Judging/Variance/Guelph

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Latest revision as of 12:25, 21 October 2008

For the first year of our school's participation in the competition, the Guelph iGEM team is building a synthetic operon for co-expression of a large number of carotenoid metabolic genes necessitating a slightly divergent cloning technique for straigtforward, multipart sequential ligations. We generated biobrick parts with appropriate restriction sites in commercial cloning vectors, but have also included non standard biobrick REs to allow us to serially ligate several genes into a synthetic operon and RNAi conformations.

Our strategy begins to generate a promoter with the start codon friendly restriction site Nde driving GFP expression in the promoter testing device, pSB1A3-E0240. The GFP is removed and the CrtE gene is inserted next to the promoter with the Nde while the Xba in the 3' end is fused to the vector's SpeI and nullified. This first gene included SpeI and XhoI sites in its 3' UTR, allowing the next gene to be inserted using Xba and Xho, while it carries SpeI and XhoI sites as well. And so on. Using this strategy we believe it possible to extend the synthetic operon to a large number of genes. Obviously if we had used a biobrick RE like Not1 instead of Xho, Biobrick parts we would've made would include the Xba or Spe sites inside the NotI sites flanking the Biobrick.

Likewise, we have used a modified RE scheme for construction of our second project - an RNAi generating Biobrick for silencing plant genes using bacteria. In order to enable use of any pre-existing Biobrick in the registry, we ordered a construct with Biobrick RE in an altered orientation. Eco and Spe together on one side of the corn actin1 intron, and Not and Xba on the other side. To the 3' end we included a synthetic terminator followed by the PstI site, and the MfeI and NdeI sites at the 5' end which allowed us to non-conservatively insert the construct into the Biobrick vector pSB1A2, eliminating the MfeI site (compatible ends with EcoRI) and allowing us again to cut with the NdeI to fuse to the strong constitutive promoter we are using. Our target vector for this project has NdeI and PstI sites we plan to use to conveniently insert this RNAi constuct into. Part of our strategy is to use a broad host range plasmid which will accept a number of different inserts, and the NdeI and PstI sites are ideal for this.

In the spirit of iGEM, we hope to share these parts with the community, and to facilitate this transfer, we have all our parts in two commercial cloning vectors, pJET from Fermentas, and pDRIVE from Quiagen. If requested of the committee, we can transfer these parts to Biobrick vectors but would also like to know if submission of these sequenced parts will be acceptable as is. Much of this is outlined on our wiki with diagrams. We hope you will accept our constructs for this year's competition and let us know what changes to our plan will be necessary.

Thank you for your consideration,

David Johnston

Guelph iGEM Team

Response

Dear David:

There are a number of issues here. First, you specifically request permission to submit your parts to the registry on the commercial cloning vectors pJET and pDRIVE that you are using in your constructs. The registry encourages the development of new standard plasmids, with two constraints: 1) they must be free of any commercial restrictions on further distribution and use; and 2) they must be thoroughly documented, similar to the documentation provided for existing plasmids. It does not appear that your proposal would satisfy those constraints, but we would welcome further information if we are wrong.

It also appears that you are using a new assembly strategy for serial ligation that includes non-standard biobrick ends. However, it is not clear that you are proposing your serial ligation strategy as a new biobrick standard, or simply want to make your parts available in that form. If you are interested in an evaluation of that assembly strategy as a new standard, please let us know. Otherwise, to avoid confusion in the absence of a new standard, you should only submit the current biobrick standard versions of your parts.

Assuming you choose not to develop new plasmid or assembly standards in the short term, then please submit your new parts as biobrick parts on an existing standard biobrick plasmid. These vectors can be found at http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=Plasmid

It would be helpful if you would indicate in the registry that you have these parts (both biobricked and with your alternate REs) available on those commercial vectors. If that generates frequent requests, then it would clearly be important to develop a new standard vector and/or cloning strategy for the registry.

Sincerely,

iGEM judging team

Response to your Response

Dear iGEM Judging Team,

Thank you for your respopnse. We will get all our genes and constructs into pSB1A2. Since they do all conform to biobrick standards, it likely doesn't matter that our submissions will also contain NdeI and XhoI restriction sites. Right?

One other thing: we are proud that the RNAi construct tool we designed (and put into pSB1A2) could allow ANY biobrick part (with the sites EcoRI, NotI, XbaI, and SpeI) to be made into an RNAi construct. It uses the Biobrick RE but not in your standard Biobrick conformationa. An approximation of its structure follows:

NdeI - EcoRI - XbaI - Intron - NotI - SpeI - Terminator - PstI

Please tell me if this posses a problem.

Oh, and one more thing. The broad host range plasmid we are using was released to us for use in this competition after our signing of an MTA. We are allowed to use it in this competition, but there are clauses in the document preventing the spread of this plasmid outside of our direct use so I don't know if we can submit it as a biobrick (although I want to). Will this pose a problem for our entry?

Sincerely,

David Johnston iGEM Guelph

Response Cubed

Hi David:

We are discussing the first two questions, but meanwhile want to be clear about the third. It sounds like you have lots of parts that you can submit on standard vectors, but are constrained regarding distribution of the broad host range assembly plasmid. While you can certainly use that commercial plasmid in the competition to demonstrate and test your device, it would not be appropriate to submit the assembled device on that plasmid to the registry. That constraint will not penalize your team in the competition as long as you submit the device components on pSB1A2 or another standard vector as you propose.

iGEM judging team

Yet another comment

Dear iGEM Judging team,

I'd like to withdraw our request for variance regarding the synthetic operon ; in cutting our genes out of the commercial cloning vectors and putting them into pSB1A2, we trimmed off the XhoI site. We are ligating each gene together by cutting the vector with SpeI and PstI, while the gene to be inserted is cut with XbaI and PstI.

We still hope for variance with our RNAi construct however.

Thanks for your patience and guidance.

Sincerely, Guelph iGEM Guru David