combined with the reverse dif-site to yield the biobrick difR-YFP, where YFP is the reporter gene.
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* Designed primers for ech and fcs genes
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A control sequence that consists of the lac promoter and YFP was made for imaging purposes.
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* Ordered ''Saccharothrix espeanensis'' from DSMZ culture collection, which contains Sam5 and Sam8 genes required for the synthetic vanillin biosynthesis pathway
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A miniF plasmid was transformed and plated.
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Primers for FtsK and XerC were ordered and said sequences amplified, digested and biobricked, but only FtsK yielded a final biobrick; cells overexpressing XerC refused to grow.
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====Mon 23rd July====
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All above constructs have undergone the same basic labwork:
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* Ran gels for ECFP mini preps
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:: Restriction digested using XbaI and PstI
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* Alfalfa growing well - now have enough leaf material from which to extract DNA
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====Tue 24th July====
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* Design forward and reverse primers for the sequence of choice
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* Annealed dif F & R oligoes, restriction digested with SacI & SpeI, ligated with BioBrick vector Edinbrick 1
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* PCR amplify the sequence with said primers
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* Double restricion digest the PCR amplified sequence and a vector (Edinbrick1) to create dissimilar sticky ends
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* Purify the reactions
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* Mix cut vector and insert with DNA ligase overnight
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* Transform the biobrick into a chemocompetent E. coli cell line
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* Plate high and low concentrations of transformed cells onto selective plates and leave overnight
 +
* Replate colonies and inoculate broths overnight with antibiotics that select for the vector
 +
* MiniPrep broths that correspond to successful replated colonies
 +
* Do analytical restriction digest and/or PCR reactions and run samples on agarose gel to check the insert size
 +
* Sequence the produced construct.
 +
* MaxiPrep correct biobricks.
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====Wed 25th July====
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It goes without saying that trial-and-error preceeded optimal PCR conditions for each designed primer. We have also employed high-resolution agarose gels when required.
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* Transformed dif F & R DNA into E.coli, incubated at 37C overnight
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* designed primers for Sam5 and Sam8 genes (part of vanillin biosynthesis pathway)
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====Thu 26th July====
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Three attempts to combine difF-PlacRev and difR-YFP have failed to date. We have employed a selection of techniques including gel band excision and purification of both vector and insert and differential restriction enzyme and ligation times. Lab work continues.
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* Prepared glycerol stock of ''Pseudomonas fluorescens''
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* Prepared miniprep of GFP and mint biobricks
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* Streaked ''Pseudomonas fluorescens'' onto a fresh LB plate
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* Revived [http://partsregistry.org/Part:BBa_I0500 BBa_I0500] and [http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510]
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====Fri 27th July====
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Up next is confocal microscopy of the control and final construct. We're eagerly anticipating the arrival of the more advanced contruct (the actual division PoPper) via GeneArt, but their email correspodence is not promising. The word is that our orders are heavily Delayed. The clock is ticking.
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* Continued miniprep of GFP and mint biobricks
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:: Restriction digest using SpeI & EccRI restriction enzymes
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* PCR of ''Pseudomonas fluorescens'' genes
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Latest revision as of 08:43, 26 October 2007

Edinburgh > Lab Work

https://static.igem.org/mediawiki/2007/f/f5/800px-Edinburgh_City_15_mod.JPG

Biobricks Revived

This is a list of the biobricks we have revived from the registry

Brick Description Well Used for
[http://partsregistry.org/Part:BBa_E0241 BBa_E0241] PoPS --> GFP Plate 2, 15L For use in dif experiment 1
[http://partsregistry.org/Part:BBa_E0422 BBa_E0422] fast degrading ECFP Plate 1, 11G For use in dif experiment 1
[http://partsregistry.org/Part:BBa_I0500 BBa_I0500] arabinose promoter Plate 2, 9I For use in dif experiment 3
[http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510] CI (Lambda) Inverter Plate 2, 13K For use in dif experiment 3

Lab Diary

Condensed LabJournal for the Division PoPper

For the Proof-of-Concept: Ordered oligonucleotides for reverse and forward dif-sites (difF and difR) were amplified, combined and biobricked. Primers for a reverse lac promoter (PlacRev) were ordered and the sequence amplified and biobricked. This reverse lac promoter was combined with the dif forward sequence to give the biobrick difF-PlacRev. Yellow fluorescent protein (YFP) was revived from the Registry and combined with the reverse dif-site to yield the biobrick difR-YFP, where YFP is the reporter gene. A control sequence that consists of the lac promoter and YFP was made for imaging purposes. A miniF plasmid was transformed and plated. Primers for FtsK and XerC were ordered and said sequences amplified, digested and biobricked, but only FtsK yielded a final biobrick; cells overexpressing XerC refused to grow.

All above constructs have undergone the same basic labwork:

  • Design forward and reverse primers for the sequence of choice
  • PCR amplify the sequence with said primers
  • Double restricion digest the PCR amplified sequence and a vector (Edinbrick1) to create dissimilar sticky ends
  • Purify the reactions
  • Mix cut vector and insert with DNA ligase overnight
  • Transform the biobrick into a chemocompetent E. coli cell line
  • Plate high and low concentrations of transformed cells onto selective plates and leave overnight
  • Replate colonies and inoculate broths overnight with antibiotics that select for the vector
  • MiniPrep broths that correspond to successful replated colonies
  • Do analytical restriction digest and/or PCR reactions and run samples on agarose gel to check the insert size
  • Sequence the produced construct.
  • MaxiPrep correct biobricks.

It goes without saying that trial-and-error preceeded optimal PCR conditions for each designed primer. We have also employed high-resolution agarose gels when required.

Three attempts to combine difF-PlacRev and difR-YFP have failed to date. We have employed a selection of techniques including gel band excision and purification of both vector and insert and differential restriction enzyme and ligation times. Lab work continues.

Up next is confocal microscopy of the control and final construct. We're eagerly anticipating the arrival of the more advanced contruct (the actual division PoPper) via GeneArt, but their email correspodence is not promising. The word is that our orders are heavily Delayed. The clock is ticking.

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