Flashcards- ABE Lab 5a-6

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LAB 5a: Transforming Bacteria with the pARA-R Plasmid

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Introduce the genetically modified plasmid into E. coli bacteria to produce the rfp protein.

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Contents of the P+ tube

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The P+ tube contains the plasmid (and E. coli).

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Contents of the P- tube

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The P- tube does not contain the plasmid (but it did contain E. coli).

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Step causing plasmid entry into bacteria (transformation step)

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Heat shock causes the plasmid to enter the bacteria.

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Identifying transformed bacteria on petri plates

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Transformed bacteria can be identified by their growth on a plate that contains antibiotics. (untransformed cells would die)

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Environmental needs for transformed bacteria to make rfp protein

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Transformed bacteria need Arabinose (an inducer) in their environment to make rfp protein.

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Description of Recombinant plasmid of interest: pARA-R

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The pARA-R construct is a recombinant plasmid with a size of 5,302 base pairs. It includes sections such as araC, Amp^R, PBAD, and rfp, with restriction sites BamHI and HindIII.

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What makes our E. coli competent?

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The have been coated in Calcium ions (which leads to adhesion zones allowing for the plasmids to potentially enter)

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Expected Growth of bacteria on plate containing only Luria Broth (LB plate)

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Both the P+ and P- sides on the plate should shown bacterial lawns (unrestricted growth; LOTS!)

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Expected Growth of bacteria on plate containing Luria Broth & Ampicillin (LB/amp plate)

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The P+ side of the plate should contain white colonies. These are only the transformed cells (they have the plasmid which contains the ampicillin resistance gene so they live in the presence of antibiotics).

The P- side of the plate should not have growth. The bacteria did not get any plasmid so they are not anti-biotic resistant, so they will all die on this plate)