Team:Cornell/Notebook/Nah operon


Revision as of 19:53, 28 June 2012 by Cpaduano (Talk | contribs)
Home Team Project Parts Modeling Notebook Protocols Safety Attributions



Project Description

The purpose of this subproject is to make the nah operon biobrick compatible for the use of future iGEM teams. The nah operon is an operon that converts napthalene into salicylate. In our system, this will allow napthalene to be detected by the salicylate reporter. There are three standard cutsites internal to the operon which we are removing - the biobrick compatible piece could be used by future teams in bioremediation projects.

June 10th-16th

June 13th, Wednesday

Set up PCR for four fragments of the nah operon as well as the pBMT-1 backbone, using primers designed to mutate cut-sites concurrently with Gibson Assembly. Also set up a PCR for the entire nah operon. Due to length of the fragments, a longer extension time was chosen
If Gibson Assembly fails, but we are able to PCR the entire 10kb nah operon using Phusion polymerase, an alternate method of mutation using Phusion will be pursued.
Work done by: Caleb, Claire, Dylan, Spencer, Steven, and Swati

June 14th, Thursday

PCR only amplified nah1, nah3, and nah4 - longer fragments (nah2, the full nah operon, and pBMT-1) were not identified on the PCR product gel. Set up another PCR with shorter extension time

June 17th-23rd

June 19th, Tuesday

Ran Gibson Assembly of nah operon fragments into pBMT-1 backbone and transformed into DH5a electrocompotent E. coli cells.
Set up PCR to amplify the Gibson Assembly products.
Work done by: Dylan and Swati

June 20th, Wednesday

Set up a digestion of the PCR of Gibson Assembly products and ran the digested products on a gel to see if Gibson worked.
We'll also check to see if we got colonies from the transformation then.
Work done by: Dylan and Swati

June 24th-30th

June 27th, Wednesday

  • Ran digest of gibson-assembled nah operon on gel

====June 28th, Thursday====