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 as a part of creating Operon 1. The clone was confirmed by sequencing and we submitted it as our only contribution to the registry this season (See: Parts page).

2. For Operon 2, HMT1 was also obtained from nature (S. pombe) but we could proceed with its site directed mutagenesis only partially as a result HMT1 could not be submitted as a part as it still had illegal sites.
   The protocol for obtaining HMT1 and mutating the illegal PstI and EcoRI sites that the ORF contains is presented in the following flowchart. To view a clearer version, please click here.
   
   We obtained the total RNA content from S. pombe from the lab of Dr. Geetanjali Sundaram at Calcutta University and the reverse transcribed it in RT-PCR to get the cDNA. The cDNA was then used in a normal PCR to yield unmutated HMT1 by amplification using primers.
   
   Four primers were designed such that they first amplify a small region on HMT1 gene to produce a few hundred basepairs long megaprimers that contain mutation at the illegal restriction site such that protein code and frame is not altered (site directed mutagenesis). The Mega primers were purified from PCR Reaction and then used again on the cDNA to give a mutated version of HMT1 lacking illegal sites but coding for same protein.

We could proceed successfully only till the step of obtaining Megaprimers containing mutation.

3. We cloned PC-CBD upstream of RBS as a part of creating Operon 2. However, this clone was found to be negative during confirmation by sequencing and hence was not submitted as a part by the team.
4. As a back up plan to amplifying HMT1 and AOX genes from nature all by ourselves, we also availed the IDT offer to obtain 1000bp long g-blocks to construct Operon 2 using Gibson Assembly Kit. But due to limitations of time we could not work on the gibson assembly to ligate the g-blocks.
5. We wanted to characterize the effect of expressing a metal chelating protein such as phytochelatin in wild type bacteria to see how the growth is affected upon exposure to heavy metals such as arsenic. For this we wanted the PC CBD part in a good expression vector such as pET28a as previous attempts of expression of this part from pSB1C3 had yielded poor results in terms of strength of the protein band in PAGE and incubation time required after induction by IPTG. Therefore, PC CBD part was cloned into pET28a vector and expressed in E. coli BL21(DE3) by IPTG induction.

The above image shows gel electrophoresis result of retardation and insert release digestion tests performed to confirm the clone of PC CBD in pET28a vector. The next step was to express PC CBD protein in BL21(DE3) strain of E. coli by inducing the cells with increasing concentrations of IPTG.

The SDS PAGE result shows that PC CBD band at 16kDa is obtained and the protein is getting expressed in lesser time and greater quantities than it did from pSB1C3 vector.