Team:IIT Kanpur/Results

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Results

Cloning of our device for optimizing extracellular expression using PelB/Ompt secretion tags under different Anderson promoters:

We used pSBIK3.m1 and pSB1C3 for cloning of our designed constructs. We synthesised our DNA construct via IDT. We added restriction enzyme site for Ecor1 and Pst1 in our synthesised double stranded DNA. We performed restriction enzyme digestion for both our double stranded DNA and backbones with Ecor1 and Pst1. These were ligated together.

These are the biobricks we submitted in iGEM registry.
BBa_K2874000
BBa_K2874001
BBa_K2874002

These are the sequences which we cloned but are not submitted with iGEM registry.
pBS1K3.m1 ( JA23100->pelB-LINKER-sfGFP-HIS-STOP)
pBS1K3.m1 ( JA23102->ompT-LINKER-sfGFP-HIS-STOP)

Furthermore we have analyzed using different techniques to detect if our construct facilitates extracellular expression of sfGFP.

Detecting protein secretion using different staining techniques in E.coli (DH5 alpha and Rosetta strain)

E.coli (DH5 alpha)

We first decided to test whether our cloned product could produce extracellular protein. We chose E.coli(DH5 alpha) as our first bacterial production system. We picked colonies and set Luria broth (LB) corresponding to the constructs as shown in the scheme below. We separated supernatant and pellet corresponding to each sample. These samples were further analyzed for protein content.

Ponceau S is a sodium salt of a diazo dye of a light red color, it is used a stain for rapid reversible detection of protein bands on nitrocellulose membrane(Western blotting).

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