Team:Cornell/project/wetlab/chassis

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Revision as of 17:07, 27 September 2013

Cornell University Genetically Engineered Machines

Chassis

E. coli DH5α

Escherichia coli is a gram-negative, facultative anaerobic, rod-shaped bacterium that is the most widely studied prokaryotic model organism for work with recombinant DNA. We are using DH5-α strains, which are electrocompetent and contain recA1 and endA1 mutations that increase insert stability and improve the quality of plasmid DNA prepared from minipreps.

E. coli BL21-A1

We are also using BL21 competent strains of Escherichia coli, which are designed for high-efficiency protein expression of any gene under control of the T7 bacteriophage promoter system. T7 RNA Polymerase is regulated by the arabinose inducible and glucose inhibited araBAD promoter in its chromosomal DNA. Proteins under control of T7 promoter driven vectors are then expressed at high levels. The cells also contain the T7 lysozyme gene within the pLysS plasmid, which suppresses the activity of T7 RNA polymerase caused by leaky basal expression.

Cochliobolus heterostrophus

Cochliobolus heterostrophus is an ascomycete fungal species widely studied for its pathogenic traits as well as its ability to uptake recombinant DNA. Typical methods to integrate recombinant DNA into Cochliobolus include flanking the gene of interest with homologous regions to the fungal genome, restriction enzyme mediated integration(REMI), and random insertion. Typically using homologous recombination offers the highest efficiency while random insertion offers the lowest. The fungal strain is transformed by protoplasting followed by PEG transformation.

References

1. http://www.mclab.com/Dh5-Alpha-Competent-E.-Coli.html
2. http://www.promega.com/
3. http://www.biodynamics.co.jp/images/prd_ds250/DS260BLysShp.pdf

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-					<h3><i>E. Coli</i></h3>
+					<h3><i>E. coli</i> DH5α</h3>
-Escherichia coli  is a gram-negative, facultative anaerobic, rod-shaped bacterium that is the most widely studied prokaryotic model organism for work with recombinant DNA. We are using DH5-α strains, which are electrocompetent and contain recA1 and endA1 mutations that increase insert stability and improve the quality of plasmid DNA prepared from minipreps. We are also using BL21 competent strains, which are designed for high-efficiency protein expression of any gene under control of the T7 bacteriophage promoter system.  Under control of the lacUV5 promoter in its chromosomal DNA, T7 RNA Polymerase is expressed upon addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG). Proteins under control of T7 promoter driven vectors are then expressed at high levels.The cells also contain the T7 lysozyme gene within the pLysS plasmid, which suppresses the activity of T7 RNA polymerase caused by leaky basal expression.
+<i>Escherichia coli</i> is a gram-negative, facultative anaerobic, rod-shaped bacterium that is the most widely studied prokaryotic model organism for work with recombinant DNA. We are using DH5-α strains, which are electrocompetent and contain recA1 and endA1 mutations that increase insert stability and improve the quality of plasmid DNA prepared from minipreps.
+                                        <h3><i>E. coli</i> BL21-A1</h3>
+We are also using BL21 competent strains of <i>Escherichia coli</i>, which are designed for high-efficiency protein expression of any gene under control of the T7 bacteriophage promoter system. T7 RNA Polymerase is regulated by the arabinose inducible and glucose inhibited <i>ara</i>BAD promoter in its chromosomal DNA. Proteins under control of T7 promoter driven vectors are then expressed at high levels. The cells also contain the T7 lysozyme gene within the pLysS plasmid, which suppresses the activity of T7 RNA polymerase caused by leaky basal expression.
 					<h3><i>Cochliobolus heterostrophus</i></h3>
 Cochliobolus heterostrophus is an ascomycete fungal species widely studied for its pathogenic traits as well as its ability to uptake recombinant DNA. Typical methods to integrate recombinant DNA into Cochliobolus include flanking the gene of interest with homologous regions to the fungal genome, restriction enzyme mediated integration(REMI), and random insertion. Typically using homologous recombination offers the highest efficiency while random insertion offers the lowest. The fungal strain is transformed by protoplasting followed by PEG transformation.