Team:Cornell/Notebook/Arsenic reporter/June10

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The purpose of this subproject is to make the nah operon biobrick compatible for the use of future iGEM teams. The nah operon codes for a pathway that converts naphthalene into salicylate. In our system, this will allow naphthalene to be detected by the salicylate reporter. There are three standard cut sites internal to the operon which we are removing - PstI, and two NotI sites. This biobrick compatible piece could be useful to future teams in projects that involve bioremediation or detection of naphthalene.
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The goal of the arsenate reporter is to detect soluble arsenic-containing compounds and produce an electrical current. In this subproject, we put control of the mtrB gene under an arsenate-inducible promoter, allowing Shewanella to activate the [[Team:Cornell/Project/Mtr pathway|Mtr pathway]], producing an electrical current.
[[Team:Cornell/Notebook/Arsenic_reporter|Back to arsenic reporter week view]]
[[Team:Cornell/Notebook/Arsenic_reporter|Back to arsenic reporter week view]]
===June 10th-16th===
===June 10th-16th===
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====June 13th, Wednesday====
 
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*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.
 
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*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.
 
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**''Work done by: Caleb, Claire, Dylan, Spencer, Steven, and Swati''
 
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====June 14th, Thursday====
 
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*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.
 
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====June 15th, Friday====
 
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*PCR of four out of five products visible on gel. Set up PCR of pBMT-1, final fragment required for Gibson Assembly of the nah operon.
 

Revision as of 21:41, 3 July 2012

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The goal of the arsenate reporter is to detect soluble arsenic-containing compounds and produce an electrical current. In this subproject, we put control of the mtrB gene under an arsenate-inducible promoter, allowing Shewanella to activate the Mtr pathway, producing an electrical current. Back to arsenic reporter week view

===June 10th-16th===