Recombinant DNA technology ( Alpha complementation and Blue white screening)

Recombinant DNA Technology including Blue white screening and Alpha complementation.

What is gene ?

It is segment of DNA which express is specific type of protein.

What is cloning?

The exact genetic copies

To produce multiple copies of gene we use gene cloning technology or techniques.

There are some methods of gene cloning -- method for molecular cloning using polymerase chain reaction and cell free gene cloning method

If we use any host cell to make multiple copies of our gene of interest then this type of methods are called cell based gene cloning method.

Now I show you some images .

In my  next article I will discuss more about the gene cloning. 

##############################

Now I will tell you some techniques which is needed for identification of recombinant DNA in host cell .

## Blue white screening 

In recombinant DNA technology there are some selection marker are found ---

1. Lac z enzyme based  selection marker are produced by beta galactosidase . Help in blue white screening .

2. Tet^r antibiotic based selection marker --- for Replica plating method. 

Assume there is a plasmid which has a lac z site . When the gene of interest and plasmid is mixed in a test tube then some of them are Self ligated or recirculated , some are recombinant and some are non transferable . 

This is the simple method by which we can easily identify which are recombinant and which are not. 

We take the mixed solution agar plate with x gal . 

Beta galactosidase in the presence of x gal produce blue coloured product.

Beta galactosidase enzyme can metabolize x gal in culture medium .

In which cells ,the gene of interest is inserted ( already has broken lac z part and entered gene of interest). This will produce natural white colour . Now these are identified as recombinant . But if doesn't inserted then it will produce blue colour in x gal media.

 

##Alpha complementation test 

In bacterial cell there is a bacterial chromosome which has lac operon which responsible for producing beta galactose by lac z gene . 

So  the gene of interest are the recombinant genome is present there but in agar plate it gives blue colour instead of white colour because the presence of bacterial chromosome so in this case we use this technique.

Now Prince please assume 2 types of vector 

1 . Self ligated 

2 . Recombinant 

Lac z gene has 4 regions two Alpha and two omega regions .

But as we use the strain of bacteria only responsible for producing two omega subunits only .

In this case of recombinant vector the inserted vector lac z only can produce two Alpha subunits . Then it will activate Beta gal.. by attachment with omega subunits of bacterial genome and then produce blue coloured product . Now it is identified as recombinant .

But if self ligated host cell , they can produce only Omega subunits so they will produce white colonies in culture medium .

...................................................................

"Anyone who has never made a mistake has never tried anything new."

                 ..... Einstein

...................................................................

If you like my article please comment in the comment box and please share and subscribe my blog ❤️