Team:BIOTEC Dresden/Biobrick Assembly

Objective

Our group works on the building of biological parts and assembling them into major functional biological system that serves a specific function. Currently, we are trying to assemble sub parts that would help recognize AHL and express a reporter protein as a function of AHL concentration.

What parts do we assemble?

We attempt to assemble the ribosome binding site (RBS) with each of the following parts: the LuxR as an amplifier and the reporter proteins GFP, YFP and CFP.

What are our approaches?

In order to determine the most convenient and optimal method of assembly, we adopted and experimented on various approaches of assembly. The following are the different kinds of approaches that we are trying to optimize: (i) three antibiotic assembly (3A assembly) (ii) Cloning of the part directly into the plasmid backbone containing the ribosome binding site (iii) Designing of primers that would eventually amplify a subpart composed of the RBS followed by the actual part.

Approach one: 3A Assembly

This approach is the standard method for assembling any two given Biobrick parts and is basically the preferred assembly method for iGeM. The left part (RBS) and the right part (specific part, say, LuxR or any desired reporter) are digested with specific restriction enzymes and cloned into a construction plasmid (plasmid backbone containing chloramphenicol in our case). The final outcome would be the desired chloramphenicol backbone with the desired insert: “RBS + part”.

Approach two

In this approach, we try to insert the desired part directly into the plasmid backbone (ampicillin) containing the RBS. The backbone with RBS is digested with specific restriction enzymes while the backbone containing the part is digested with compatible enzymes so that the end product would contain the RBS followed by the desired part as the insert. This entire insert could then be ligated into a plasmid backbone containing chloramphenicol. This method is simple but the efficiency might be a bit lower than that of the other strategies.

Approach three

Here, we designed appropriate primers whose sequence is composed of the RBS sequence and the corresponding part. This would enable us to acquire a PCR product consisting of the RBS and the part which could be inserted into the backbone with chloramphenicol. This method, as of now, appears to be straightforward with lesser complications and drawbacks than the other two.

Which approach is feasible?

Considering all the above points from all the three methods, we can claim that the third approach seems feasible and simple. We are trying to optimize this method and troubleshoot the problems faced so far.

What techniques we used?

Restriction digest, Ligation, Transformation (electroporation), Colony PCR, Gel purification, PCR purification, Gradient PCR.