From 2009.igem.org

Team Request

Dear iGEM Headquarters,

Our project this year uses mammalian microglial (BV-2) cells. As there is no current standard for mammalian plamsids, we are requesting to use pcDNA3 mammalian expression vectors. We are ordering ours from GeneScript, following their standards. They use a fairly common cloning technique, similar to iGEM. We will send our completed parts into the registry. Will you allow us to use this alternate system?

Thanks for your consideration.

Sincerely, Janie Stine and Jessamine Osborne Purdue University iGEM Team

Judges' Response

Janie & Jessamine,

Thanks for your email.

We don't have enough information to fully consider your request.

First, do the pcDNA3 expression vectors have a bacterial replication origin (i.e., can they serve as shuttle vectors via E.coli)?

Second, is there any particular reason(s) why this vector(s) should become a standard for mammalian systems? If so, what are they?

Third, are they any mammalian vectors in the Registry (i.e., already in use) and if so what is wrong with them?

Fourth, what is the GeneScript standard?

Fifth, does the "fairly common cloning technique" that you allude to support idempotent assembly?

Sixth, is the "fairly common cloning technique" that you allude to compatible with any of the existing BioBrick physical assembly standards?

Sorry to ask so many questions, but I wanted to quickly detail the underlying issues that we need to consider.

Very happy to discuss or talk more!

All best, Drew

Team Response

Hi Drew,

We are happy to provide you with all the specifics. Hopefully we will answer your questions.

The pcDNA3 expression vector contains both the f1 ori (for single-stranded prokaryotic replication) as well as a ColE1 ori (for double-stranded prokaryotic replication). Therefore, it could be used by bacterial systems.

The pcDNA3 vector (see http://www.molecularinfo.com/MTM/K/K2/K2-1/pcdna3.pdf) is a common mammalian expression vector system, and is available commercially from vendors such as Invitrogen. Although this is a convenient system for us to use, to be honest, we ran out of time to be able to correctly convert our genes to the Registry standards. We would ultimately like to convert our genes into a more standard expression vector to be used in the Registry.

We unfortunately mis-typed part of our email earlier when we said there are no mammalian standards. What we meant is that for our project, we felt we needed a more professional design to make sure our parts came out correctly. One of our genes is only 50 bp in length, and would be difficult to clone with standard methods without professional help. As I'm sure you know, in the Registry, there are quite a few parts that are yeast-derived; some are based on the original Registry standard and some conform to the new Aar1 system recently approved as RFC 28. As we stated previously, we plan on converting our parts to a Registry standard in the future; however, for the present we do not have sufficient time to do so and still complete the main goal of our project.

GenScript uses their trademarked CloneEZ system to make your specified part. Here is the website with their information: http://www.genscript.com/cloneez_PCR_Cloning_kit.html. Our parts will contain (along with multiple other cloning sites) an EcoRI and an XbaI site to either side of our genes. These restriction sites could be used to convert our parts to Registry standards.

If you have any further questions, please feel free to ask. Best of luck getting the Jamboree ready to go!

Thanks very much, Janie Stine and Jessamine Osborne Purdue University iGEM Team