1. SIMPLE CLONING EXERCISE In the genome sequence of Propionibacterium acnes (ba
ID: 278420 • Letter: 1
Question
1. SIMPLE CLONING EXERCISE In the genome sequence of Propionibacterium acnes (bacterium that causes acne), you have discovered a gene predicted to encode a lipase. This is an enzyme that breaks down lipids in the skin, and if an inhibitor could be developed, it might lead to a therapy for acne. You want to find out more about this enzyme. You plan to clone the gene, express it in E. coli, make lots of the protein, and study its propertics How would you clone this gene into E. coi? (15 points, 2-page limit, including figures and references) - assume you have a suitable cloning vector (pCR2.p from Invitrogen; below) assume you can use the techniques you learned about so far Lipase gene (743 bR) as positioned in the P. acnes chromosone 2 PCR2.1 020Explanation / Answer
Experimental steps for cloning lipage gene into pCR2.1 vector:
1. Amplify the lipage gene from P. acnes chromosome using suitable PCR primers containing KpnI and EcoRI complementary sequences.
2. Purify the PCR product and sequence it to verify the amplication.
3. Restriction digestion of purified DNA (ligase gene) and pCR2.1vector with KpnI and EcoRI.
4. Ligate digested gene and vector using DNA ligase.
5. Purify the Ligate products. Check the DNA insertion (recombinant vector) using PCR.
6. Transform competent E. coli with the recombinant vector. Use medium containing ampicillin to grow the transformed colonies.
7. Transformed cells will form colonies on medium containing ampicillin because they have ampicillin resistance gene in thier recombinant vector.
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