Team:Northwestern/Project/Chassis

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Modeling	Chassis	Induction	Chitin	Apoptosis

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 Protocol

http://www.wired.com/wiredscience/2009/07/cellfactories/

http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html

@@ Line 155: / Line 155: @@
 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).
+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.
+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.