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PCB genes, Shipment & LovTAP frameshift
I'm forwarding the e-mails that we have sent to Dr. Takayuki Kohchi in order to get his plasmid that enables the PCB biosynthesis in E.coli.
His e-mail address is:
If you could get the plasmid first, it will be great if you could send it to us.
About the synthesis, we are doing our best in order to send your team the parts as soon as possible, we are planning to send the parts inside plasmid pSB1C3.
The process to extract the designed parts from Mr. Gene plasmid has been very laborious because it seems to contain some of the four restriction enzyme sites used to assemble biobricks.
Today we finally contacted our advisor that will help us with luciferase experiments and Mariana will test the luciferase on Tuesday. We are very excited, I really hope that it works out.
About LovTAP, I was wondering where exactly you found the frameshifth. When I was designing LovTAP for synthesis, the sequence reported at the registry was very confused for me. It didn't have a clear start codon and there were like 15 extra nucleotides in the middle of the protein that didn't make sense considering the original LovTAP sequence reported in the entry of LovTAP:Design
So that, after several alignments using BLAST and ClustalW I decided to remove the approximately 15 extra nuCleotides and include the proper start codon in order to have a functional ORF.
This is all for now.
Please send the request again through the faculty member who is responsible for your biological experiments and materials in the Center for Genomic Sciences in UNAM.
I am an undergraduate student majoring in Genomic Sciences at the Center for Genomic Sciences (CCG) from the National Autonomous University of Mexico (UNAM).
Actually, I am a member of the team that will take part in the next International Genetically Engineered Machine (IGEM) competition at MIT in November 2010.The aforementioned competition consists of the development of a project in the area of Synthetic Biology.
In planning our project, we are going to use a biological device that involves a transcriptional response directed by the chimaeric photoreceptor “Cph8”–the cyanobacterial phytochrome (Cph1) fused to the EnvZ histidine kinase domain from E.coli- but we have not been able to get the producing phycocyanobilin (PCB) genes, necessary for the photoreceptor to sense red light. As well, we are working with CcaS green light sensor, thus we also need PCB producing genes to make it functional.
So, I am contacting you to ask for your help. It would be really very useful and valuable for our project if you could kindly provide us the plasmid pKT271, which harbors the gene ho1 and the pcyA of Synechocystis to enable the PCB biosynthesis in E.coli, according to the article “Metabolic engineering to produce phytochromes with Phytochromobilin, Phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli”.
Thank you so much for your attention, and I remain at your disposition in order to work out the necessary details in order to mail the strain to us.
Claudia Hernandez Armenta
Undergraduate Program on Genomic Sciences
National Autonomous University of Mexico UNAM