Judging/Variance/Warsaw

Request
Dear iGEM Judges,

During this year's iGEM competition our team is building a system of biological machines allowing to investigate protein interactions and simultaneously change sequence of one of them in order to achieve the strongest possible interaction. The character of our project requires stable single or low copy number vector carrying part of our system which we call 'one-armed bandit'. Low copy number vector is a template for random mutagenesis which is crucial part of this system. For this reason we would like to use pACYC177 and pMPM-T5 derivatives as a backbones for our parts. We have tried to use vector BBa_I739204 (derivative of pACYC177), but we weren't able to recover it from DNA Kit of Parts.

Another thing is as our pACYC177 already contained RBS and lactose promoter, constructing such vector from the beginning would be redundant. In the case of pACYC177 backbone we've had also to use cloning sites different from standard because it doesn't contain standard restriction sites and changing multicloning site would be time-consuming. Another simple reason is that in few cases our parts contain standard sites inside their sequence. We tried to put our tools into parts with standard cloning sites, but we are already sure that for a few of them it will be impossible.

We also used some non-standard vectors in the section of our system which requires purification of recombinant fusion (protein A + part of beta-lactamase) proteins. Sequences of mentioned fusions were cloned into pet15b vector from Novagen, giving us an opportunity to purify his-tagged proteins easily. We want to mention here that prepared parts will be sent on standard BioBrick vectors unless it will be impossible because of different restriction sites.

All mentioned variances are required for our system to work. In our set-up of vectors and strains everything has to be fully adjusted and change or removal of any part will cause damage of the whole system. That's why we are requesting permission to submit non-biobrick parts to the registry.

We will make sure that our parts can be easily extracted from the plasmid either through digestion with commercially available enzymes or through PCR (and we will also make sure that all non-standard parts are fully described and documented to ensure that they are useful for the community.

Thanks for your time and consideration

Pawel Krawczyk

University of Warsaw iGEM'08 team

Response
Dear Pawel:

We fully appreciate the challenges you describe, and are pleased to see that you will be submitting prepared parts on standard BioBrick vectors whenever possible. Please do clearly explain the reasons in those cases that you feel are impossible, and submit those parts clearly identified as being on a different vector. While we regret that you were unable to extract BBa_I739204, it would have been good to try the pSB3 series which has the copy number range and hopefully stability you need.

It seems like you are also asking for a variance to submit your assembled device on a non-standard plasmid. This would be fine if you are willing to propose and document a new assembly standard and submit the vector in that format. Please look at the existing documentation of existing standard biobrick assembly plasmids in the registry to see what would be expected.

The Novagen vector presumably has restrictions on distribution, in which case you also need to avoid submission on that vector.

Sincerely,

iGEM judging team

Response^2
Dear iGEM Judges,

Thank you for your quick reply to our request. We hope to answer all the questions you asked us. For the moment we have put 7 out of 13 of our parts into BioBrick standard. We're working hard to finish the remaining 6, but we feel that given all other requirements we have to fulfill (and regular student duties) we might be unable to do it in time. One of the reasons is complexity of these parts, i.e. several XbaI, EcoRI or NotI sites, all of which should be removed. Discussed parts are located either on pMPM vector, or on our derivative of pACYC177. We are now working on the documentation of our new assembly standard and we will make sure that it will help the Community to use our device and a new standard. Briefly writing – our standard uses NdeI or NcoI as a beginning of part. Sequences recognized by these enzymes include ATG start codon, which allows to easily exchange ORFs in a given part. This is very useful in devices generating proteins on a high-throughput scale (which is the case of our device). For protein fusions we use BamHI or SacI restriction sites because they allow to introduce linkers between fused proteins, containing only glycine and serine. Gly-Ser linkers are in our opinion the best general-purpose protein linkers, but their introduction is impossible with currently available standards. We want to point out that available standard didn't suit our parts because it introduces 'scars' (TACTAG sequences) between each two parts. That would disrupt reading frame of our fusions or introduce wrong amino acids to the linker and all our parts need to be 'scarless'.

We are aware that this promise cannot have any influence on your decision concerning our request nor on the final judgment, but if you feel it is desirable for the community that we submit all the remaining parts, we will commit ourselves to do it after the Jamboree. We'd rather use the little time that is left to test and document our device since we believe it is worth putting it to work in the first place. I hope I've answered all of your doubts. Sincerely

Pawel Krawczyk

University of Warsaw iGEM team