Edinburgh/Lab

From 2007.igem.org

< Edinburgh(Difference between revisions)
 
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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 [https://2007.igem.org/Edinburgh/Counter#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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|[http://partsregistry.org/Part:BBa_E0422 BBa_E0422] || fast degrading ECFP || Plate 1, 11G || For use in [https://2007.igem.org/Edinburgh/Counter#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]]
|}
|}
==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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* PTG 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
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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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For the Proof-of-Concept:
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* Alfalfa Transferred to compost
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Ordered oligonucleotides for reverse and forward dif-sites (difF and difR) were amplified, combined and biobricked.
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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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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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A control sequence that consists of the lac promoter and YFP was made for imaging purposes.
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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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* DNA containing GFP and mint genes extracted from E.Coli
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All above constructs have undergone the same basic labwork:
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====Wed 18th July====
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* Design forward and reverse primers for the sequence of choice
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* Digest GFP and mint mini preps and ran on gel
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* PCR amplify the sequence with said primers
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* Biobrick [http://partsregistry.org/Part:BBa_E0422 BBa_E0422] (ECFP) revived
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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
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* MiniPrep broths that correspond to successful replated colonies
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* Do analytical restriction digest and/or PCR reactions and run samples on agarose gel to check the insert size
 +
* Sequence the produced construct.
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* MaxiPrep correct biobricks.
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====Thu 19th 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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* Repeat digest of  GFP and mint mini preps with more DNA
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====Fri 20th 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 GFP and winter green biobricks for sequencing
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* Revived Pseudamonas Fluorescens from freeze dried culture
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:: Used to retrieve frs and ech genes in vanillin pathway
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* Prepare 4 mini preps of ECFP
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====Mon 23rd 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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* Ran gels for ECFP mini preps
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:: Digested using Xba1 and Prt1
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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.