the Heidelberg iGEM team has been focusing on the shuttling of DNA via conjugation. The conjugation origin of replication (oriT) is inserted into a plasmid or genome of interest; in the presence of a (non-modifiable) conjugation plasmid as a chassis the DNA of interest is transfered to cells of a second E. coli strain.
1) We would like to add this conjugation plasmid to the Registry as a chassis system. The diverse genes are essential for effective conjugation and are not supposed to be modified. However, in the Registry there are only chassis systems for bacterial strains and cell-free systems yet. Where should we best place this 60 kb helper plasmid? We think it would be suitable to introduce a category for "Chassis Plasmids"!
2) We have inserted the oriT into the lambda phage genome and used the above mentioned conjugation/oriT system to transfer the phage via conjugation. Since the phage genome is not a standard vector, we have been deliberating over an appropriate standard, but we had to conclude that - in terms of time and money - this is out of our scope:
The oriT fragment in the lambda phage genome cannot be exchanged via standard prefix and suffix sites, since the corresponding restriction sites occur too frequently in the >40 kb lambda phage genome to be eliminated; indeed, we were lucky to even find few single-cutting restriction sites to make an insertion of the oriT fragment possible! There is still no simple cloning strategy yet to exchange the oriT in the phage genome, because the smallest embracing fragment that is flanked by single-cutting restriction sites contains genes essential for the phage (e.g. GAM). Since mutagenesis would be inefficient due to the large size, the introduction of standard sites would be possible only in a synthesis approach with costs of estimated 25,000 USD - and this is not affordable from our team budget.
The phage, however, is an essential part for the dynamics of our overall population system (hunter/prey), and we are convinced of its value for the Registry: The system will provide a novel approach to effectively eradicate a specific target strain. A category for T7 phages - containing only few constructs yet - already exists in the Registry, and we suggest to declare it as a more general category for "bacteriophages"; our modified lambda phage could then be placed there. To account for consistency with Registry standards, however, we will in addition provide the oriT with standard restriction sites in a standard vector.
We would therefore like to ask, if you consider the introduction of the two categories as appropriate.
We sincerely look forward to your judgement!
the Heidelberg iGEM team.
____________________________________ Jens Keienburg, M. Sc. Bioquant, AG Eils Ruperto-Carola University INF 267 (R. 355), 69120 Heidelberg, Germany Office: +49 (0) 6221 54 51 305 Mobile: +49 (0) 0176 6201 0563 Fax: + 49 (0) 6221 54 51 488