GROUP 1: Fluorescent Proteins
Date: Oct 19, 2008
Status report by: Tejas
Part no.: BBa_K110017 -> BBa_K110023
Part Description: yESapphire , mCherry, venusYFP, and Citrine
Work on YFP has progressed, though the biobrick system is giving diffiulty in obtaining a useful
construct. Moreover, the YFP sequence may not fully match the expected sequence. However,
fluoroescent proteins (yeast optimized) sent us as STABS from MIT are showing promise, and may be
ready for some use by the Jamboree date if all goes well. Work will continue on venus YFP and
mCherry (though mCherry unfortunately contains a restriction site). Sapphire must be started over
sue to technical difficulties getting a clone.
Date: July 22, 2008
Status report by: Tejas, Ingrid
Part no.: BBa_K110017 -> BBa_K110023
Part Description: yESapphire , mCherry, venusYFP, and Citrine
Work on yESapphire was gratiously done by James. Primers were designed. Restriction site
ends have been added through PCR. The product was cloned into JM109. Colonies were picked
and an inoculation was grown. Miniprep was performed and subsequent DNA was CS PCR'd
and run on a gel to verify contents.
([http://www.jhu.edu/iGEM/Group3:ShortTwo-wayStops/2008-7-22.Short%202Way%20Stop,%20Alpha%20Promoters,%20&%20Sapphire%20FP.Allison%20Suarez%20and%20Nate%20Sotuyo%20.html Short 2Way Stop, Alpha Promoters, & Sapphire FP]) BioBrick is currently being sequenced.
Work on mCherry and venusYFP (BBa_K110018 -> BBa_K110021) is currently being done. Primers were designed.
Restriction sites have been added through PCR. ([http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-23.PCR%20Products:%20mCherry%20and%20Venus%20YFP,%20both%20RtL%20and%20LtR.Ingrid,%20Tejas.html PCR Products: mCherry and Venus YFP, both RtL and LtR]) The
products were cloned into JM109 and plated. 3 colonies per 4 BioBricks (total 12) were picked, grown out,
mini prepped, and digested for verification on a gel.
According to the results, ([http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-22.Venus%20YFP%20and%20mCherry%20Miniprep%20check%20via%20digest.Ingrid.html Venus YFP and mCherry Miniprep check via digest]) , only one of the mCherry's
(BBa_K110019) is the correct product. A second Digest was preformed to check this, meanwhile new colonies
have been picked on 7.22.2008 and are being grown out for a higher yield of DNA so that it can be sent off
for sequencing. [http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-23.Re-Digest%20of%20three%20samples%20of%20mCherry%20BBa_K110018.Ingrid%20.html Re-Digest of three samples of mCherry BBa_K110018]
Work on Citrine has not yet begun. Primers have been designed and ordered, but template DNA must be grown out.
Template DNA will be grown and extracted by James should we later decide that Citrine will be needed.
*Note that mCherry and Citrine both have restriction sites within the coding region, and are therefore not
optimal. Advice/help on this issue would be appreciated.
Another Digest was done [http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-8-19.mCherry%20and%20YFP%20Digest%20Gel.Ingrid%20Spielman.html mCherry and YFP Digest Gel] and weird lines exist.
Date: July 17 2008
status report by: Ingrid
Part no.: BBa_K110018
Part Description: mCherry:
Part Location (in build a genome lab): In silver fridge by door
PCR successful?; Yes
[http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-22.PCR%20Products%20of%20mCherry%20and%20vYFP.Ingrid%20&%20Tejas.html PCR Products of mCherry and vYFP]
Cloning of PCR product successful: Inconclusive
[http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-22.Venus%20YFP%20and%20mCherry%20Miniprep%20check%20via%20digest.Ingrid.html Venus YFP and mCherry Miniprep check via digest]
Sequencing of cloned PCR product successful: Not done
Joining of validated part to adjacent part(s) status: Not done
Problems to be solved: Had some extra unknown products, with unknown bands in the gel
Current status of this part: Done with touchdown PCR and gel. Next step is to clone product.
Date: July 17 2008
status report by: Ingrid
Part no.: BBa_K110020
Part Description: Venus Enhanced YFP
Part Location (in build a genome lab): In silver fridge by door
PCR successful?; Yes
[http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-22.PCR%20Products%20of%20mCherry%20and%20vYFP.Ingrid%20&%20Tejas.html PCR Products of mCherry and vYFP]
Cloning of PCR product successful: inconclusive
[http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-22.Venus%20YFP%20and%20mCherry%20Miniprep%20check%20via%20digest.Ingrid.html Venus YFP and mCherry Miniprep check via digest]
Sequencing of cloned PCR product successful: No
Joining of validated part to adjacent part(s) status: Not done
Problems to be solved: Had some extra unknown products, with unknown bands in the gel
Current status of this part: Done with touchdown PCR and gel. Next step is to clone product.
Date: July 1 2008
status report by: Ingrid (work done by James)
Part no.: BBa_K110017
Part Description: yESapphire
Part Location (in build a genome lab): In James and Jasper's PCR product Box,
Stainless Steel 4 degree
PCR successful?; Yes
Cloning of PCR product successful: Y/N
Sequencing of cloned PCR product successful: Not done
Joining of validated part to adjacent part(s) status: Not done
Current status of this part: This part is being Sequenced
Date: August 27 2008
status report by: Ingrid
Part no.: BBa_K110018, 19, 20, 21
Part Description: mCherry, YFP
Part Location (in build a genome lab): In James and Jasper's PCR product Box,
Stainless Steel 4 degree
PCR successful?; Yes
Cloning of PCR product successful: Y/N
Sequencing of cloned PCR product successful: Successful for YFP LtR and RtL
Joining of validated part to adjacent part(s) status: In progress
Current status of this part: Ligation into vector
Digest of colonies repicked from mCherry and YFP
Aug 29, 2008
Lane 1: 18.1 mCherry LtR
Lane 2: 18.2 mCherry LtR
Lane 3: 19.1 mCherry RtL
Lane 4: 20.1 Venus YFP LtR
Lane 5: 20.2 Venus YFP LtR
Lane 6: 21.1 Venus YFP RtL
Lane 7: 21.2 Venus YFP RtL
Lane 8: Ladder
Used digest protocol. Sequences have been made, YFP and mCherry have mutations.
mCHerry mutations seem deleterious = discontinued use.
YFP mutations are not essential and therefore the RtL
Venus YFP will be ligated into final vector product!
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