Team:Washington/Gram Negative/Build

From 2010.igem.org

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(Creating the Tse2/Tsi2 Toxin/ Antitoxin system)
(Engineering the Toxin Antitoxin HSL-inducible circuit)
 
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=Building a Type VI Dependent Probiotic=
 
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One of the major hurdles of creating our probiotic is the high level of regulation that this system is under in its native host.  Several transcriptional and post-transcriptional regulators are present in both promoter regions, which prevented the system from expressing in ''E. coli''. 
 
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=Creating the Tse2/Tsi2 Toxin/ Antitoxin system=
 
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[[Image:Washington_Building_Tse2_circuit.png|800px|]]
 
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The Tse2/Tsi2 locus was amplified using PCR with primers that amplified the region from the start codon of Tse2 to the stop codon of Tsi2.The primers were designed to flank the Tse2/Tsi2 product with approximately 40bp regions of homology to the psb3k3-F2620 plasmid. The forward primer added 44 basepairs of homology to the 3' end of F2620 to the 5' end of the construct. The reverse primer added 38 basepairs of homology to the suffix end of psB3k3 to the 3' end of the construct. The Tse2/Tsi2 locus was then placed downstream from F2620 via [https://2010.igem.org/Team:Washington/Protocols Gibson Cloning]. Gibson cloning is a method that joins regions of homology found on the 5' and 3' ends of linearized molecules.  The end result of this Gibson cloning reaction was a circularized plasmid containing the final F2620-Tse2/Tsi2 construct in psb3k3. The Gibson reaction mixture was transformed into electrocompetent Dh5a, and the resulting colonies were screened for insert of expected length via double restriction digest followed by agarose gel electrophoresis. The plasmids that showed inserts of expected length were sequenced, and it was determined that multiple plasmids had F2620-Tse2/Tsi2 inserts with expected sequences.
 
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=Recombineering the type 6 secretion system=
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=Recombineering the Promoter Region of the Type VI Secretion System=
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[[Image:Washington_Recombineering_basic.png|800px]]
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[[Image:Washington_Recombineering_basic.png|750px|center|thumb|Schematic Showing the Engineering of the Fosmid to Contain a Divergent T7 Promoter Instead of the Native ''Pseudomonas'' Promoter]]
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Our goal was to replace the native ''P. aeruginosa'' regulation of the Type VI Secretion System with one that can be utilized by the ''E. coli'' transcriptional machinery.  In order to replace the native promoters with more robust T7 promoters we used a technique called [https://2010.igem.org/Team:Washington/Protocols/Recombineering Recombineering] to flip out the region containing both promoters and replace it with a gene cassette as follows.  We designed primers to create a cassette using PCR, containing the ''galK'' galactose metabolism gene flanked by 50 base pairs of homology just outside of the promoter region in the fosmid.  The cassette was inserted, giving us a selection factor for our final insertion of our new promoters.  A new promoter cassette was designed in total using oligos that encoded the two T7 promoters flanked by the same 50 base pairs of homology used previously, and inserted using recombineering into the fosmid that contained ''galK'' in the promoter region.
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=Engineering the Toxin Antitoxin HSL-inducible circuit=
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[[Image:Washington_Building_Tse2_circuit.png|center|750px|thumb|Schematic Detailing the Transfer of Tse2 and Tsi2 from ''P. Aeruginosa'' into pSB3k3]]
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The Tse2/Tsi2 locus was amplified from ''P. aeruginosa'' genomic DNA using PCR.  Primers were used that amplified the region from the start codon of Tse2 to the stop codon of Tsi2.  The Tse2 and Tsi2 locus was then placed downstream from F2620 via [[Team:Washington/Tools_Used/Next-Gen_Cloning|Gibson Cloning]]. Gibson cloning is a method that joins regions of homology found on the 5' and 3' ends of linearized molecules.  The end result of this Gibson cloning reaction is a circularized plasmid containing the final F2620.Tse2.Tsi2 construct in pSB3K3. The circularized plasmid is then transformed into electrocompetent ''E. coli'' Dh5a and the resulting colonies were screened for the insert of expected length via double restriction digest followed by agarose gel electrophoresis. The plasmids that showed inserts of expected length were sequenced, and it was determined that multiple plasmids had correct F2620.Tse2.Tsi2 inserts.
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'''&larr; [[Team:Washington/Project/Mougous/Design|Designing the Gram(-) Therapeutic]]'''
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'''&larr; [[Team:Washington/Gram Negative/Design|Designing the Gram(-) Therapeutic]]'''
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'''[[Team:Washington/Project/Mougous/Test|Testing the Gram(-) Therapeutic]] &rarr;'''
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'''[[Team:Washington/Gram Negative/Test|Testing the Gram(-) Therapeutic]] &rarr;'''
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Latest revision as of 16:49, 26 October 2010


Recombineering the Promoter Region of the Type VI Secretion System

Schematic Showing the Engineering of the Fosmid to Contain a Divergent T7 Promoter Instead of the Native Pseudomonas Promoter

Our goal was to replace the native P. aeruginosa regulation of the Type VI Secretion System with one that can be utilized by the E. coli transcriptional machinery. In order to replace the native promoters with more robust T7 promoters we used a technique called Recombineering to flip out the region containing both promoters and replace it with a gene cassette as follows. We designed primers to create a cassette using PCR, containing the galK galactose metabolism gene flanked by 50 base pairs of homology just outside of the promoter region in the fosmid. The cassette was inserted, giving us a selection factor for our final insertion of our new promoters. A new promoter cassette was designed in total using oligos that encoded the two T7 promoters flanked by the same 50 base pairs of homology used previously, and inserted using recombineering into the fosmid that contained galK in the promoter region.

Engineering the Toxin Antitoxin HSL-inducible circuit

Schematic Detailing the Transfer of Tse2 and Tsi2 from P. Aeruginosa into pSB3k3

The Tse2/Tsi2 locus was amplified from P. aeruginosa genomic DNA using PCR. Primers were used that amplified the region from the start codon of Tse2 to the stop codon of Tsi2. The Tse2 and Tsi2 locus was then placed downstream from F2620 via Gibson Cloning. Gibson cloning is a method that joins regions of homology found on the 5' and 3' ends of linearized molecules. The end result of this Gibson cloning reaction is a circularized plasmid containing the final F2620.Tse2.Tsi2 construct in pSB3K3. The circularized plasmid is then transformed into electrocompetent E. coli Dh5a and the resulting colonies were screened for the insert of expected length via double restriction digest followed by agarose gel electrophoresis. The plasmids that showed inserts of expected length were sequenced, and it was determined that multiple plasmids had correct F2620.Tse2.Tsi2 inserts.


Designing the Gram(-) Therapeutic       Testing the Gram(-) Therapeutic