Team:Johns Hopkins/Notebook/GROUP 5: MATa Specific Promoters II

GROUP 5: MATa Specific Promoters II
Summary for MATa Specific Promoters II Group Part Description: Ste2 Promoter Reverse Summary here: We were able to grow colonies with sequence verified Ste2 promoters. The promoter is currently located in a glycerol stock in pGEM vector. One attempt to    transfer to the iGEM vector has been made, unsuccessfully. This was due to a mistake in protocol however, so final transfer to the iGEM vector should still be straightforward. Progress on this bio-brick has been paused in order to focus on MFA1, which is more applicable to our project design because this Ste2 designed for the wrong direction. Part no.: BBa_K110015 Part Description: MFA1 Promoter Forward Summary here: We have been unable to get successful, sequence verified clones with our MFA1 promoter thus far. We currently have a few samples of DNA ready to be tested by restriction enzyme digest, and then, hopefully, sequencing.

For more information about the parts go to: http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins

Up to today... Since then -- we have been troubleshooting and working on getting higher concentrations and using the correct stock plates. Several delays have occurred,and we should be close to ligating into the iGEM vector from the pGEM vector for Ste2 promoter. Glycerol stocks of the Ste2 are currently available. Verification of clones of the MFA1 promoter are still in progress. Status Report by: Alexandra McMillan

8/24  Restriction Enzyme Digested and the insert appears promising. Ready for sequence submission for Ste 2 promotor.

8/21  Due to low concentrations, we needed to concentrate our DNA before preceeding. Ethanol precipitation using EtOH/NaAc and 100 ul of miniprep mix was complete of the BBa_K110015 and BBa_K11009 minipreps.

8/18 Miniprep products were < 100 ng/ul. Restriction digest. There appear to be correct size inserts. Gel image coming. Results look promising.

8/15 DNA miniprep of BBa_K110015 and BBa_K11009 cultures

8/13 Incubated inoculations of old colony plates for approximately 12 hours.

By 7/19/08 We successfuly transformed both BB 15 and 9 and ran a gel that came out with faint bands. We digested BBa_K110015 and BBa_K11009.

Date: 7/11/08 status report by Rick Carrick Part no.: BBa_K110015, Part Description: MFA1 Part Location (in build a genome lab): PCR successful?; Y (on moodle somewhere) Cloning of PCR product successful: Y Sequencing of cloned PCR product successful:N Joining of validated part to adjacent part(s) status: Not done Problems to be solved: None so far Current status of this part: This parts must be restriction enzyme digested and sequenced next.

Date: 7/11/08 status report by Rick Carrick Part no.: BBa_K110009 Part Description: Ste2 Part Location (in build a genome lab): PCR successful?; Y (on moodle somewhere) Cloning of PCR product successful: Y Sequencing of cloned PCR product successful:N Joining of validated part to adjacent part(s) status: Not done Problems to be solved: None so far Current status of this part: This part must be restriction enzyme digested and sequenced next