The cold resistance BioBricks - or CryoBricks - we've developed for our project are classified as basic or composite parts. Composite parts are further grouped into finalized parts, containing a promoter, RBS and coding region, and construction intermediates, containing either a promoter and RBS, or an RBS and coding region. Note that some of the parts listed below were submitted to the registry by other teams. Our own parts are coded K538###. Some of them are submitted as physical DNA, where others are theoretical submissions that never got past the planning stage.
We use 3 different types of basic parts in the construction of our CryoBricks: promoters, RBSes, and protein coding regions. More detailed information about their purpose can be found on the basic parts page.
Based on literature study and safety considerations, we had planned to incorporate four different promoters in our project. The selection comprised the lac operon's promoter (pLacI, BBa_R0010), two arabinose-induced promoters (pBAD derivatives - strong, BBA_K206000, and weak, BBa_K206001) and the hydrogenase II operon's promoter (hybB, BBa_J45503). While the selected promoters are relatively well characterized, little to no information is available about the effect of low temperature on their activity. For our project, investigating this effect would have been of great value to fine-tune protein expression at different temperatures. Unfortunately, due to facing many difficulties with assembly, we weren't able to characterize the promoter activities, and we ended up working with only two of the selected promoters: pLacI and the strong pBAD promoter.
Many iGEM teams before us have characterised the strength of one or several ribosome binding sites, and this information is valuable for the same reasons as characterising promoter activities: fine-tuning protein expression. Again, no information was available of RBS activity at different temperatures. We would have liked to carry out characterisation experiments, but failing assemblies and constraints on time prevented this. In the end, we simply decided to work with one of the strongest RBSes in the Community Collection: BBa_B0034.
Our project aims to enhance E. coli's growth rate at low temperatures, by heterologously expressing chaperones from cryophillic bacteria. Their effect may possible be further exacerbated by adding genes encoding anti-freeze proteins (AFPs). Three different AFPs we'd hoped to use for this were generously provided by the team that represents Yale this year. Unfortunately, we received their shipment too late to incorporate their parts in our project.
Selection of chaperones suitable for our project was done based on literature study. We have selected five, the first two of which depending on eachother to function. The selected chaperones are: Cpn10 and Cpn60, forming Oleispira antarctica 's Cpn60/10 system (BBa_K538000 and BBa_K538001); SheDnaK, from Shewanella sp. AC10 (BBa_K538002); CspA, from Polaribacter irgensii (BBa_K538003); and CspC, also from Polaribacter irgensii (BBa_K538004). GeneArt synthesized these parts for us de novo, from sequences that were taken from online repositories and recoded for compatibility with assembly standards.
The composite parts we submitted to the registry are either construction intermediates or finalized parts. The construction intermediates consist of all possible combinations of our selected promoters (BBa_R0010 and BBa_K206000) and RBS (BBa_B0034), and all combinations of that same RBS with our coding regions (BBa_K538000 through BBa_K538004). Note that the promoter + RBS parts contain no bricks that weren't already in the registry.
The finalized parts all comprise a promoter, an RBS and a coding region. They are made either by appending a coding region to the construction intermediates containing a promoter and RBS, or by prepending a promoter to the construction intermediates containing an RBS and a coding region. An additional four final constructs are designed as 'operons' comprising Cpn10 and Cpn60. They are made by appending either the RBS + Cpn10 or the RBS + Cpn60 construction intermediates to the finalized parts that contain the other.
None of these bricks contain a terminator sequence; we have considered appending BBa_B0014 to them, but the tightly constrained time we had for our project, and what little of it remained after sorting out (part of) the problems with assembling BioBricks, caused us to decide against this and relying instead on the terminator sequence in the standard plasmid backbones.
All parts we submitted to the registry are listed in the table below. The BBa_* links will take you to their respective pages on the parts registry. To view their information on this wiki, refer to our basic and composite part pages instead. Our newly synthesized chaperone genes (basic parts) are labelled K5380##. Construction intermediates are labelled K5381##. Finalized CryoBricks are labelled K5382## or K5383## depending on their promoter: 2## for parts containing pLacI, 3## for parts containing pBAD.