Question

How would you clone the Eukaryotic gene into the plasmid

How would you transform bacteria with that engineered plasmid

How would you select for the bacteria that will take up the plasmid with integrated Eukaryotic gene

Answer 1

Steps-

1) First we have to know the sequence of the eukaryotic gene to be cloned. The gene sequence of the eukaryotic gene is taken from NCBI or UniProt.

2) Design a forward primer and reverse primer which will have some portion of vector sequence and some portion of sequence from the eukaryoric gene. The vector chosen has to be bacterial vector which has an 'origin of replication' – a stretch of DNA that ensures it gets replicated (copied) by the host bacterium.

3) The first PCR is carried out to amplify the gene of interest from the eukaryotic genome. The amplified product has the gene of interest and overhangs of few base pairs which correspond the vector sequence between the multiple cloning site.

4) Restriction free (RF) cloning is done where the PCR uses bacterial plasmid as the template and the gene of interest as a mega primer. This amplifies the whole plasmid. Use the enzyme DpnI to cleave the methylated plasmid (only vector) so that only the amplified vector (vector plus insert) stays in the solution.

or

We can do digestion ligation instead of RF cloning. First digest the insert and the vector with the same restriction enzymes and then ligate it using ligase.

5) Transform the cloned product into bacteria using heat shock method where bacterial cells are made permeable using calcium chloride so that it takes up the plasmid by giving heat shock.

6) To ensure that the cloning has worked, the transformed bacteria is plated onto antibiotic resistant plate like ampicilin (if the vector has ampicilin resistant gene) and let it grow.