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Team:Northwestern/Project/Chassis
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To prevent bacteria from digesting synthesized Chitin, as it normally would, the ΔChiA strain was also taken from the Keio Collection. Because this strain lacks Chitinase, a digestive enzyme that breaks the glycosidic bonds in Chitin. | To prevent bacteria from digesting synthesized Chitin, as it normally would, the ΔChiA strain was also taken from the Keio Collection. Because this strain lacks Chitinase, a digestive enzyme that breaks the glycosidic bonds in Chitin. | ||
| - | + | We also attempted to perform the MAGE protocol on EcNR2 lambda-red competent cells to obtain double knockouts, yet after 15 rounds of electroporation and subsequent extensive culture screening, we found that the ShockPod on our BioRad electroporator had shorted, meaning no electroporation was ever performed. We were unable to attempt this again due to time and equipment restraints. According to Wang et al., this experiment should yield approximately 30% efficiency when performed properly. | |
Revision as of 17:38, 23 October 2010
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Our team primarily used the high transformation efficiency top10 cells from invitrogen for most protocols. In order to ensure lawn formation, the TqsA (also known as the YdgG) knockout were taken from a copy of the Keio Collection with permission from Professor Margaret Saks of Northwestern University. The ΔTqsA strain increases the thickness of biofilm by interfering with the quorum sensing mechanism (more specifically, the autoinducer-2 transport system). To prevent bacteria from digesting synthesized Chitin, as it normally would, the ΔChiA strain was also taken from the Keio Collection. Because this strain lacks Chitinase, a digestive enzyme that breaks the glycosidic bonds in Chitin. We also attempted to perform the MAGE protocol on EcNR2 lambda-red competent cells to obtain double knockouts, yet after 15 rounds of electroporation and subsequent extensive culture screening, we found that the ShockPod on our BioRad electroporator had shorted, meaning no electroporation was ever performed. We were unable to attempt this again due to time and equipment restraints. According to Wang et al., this experiment should yield approximately 30% efficiency when performed properly.
Invitrogen Top10 Cells:Purchase and Description: http://products.invitrogen.com/ivgn/product/C404010 TqsA aka YdgG Knockouts:http://jb.asm.org/cgi/content/short/188/2/587 http://ecoli.aist-nara.ac.jp/GB5/info.jsp?id=JW1593 http://cgsc2.biology.yale.edu/Mutation.php?ID=106356 ChiA Knockouts:http://cgsc2.biology.yale.edu/Mutation.php?ID=100308 http://www.ncbi.nlm.nih.gov/gene/947837 http://ecoli.aist-nara.ac.jp/GB5/info.jsp?id=JW3300 MAGE Protocolhttp://www.wired.com/wiredscience/2009/07/cellfactories/ http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html | |||||||||||
