Assembly

All cloning steps in the project were carried out following a general scheme as presented below:

The assembly mechanism followed is called 2 way or standard assembly, whose pictorial representations are shown below:

1. Cloning Biobrick A upstream of another Biobrick B with the former as vector.

2. Cloning Biobrick A upstream of another Biobrick B with latter as the vector.

The circuit design of BacMan requires two protein generator parts or operons:

Operon 1 that will produce T7 RNA Polymerase in response to exposure to arsenic.

To obtain the above composite part, J33201, B0030 and K145001 biobricks supplied through the DNA distribution kit plate have to be cloned together in a single vector in correct sequence. This requires the following clonings:

1. Cloning ArsR upstream of RBS.

2. Cloning the part obtained from above step upstream of T7 pol.

Operon 2 that will in response to T7 Pol production start expressing the metal chelating and other proteins required for effective sequestration of the ion.

Creation of the above part will require the following sequential steps:

1. Cloning PC CBD upstream of RBS.

2. Amplifying and obtaining HMT1 from the wild (S. pombe) with appropriate restriction sites at the ends incorporated by specific primers and site directed mutagenesis to eliminate illegal sites present within the ORF.
3. Cloning obtained HMT1 downstream of PC.CBD + RBS clone obtained in step 1.

4. Cloning the entire part obtained in step 3 upstream of a RBS biobrick.

5. Amplifying and obtaining AOX from the wild (Beta-Proteobacteria like Thermus thermophilus).
6. Cloning the obtained AOX downstream of the part obtained in step 4 to finally get the entire composite part.

Of the planned steps presented above, during the course of this iGEM Project, Team IISER-Kolkata accomplished the following:

1. We successfully cloned ArsR upstream of RBS.