Team:USP SaoCarlos-Brazil/Composite Part

IARA

COMPOSITE PARTS

Composite part: MermAID (BBa_K3280007)

Our work plan was basically based on combining two biobricks in order to capture mercury (BBa_k3280005 and BBa_k3280006, respectively the MerR domain with a bidirectional, a promotear and a terminator and the metal binding peptide), a double cellulose binding domain (dCBD), which consisted in two cellulose binding domains from Trichoderma reesei cellobiohydrolases completed with linkers (BBa_k3280006) and a protein domain that increases levels of c-di-GMP to induce biofilm formation (BBa_k3280000). We wanted to achieve a system that could capture mercury from contaminated samples and from that, induce biofilm. We also wanted the captured metal to stay attached to a substrate, which was coconut fiber, therefore the cellulose binding domain (BBa_k1321340).

In addition to our main parts, we also had to use other biobricks to complete the system, such as linkers (BBa_k243005), a terminator (BBa_B0015) and a signal peptide, HlyA, used to target proteins for secretion via the Type I secretion pathway of gram-negative bacteria (BBa_B0015).

Below we have a more detailed description of the parts we used:

Terminator tpr + MerR+ bidirectional Promotor + RBS (Mer operon): BBa_K3280005

Segment of BBa_K1355001, a biobrick designed to be the central piece of different genetic constructions related to mercury, which is composed by a bidirectional promoter, a operton expression regulator (MerR), a carrier protein (MerP) and a mercury transport protein (MerT).

BBa_k3280005 has only a terminator, a RBS, MerR domain and a bidirectional promoter that has a dual function: in reverse, transcription of the MerR regulator protein and in forward, it is opened for insertion.

In the absence of mercury MerR represses transcription by binding tightly to the mer operator region; when mercury is present the dimeric complex binds a single ion and becomes a potent transcriptional activator, while remaining bound to the mer site.

Double cellulose binding domain (dCBD): BBa_k1321340

This biobrick uses two cellulose binding domains from Trichoderma reesei cellobiohydrolases combined with an N-terminal linker and an internal linker sequence between the two domains.

MBP (Metal Binding Peptide) + Linker: BBa_k3280006

Metal binding peptide engineered from PbrR combined with a short linker. It is one of the parts of our construction responsible for the metal capturing process.

Middle linker: BBa_k243005

Linker that can be used to connect two parts and add additional space between them, which can be necessary sometimes to avoid interactions between those parts. It is important that the linker itself has no influence on the connected parts, therefore its sequence is designed with amino acids that don’t interact with other amino acids residues.

The linker’s sequence produced the amino acids Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly. Glycine and Serine are both zwittetionic and hydrophile, and those properties make them good choices for the linker’s repetitive sequence. The linker’s length is also important, because it is responsible to guarantee the independent functioning of the connected parts. Too short linkers can cause sterical interference between the connected parts, and too long linker can cause instability inside the construction.

tDGC + RBS: BBa_k3280000

Protein domain from Thermotoga maritima, a species of hyperthermophilic bacteria that belongs to the order Thermotogales. It increases the levels of c-di-GMP to induce biofilm formation.

Double terminator: BBa_B0015

Consists of the biobricks BBa_B0010 and BBa_B0012. It is the most commonly used terminator.

Signal peptide HlyA: BBa_k208006

It is used to target proteins for secretion via the type I secretion pathway of gram negative bacteria. The fusion of the HlyA signal peptide to the target protein may result in transport of the protein from the cytoplasm to the extracellular medium in a single step. This biobrick should be appended to the C-terminus of the target protein.