Team:UrbanTundra Edmonton/Experiments
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Perchlorate Metabolizing E.coli
CPB-38-441 Expression Vector
Our cloning strategy was to replace the “Tinsel” chromophore gene cassette contained in the host plasmid CPB-38-441,with the G-Blocks for Cld(SP+) and Cld(SP-) using the compatible BsaI sticky-ends located at the ends of our G-Blocks and the Tinsel gene cassette as shown in Figure_ below. When transformed and plated, we expected that ligation mixtures containing equal molar concentrations of digested G-Block and plasmid would produce an equal number of white colonies (Cld-containing plasmids) and purple colonies (Tinsel containing plasmids)in the presence of kanamycin. We also plated transformations that contained either no DNA or the host plasmid CPB-38-441. The absence of colonies on the former would verify that resulting colonies are not due to contamination. The presence of purple colonies on the latter would demonstrate that the lack of colonies on either Cld plate was not due to failed transformation but rather a problem upstream in the cloning procedure.
Full CLD± Constructs
LacI Operators and Repressor
Transformation
Plating
Our cloning strategy was to replace the “Tinsel” chromophore gene cassette contained in the host plasmid CPB-38-441,with the G-Blocks for Cld(SP+) and Cld(SP-) using the compatible BsaI sticky-ends located at the ends of our G-Blocks and the Tinsel gene cassette as shown in Figure_ below. When transformed and plated, we expected that ligation mixtures containing equal molar concentrations of digested G-Block and plasmid would produce an equal number of white colonies (Cld-containing plasmids) and purple colonies (Tinsel containing plasmids)in the presence of kanamycin. We also plated transformations that contained either no DNA or the host plasmid CPB-38-441. The absence of colonies on the former would verify that resulting colonies are not due to contamination. The presence of purple colonies on the latter would demonstrate that the lack of colonies on either Cld plate was not due to failed transformation but rather a problem upstream in the cloning procedure.
Gel Analysis
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