Question

Pretend you are trying to get E. coli to express a gene of interest. You plan to insert the gene into a plasmid, transform your plasmid into the bacteria, and verify your results with blue-white screening. Describe three things that you would need the plasmid to have (6 pts), what processes you would have to do to get a finished plasmid (2 pts), what results you expect to see (2 pts) and the cause of those results (2 pts).

Answer 1

3 things that plasmid need to have required to facilitate cloning are:

1. Origin of replication (ori) ​​​​​​- This is sequence from where replication starts and any piece of DNA when linked to the sequence can be made to replicate within the host cell. This sequence is also responsible for controlling the copy number of the linked DNA.

2. Selectable marker- Selectable marker helps in identifying an eleiminating non-transformants and selectively permitting the growth of transformants. Normally, the genes encoding resistance to antibiotics such as ampicillin, tetracycline, kanamycin etc., are considered useful selectable markers for E.coli.

3. Cloning sites- To link the gene of interest, the plasma needs to have few or a single recognition site, for the commonly used restriction enzymes. Presence of more than one recognition sites within the plasmid regenerate several fragments, which will complicate the gene cloning.

The processes needed to be done to get a finished plasmid are:

Gene cloning, refers to the process of isolating a gene of interest for the purpose of making multiple copies of it. So the processes needed are

1. Isolation of gene of interest-

A restriction enzyme (restriction endonuclease) is an enzyme that cuts double-stranded DNA at a specific sequence producing either overlapping ends (also known as sticky ends) or blunt ends.

The plasmid or vector is digested with restriction enzymes, opening up the vector to allow insertion of the target DNA.

2. Ligation-

Restriction fragments ( DNA of interest and a plasmid/vector) can be spliced together, provided their sticky ends are complementary. Blunt end ligation is also possible.

The two DNAs are then incubated with DNA ligase, an enzyme that can attach together strands of DNA with double strand breaks. This produces a recombinant DNA molecule.

Recombinant plasmids are combined with a culture of living bacteria. Bacteria may take up the plasmid with or without the insert or may not take up plasmid at all. So the results expected to be seen and their causes are:

Non- Transformants- Are those bacteria which do not take plasmids into their cells.

Transformants- Are those bacteria that may take plamids, but do not have foreign DNA into them.

Recombinants- Are those plamids which take up the recombinant plasmid i.e. plasmid with foreign insert.

These can be differentiated by the process of blue- white screening.

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