Team:IONIS-PARIS/project/definition

Back to top

Definitions

Biobricks

A BioBrick is a DNA sequence which conforms to a standard of assembly by restriction enzymes. A BioBrick can be a promoter, a ribosomal binding site, the desired coding sequence, or a terminator. A part is the assembly of those BioBricks. It is framed with prefix and suffix standardised sequences, containing restriction sites for EcoRI, PstI, XbaI and SpeI.
These DNA sequences are considered as building blocks and are used to design and assemble larger synthetic biological circuits, made of several individual or combinations of parts with defined functions. The circuits can then be incorporated into microorganisms to perform their respective functions. Different strains can be modified, but in the iGEM competition, Escherichia coli is the most used!

Our components

Cold shock proteins (Csp): a wide family of proteins acting as chaperone for DNA and expressed at low temperatures. This family of proteins include the CspA and CspB, each associated with similar features that permit the protein expression at low temperatures only. These features include a long 5’UTR and a DownStream Box, as well as a specific promoter, all believed to encourage the cold-only protein induction.

amilCP: Chromoprotein from the Acropora millepora coral, exhibiting a strong blue color. Its absorption maximum is 588nm.

cI857: Temperature sensitive repressor of the pL promoter. It is inactivated at 37°C, and so lets the pL promoter accessible for the RNA polymerase. Below 30°C, cI857 dimerization makes it functional, and it subsequently turns off the pL regulated gene expression.

mRFP: Monomeric Red Fluorescent protein, which have excitation and emission peak at 584nm and 607nm. It is an engineered mutant of red fluorescent protein from the Discosoma striata coral. We used it as a visual indicator of expression at high temperatures.

pL promoter: Lambda phage promoter, originally used to express heterologous recombinant protein in E. coli. As it is a strong promoter, it is very efficient for the characterisation of protein expression. It is repressed by the cI857 repressor and fully functional at 37°C.

5’UTR of CspA: This long (159bp) sequence is a master regulator of most of the Cold-shock proteins mRNA and is believed to be very unstable at high temperatures, making the expression of the protein coding sequence it precedes possible at low temperatures only.

CspA promoter and UP element: The CspA promoter is a strong constitutive promoter active at all temperatures. It is supplemented by an Up element that is believed to enhance the promoter strength at low temperatures (although this point is still discussed).

Downstream Box of the CspA coding sequence : This five amino-acids sequence is located downstream the START codong of the Cold-shock protein A mRNA. It is believed to be a 16'S ribosomal subunit binding site. It role is to enhance the translation of the Csp1 mRNA at low temperature by assisting the ribosome and stabilizing the mRNA folding.