Team:Amsterdam/Biobricks/Composite Parts


Composite Parts

Two types of composite parts were submitted to the registry: construction intermediates, and finalized CryoBricks. The construction intermediates contain either a promoter and an RBS, or an RBS and a coding region. The finalized CryoBricks are all protein generators with a classic PRC (Promoter, Ribosome binding site, Coding region) structure, except for 4 operon-like parts which are structured PRCRC. No terminators were used in any of our parts; termination of transcription relies on the terminators encoded in iGEM's standard plasmid backbones.

Construction Intermediates

The overview below lists all parts that were used as intermediates in the construction of our finalized CryoBrick protein generators.

By prepending an RBS to our coding regions and appending an RBS to our selected promoters, we pursued two different assembly strategies for the construction of the PRC protein generators: add a promoter to an RC part, or add a coding region to a PR part. Note that these promoter + RBS parts contain no bricks that weren't already available from the registry. Construction of all the parts listed above succeeded, but transferring it from the pSB1A3 backbone to the pSB1C3 backbone proved too difficult, only succeeding with BBa_K538101 and BBa_K538104. As a result, the remaining intermediates are uneligible for submission to iGEM and won't be available from the registry.

Finalized CryoBricks

The purpose of our finalized CryoBricks is to enhance E. coli 's cold resistance, by expressing chaperones from psychrophillic bacteria. Information regarding how these bricks achieve this can be found on the pages of the individual coding regions, or on this wiki's basic parts page. The following list contains all CryoBrick generators designed by our team:

The four parts containing 2 RBSes and coding regions were designed as such, because Cpn10 and Cpn60 should be coexpressed for them to be functional according to literature. Coexpression can be achieved by transforming E. coli with two plasmids containing separate Cpn10 and Cpn60 generators, but we feel this operon-like structure is also an elegant solution. BBa_K538210 should be functionally identical to BBa_K538211, as should BBa_K538310 and BBa_K538311 be. They only differ in the order of their coding regions, which resulted from us planning to assemble the operons by appending BBa_K538100 to BBa_K538201 and BBa_K538301, or by appending BBa_K538101 to BBa_K538200 and BBa_K538300.

Even though we tried many different assembly protocols to attain complete CryoBricks, our progress was impaired by technical difficulties. Dealing with these issues was complicated further because we are the first team to represent the VU and UvA, meaning there are no preceding iGEM participants from our universities that could share their experience with us.

At the time of this writing, only two protein generators - the Cpn10 and CspC generators under control of pLacI - are assembled and characterised, and because neither of them could be transferred to the pSB1C3 backbone succesfully, none of the CryoBrick generators are available from the registry for the time being. Fortunately, we were able to characterise the two generators we assembled. Refer to the characterisation page for further information on their characterisation.