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Judging/Variance/Warsaw
From 2008.igem.org
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 Paul:
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 varience to submit your assembled device on a non-. This would be fine if you are willing to propose and document a new assembly standard. 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
