Team:Nevada/iGEMcollaborators

The iGEM team Nevada contributed with correction of pSB1C3. Megan Tabor detected an error in pSB1C3 (the chloramphenicol resistant backbone). pSB1C3 is the submission plasmid for iGEM, and was also used for a thiamine knockout construct to make Synechocystis PCC 6803 an auxotroph. When trying to make the primers for isolation of the chloramphenicol resistance protein from the plasmid using PCR, the mapped out section did not blast as chlormamphenicol resistance and did not fit with the ORF. Megan contacted the MIT team that created the pSB1C3 to find more information and a correction. Below are the emails of the conversation.

From: Megan Tabor

Date: Mon, Aug 1, 2011 at 4:53 PM

Subject: iGEM Team Nevada 2011

To: webmail@austinche.name

Hello Austin,

My name is Megan Tabor and I am a currant iGEM member.  I am trying to work with the plasmid backbone pSB1C3 (the chloramphenicol resistant backbone).  I would like to isolate it out of the plasmid but there does not seem to be a start or stop codon associated with where on the plasmid the resistance is supposed to be.  I was wondering if you know for sure that is resistance is where it was stated.  Thank you so much for your time.

Megan Tabor

---------- Forwarded message -------------------

From: Austin Che

Date: Tue, Aug 2, 2011 at 6:06 AM

Subject: Re: iGEM Team Nevada 2011

To: Megan Tabor

 The annotation on the registry was off by 2 bases on either side. I've updated the registry info.

Collaborations With 2011 Utah State iGEM Team

We have developed a collaborative relationship with the 2011 Utah State University Team. They have generously supplied us with promoter construct for transgene expression in Synechocystis. We have had back and forth communications throughout the summer. Since Dr. Shintani has had past experience transforming Synechocystis, he was able to offer assistance to Dr. Miller of the Utah State Team on Synechocystis transformation protocols. See the e-mail correspondence below:

From: Charles Miller [mailto:Charles.Miller@usu.edu]

Sent: Wednesday, June 22, 2011 8:38 AM

To: David K Shintani

Subject: RE: iGEM Collaborations

Dave:

I would be interested in info on another insertion vector. As you are probably aware, while we have worked with E. coli and other bacteria previously, we have only recently started trying to transform Synechocystis. Do you have a standard protocol for transformation and selection of Synechocystis that works routinely?

Thanks,

Charlie

From: David K Shintani [mailto:shintani@unr.edu]

Sent: Friday, June 24, 2011 10:55 AM

To: Charles Miller

Subject: RE: iGEM Collaborations

Hi Charlie,

As a post-doc, I did some work with Synechocystis PCC6803. We were making KO mutants in targeted Vitamin E biosynthetic genes (Shintani and Dellapenna (1998) Science282: 2098-2100). As I remember it was not too difficult, but that’s looking back on it with PI-eyes. We used the protocol described in Williams JGK (1988) Methods in Enzymology 167: 766-778. While this worked well with the insertion of a single antibiotic resistance cassette, it may prove more difficult with a larger construct. We need to generate a KO in the ADP glucose pyrophosphorylase gene so we are going to insert one of our constructs into that locus. We are also going to insert a second transgene into a thiamin biosynthetic gene. This insertion will be easy to score using thiamin auxotrophy as a phenotypic marker. The thiamin gene insertion site target may be more useful to you. Is your team interested in a video conference? If not no big deal, but it might be interesting to talk. I saw that Stanford and Brown are teaming up on a project similar to ours. Maybe there is a way we could work together also.

Dave

From: Charles Miller [mailto:Charles.Miller@usu.edu]

Sent: Fri 6/24/2011 12:15 PM

To: David K Shintani

Subject: RE: iGEM Collaborations

Dave:

Thanks for the information. I video talk would be good. I am going to be away at meetings for the next week and a half so maybe right after I get back we can set something up.

Thanks again,

Charlie