Genetic Recombination Technology

[dttom]

The basic principle in this biotech is the cut-open of a plasmid which acts as a cloning vector, better work with the one containing antiobiotic favorable for later selection process, followed by insertion of desired gene or gene fragment. After amplification and proper induction in gene expression, the protein product could be harvested. If it is not the protein wanted, simple amplification is enough for harvesting of the desired gene or gene fragment.

 

However, there are instances when the sequence in the vector would be incorporated into the bacterial host genome, which I can't see to cause a trouble in the amplification process, but this makes the bacterial composition differential. This makes me to have a concern in under what circumstance would a plasmid be integrated into host genome with a comparatively high probability.

 

Besides, one of the method in selecting those bacterial having absorbed the cloning vector is by alpha-complementation of an enzyme (adding a second part of an enzyme with a polyclonal site to an antibioticase-containing vector), which, though, is a rather old method. So there should be some more recent and more efficient method doing the same job. I wonder what would they be. I am actually considering ligating the desired gene fragment with an antiobioticase gene, forming an operon with single promotor. In a medium with suitable transcription factors and antibiotic, theoretically only those which have absorbed the desired gene should survive. But I am not sure of the actual feasibility of this method.

 

Welcome discussion ^^

[Mr Skeptic]

The thing though, is that usually the gene you want to add has no or negative survivability effects on the bacteria. Hence, the need to include an antibiotic resistance in the plasmid. However, not all of your plasmids will contain the gene you wanted, since adding a gene into a plasmid is not exact. You could separate this out by running an electrophoresis gel with the plasmid and picking out one such that it has a copy of your gene in it. What is usually done instead, is to have the plasmid designed such that inserting a gene will disrupt a gene with noticeable effects, such as producing color given certain metabolites. Then the colonies with this gene active don't have your gene of interest but do have the plasmid (since they survived the antibiotic).

[CharonY]

After amplification and proper induction in gene expression, the protein product could be harvested. If it is not the protein wanted, simple amplification is enough for harvesting of the desired gene or gene fragment.

 

There is some weird use of amplification here. Once inserted the plasmid may or may not be replicated in a given cell. If the gene product is not the desired one, generally a different strategy (e.g. different vector) has to be employed. Amplification generally for PCR reactions of particular DNA sequences which does not make sense in this context. Again, you amplify the gene you want to clone, put it into the vector, then into the cell. There is no need for further amplification unless you want to verify the gene inside a given cell.

 

However, there are instances when the sequence in the vector would be incorporated into the bacterial host genome, which I can't see to cause a trouble in the amplification process, but this makes the bacterial composition differential. This makes me to have a concern in under what circumstance would a plasmid be integrated into host genome with a comparatively high probability.

 

Generally integration occurs via homologous recombination. If there are sequence similarities between vector and chromosome (regardless of whether it was in the cloned fragment or the somewhere else on the vector) it can integrate as a whole into it. Depending on whether a second event occurs the whole plasmid or parts of it can be integrated. Additionally the plasmid can be lost again with, or without the exchange of the homologous region. The rate of the event depends on the similarity and the length of the respective sequence.

 

Besides, one of the method in selecting those bacterial having absorbed the cloning vector is by alpha-complementation of an enzyme (adding a second part of an enzyme with a polyclonal site to an antibioticase-containing vector), which, though, is a rather old method. So there should be some more recent and more efficient method doing the same job. I wonder what would they be. I am actually considering ligating the desired gene fragment with an antiobioticase gene, forming an operon with single promotor. In a medium with suitable transcription factors and antibiotic, theoretically only those which have absorbed the desired gene should survive. But I am not sure of the actual feasibility of this method.

There are several counter-selection methods out there. It works by disrupting a given gene due to the successful integration of a fragment into the MCS. In other words, the loss of the function of the counter selective gene, you can determine which of those which have been selected for the presence of the plasmid also have an insert. It does, of course not tell you whether it is the correct fragment so that you would have to make a another analysis (usually analytical PCR , sometimes with digestion or Southern).

Fusing the fragment in question with another functional gene is more cumbersome and then you have the problem that you still have to verify if your fusion-construct is correct. This kind of defies the convenience that you get from the standard cloning vectors. Counter selection can be based on the classic blue-white selection or what many have, based on suicide genes. That is, genes without a disruption of the gene (i.e. with an insertion of a desired fragment) will die under selective conditions. So by including the selective properties of the vector only those cells survive that a) have the vector (e.g. selected by AB resistance) as well as a disruption in the suicide gene by a cloned fragment.

[dttom]

Thanks for the details explanation from Mr Skeptic and CharonY, I got the idea now.