Team:Johns Hopkins/Notebook/GROUP 1: Fluorescent Proteins

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. (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. (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, (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. 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 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 PCR Products of mCherry and vYFP Cloning of PCR product successful: Inconclusive 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 PCR Products of mCherry and vYFP Cloning of PCR product successful: inconclusive 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!