Parts

Team IISER Kolkata’s contribution to this year’s 2018 iGEM registry are the following parts:

Bba_K2773000 is a composite biobrick part designed by Mr. Diptatanu Das and Miss. Hrishika Rai. The part comprises of an Arsenic responsive promoter and autorepressor unit (Bba_J33201) upstream of a strong ribosome binding site (Bba_B0030).

When cloned upstream of a reporter or any other protein coding gene, the part will cause strong expression of the protein in response to arsenate and/or arsenite ions. As per characterization by team Edinburgh 2006, the threshold for derepression of the promoter is 1μM of arsenate or arsenite salts in the external growth media of the chassis organism.

In an ideal scenario the best way to characterize this part would have been to clone a reporter biobrick such as RFP or a chromoprotein downstream to it and quantify expression upon induction with increasing concentrations of arsenate or arsenite salts. However, despite several trials, such a characterizable clone could not be obtained. The basic parts used from the registry to produce this composite clone are both very well characterized and used by several iGEM teams in different iGEM seasons before. As a result, we very strongly believe that this part is most likely to work as expected. Nonetheless, as a part of its characterization, we could show the expression of ArsR regulatory protein from the biobrick part indicating that cloning an additional RBS downstream does not affect the ArsR expression, which can be considered as an additional testimony that this biobrick is expected to work.

The above SDS PAGE image shows expression of ArsR regulatory protein with induction by arsenate salts. The characterization is not full proof yet, but can be considered as satisfactory to the conviction, that the part works.