MBIO 3410 - Lecture 12 (part 6: cloning a gene)

Want to clone rhaM into a given plasmid and express it in E.coli: FIFTH STEP

1.) Cut the PCR product and plasmids with the same restriction enzymes

2.) This creates compatible sticky ends that allows the PCR product to be inserted into the plasmid, in a specific direction

Problems that may arise with inserting the PCR product into the plasmid

1.) Small piece taken off the plasmid may insert back in

2.) Or it can ligate with the PCR product

3.) Or two copies of the plasmid join together (most problematic)

Problem with two copies of the plasmid joining together

They still allow the cell to grow on ampicillin and the lacZ gene still gets interrupted, therefore resulting in a white colony to still go

Want to clone rhaM into a given plasmid and express it in E.coli: SIXTHH STEP

Add ligase to the mixture, which seals the nicks in the backbones of the PCR product and plasmid

Want to clone rhaM into a given plasmid and express it in E.coli: SEVENTH STEP

Transform the plasmid + PCR product into the chemically competent E.coli

Chemically competent E.coli

They have been chemically altered to take up foreign DNA more easily, specifically by col CaCl2 and heat shocking, which makes the membrane permeable

What is different about the E.coli you’re putting the plasmid in?

1.) Chemically altered

2.) Does not have many of its natural host defences (so it does no degrade the foreign DNA)

3.) It does not have recA

4.) Does not have the wild-type lacZ gene, instead only has half of the lacZ gene in its chromosome

5.) Does not have antibiotic resistance genes (especially ampicillin)

Want to clone rhaM into a given plasmid and express it in E.coli: EIGHTH STEP

Grow the E.coli on LB + Ampicillin + X gal

Why LB

Has all the things E.coli needs to grow

Why ampicillin

Selects for cells that have taken up the plasmid

Why X-gal

Differentiates between cells that have the plasmid without an insert and cells that have the plasmids with an insert

Blue vs. white colony

Blue: no insert (lacZ gene was not interrupted = cleavage of X-gal)

White: yes insert (lacZ was interrupted = cleavage of X-gal)

Want to clone rhaM into a given plasmid and express it in E.coli: NINTH STEP

Isolate the plasmids from the white colonies and sequence/do a gel on them, to ensure that they’re actually carrying what you want

Doing a PCR/gel on your isolated plasmids (with suspected insert) and sequencing them

1.) Cut it with the same restriction enzymes from the beginning, to separate the plasmid and insert

2.) Find out how many bp are in the plasmid and insert, so you know where to see suspected bands

3.) Take the band of the insert and sequence it

Why sequence the insert?

You want to sequence it to ensure that it perfectly matches the sequence you’re trying to clone, in the event that taq or pfu made a mistake.

Diagnostic cutting of the plasmid + insert

1.) Along with cutting with the two initial restriction enzymes, you also want to cut it using a third RE

2.) Specifically one that cuts the insert in the middle

3.) Therefore, you get three bands in total (one of the plasmid and two of the cut insert)

If you’re sequencing it anyway, why do a diagnostic cutting of the insert?

1.) It ensures that you don’t have plasmid + plasmid product

2.) If you do, then you know not to sequence it, which saves you money

Two types of sequencing methods

1.) Sanger sequencing (common for simple procedures like this)

2.) New generation sequencing (more expensive and complex)