Edinburgh/Lab

From 2007.igem.org

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==Lab Diary==
==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.
 +
 +
 +
===Design Yoghurt===
 +
====Thu 12th July====
 +
* IPTG induced GFP revived ([http://partsregistry.org/Part:BBa_J04430 BBa_J04430])
 +
::Test biobrick revival process
 +
====Thu 12th July====
====Thu 12th July====

Revision as of 18:20, 5 October 2007

Edinburgh > Lab Work

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

Contents

Biobricks Revived

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

Brick Description Well Used for
BBa_J04430 IPTG induced GFP Plate 2, 1L Test biobrick revival process
BBa_E0241 PoPS --> GFP Plate 2, 15L For use in dif experement 1
BBa_J45700 Wintergreen Scent Plate 3, 7B Place into lacto bacillus
BBa_E0422 fast degrading ECFP Plate 1, 11G For use in dif experement 1
BBa_I0500 arabinose promoter Plate 2, 9I For use in dif experement 3
BBa_Q04510 CI (Lambda) Inverter Plate 2, 13K For use in dif experement 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.


Design Yoghurt

Thu 12th July

Test biobrick revival process


Thu 12th July

Test biobrick revival process

Fri 13th July

  • Alfalfa Seeds Planted (in Agar)
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)

Mon 16th July

  • Begin DNA extraction from Bamboo
Test DNA extraction process from plants
  • Prepared mini preps for GFP and mint genes

Tue 17th July

  • Alfalfa Transferred to compost
  • DNA containing GFP and mint genes extracted from E.Coli

Wed 18th July

  • Double digest GFP and mint mini preps, using restriction enzymes SpeI and EcoRI. These were then run on a polyacramide gel
  • Biobrick ECFP BBa_E0422 revived and transformed into E.coli JM109 competent cells

Thu 19th July

  • Repeat of double digest (using SpeI & EcoRI) of GFP and mint mini preps with increased concentration of DNA
  • Replate colonies of ECFP, 4 overnight cultures prepared for making DNA mini preps
  • designed primers for COMT gene (part of vanillin biosynthesis pathway)

Fri 20th July

  • Prepared GFP and winter green biobricks for sequencing
  • Revived Pseudamonas fluorescens from freeze dried culture (P. fluorescens contains two genes, ech and fcs, which are part of the synthetic vanillin biosynthesis pathway)
  • Made 4 mini preps of ECFP
  • Designed primers for ech and fcs genes
  • Ordered Saccharothrix espeanensis from DSMZ culture collection, which contains Sam5 and Sam8 genes required for the synthetic vanillin biosynthesis pathway

Mon 23rd July

  • Ran gels for ECFP mini preps
Restriction digested using XbaI and PstI
  • Alfalfa growing well - now have enough leaf material from which to extract DNA

Tue 24th July

  • Annealed dif F & R oligoes, restriction digested with SacI & SpeI, ligated with BioBrick vector Edinbrick 1

Wed 25th July

  • Transformed dif F & R DNA into E.coli, incubated at 37C overnight
  • designed primers for Sam5 and Sam8 genes (part of vanillin biosynthesis pathway)

Thu 26th July

  • Prepared glycerol stock of Pseudomonas fluorescens
  • Prepared miniprep of GFP and mint biobricks
  • Streaked Pseudomonas fluorescens onto a fresh LB plate
  • Revived BBa_I0500 and BBa_Q04510

Fri 27th July

  • Continued miniprep of GFP and mint biobricks
Restriction digest using SpeI & EccRI restriction enzymes

Monday 30th July

  • PCR of fcs and ech genes from Pseudomonas fluorescens was carried out
when pcr reaction was run on gel there was no product present, therefore we decided to carry out the pcr at a lower annealing temperature

Tuesday 31st July

  • Prepared chloramphenicol plate to test if the revived wintergreen biobrick with the duel resistance plasmid was present
  • Streaked out glycerol stock of Pseudomonas fluorescenes to determine if viable
  • Carried out a second PCR of the ech and fcs genes required for vanillin biosynthesis pathway
lowered the annealing temperature to 54'C
  • extracted DNA from alfalfa leaves ready for PCR amplification of COMT gene required for vanillin biosynthesis pathway
used the extract and amp kit provided by SIGMA

Wednesday 1st August

  • PCR of ech and fcs was run on an agrose gel
results!! but not what we hoped for
fcs had several amplified fragments with one being present at the 1700bp mark which could represent the fcs gene
there was a vague band present at the 800bp mark, which possibly represents the ech gene
  • decided to carry out pcr for a third time at a lower annealing temperature to suit the ech gene
fcs requires a higher annealing temperature to reduce the number of fragments amplifed
  • streak plate of Pseudomonas fluorescens showed up positive
good news means our glycerol stock is viable :)
  • chloramphenicol blue white selection plate came up positive for white colonies
shows that the transformed insert contains a plasmid with the chl resistance just like the wintergreen biobrick

Thursday 2nd August

  • repeated ech PCR at a lower annealing temperature (reduced to 50'C)
gel showed that there was no improvement in the amplification of the ech gene
problem could be due to long extention time as ech PCR was ran at same time as a PCR for a larger gene
  • PCR of COMT genes required for the vanillin biosynthesis pathway
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