Team:Cornell/Notebook/Nah operon

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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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==June==
 
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===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.
 
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===June 17th-23rd===
 
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====June 17th, Sunday====
 
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*Successful PCR of pBMT-1 [[Gel Purification | gel purified]].
 
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====June 19th, Tuesday====
 
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*Ran [[Gibson Assembly]] of nah operon fragments into pBMT-1 backbone and [[Transformation | transformed]] into DH5a electrocompotent ''E. coli'' cells.
 
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*Set up [[PCR]] to amplify the Gibson Assembly products.
 
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**''Work done by: Dylan and Swati''
 
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====June 20th, Wednesday====
 
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*Set up a [[digestion]] of the PCR of Gibson Assembly products and ran the digested products on a gel to see if Gibson worked.
 
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*Three colonies on plates of DH5a transformed with Gibson Assembly product. Made [[liquid cultures]] to miniprep and sequence.
 
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**''Work done by: Dylan and Swati''
 
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====June 22nd, Friday====
 
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*[[Miniprep | Miniprepped]] directly transformed Gibson Assembly product for sequencing using the the Gibson nah1F and nah4R primers (each w/ 20 bp overhangs).
 
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*Ran undigested miniprep with [http://2011.igem.org/Team:Cornell/Protocols gel electrophoresis], looking for large bands corresponding to supercoiled DNA. The gel shows distinct bands for all three lanes. We interpreted this to mean that we got product. Submitted for [[sequencing]]
 
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::[[File:Cornell2012_0621_Gibson-entire-plasmid.jpg|600px]]
 
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===June 24th-30th===
 
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====June 27th, Wednesday====
 
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[[Image:2012_627_RafDylanGelMaking.jpg|thumb|right]]
 
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*Ran digest of gibson-assembled nah operon with [[gel electrophoresis]]
 
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====June 28th, Thursday====
 

Latest revision as of 06:40, 26 October 2012