ligation Archives - Revolution Bioengineering

Tag Archives: ligation

04 Jun

Getting Started

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Look at those beautiful little tubes of DNA sitting on ice – we’ve gotten our regulatory approval, we have our genetic components, the enzymes to put them together and the cells they’ll go into. We’re all set for some molecular biology!

Here’s a quick run-down of our work today:
A month ago, we synthesized 1000 basepair pieces of DNA out of our list of color-changing flower components. On their own, these strings of A,T,C,and G won’t do anything – we need to arrange them in the proper context.

First, the DNA fragments need to be inserted into a plasmid. This is a circular piece of DNA that, when inserted in a bacteria, will be replicated and maintained with very little error. The plasmid Nikolai is building will contain our synthesized DNA, the replication instructions, and a way to identify bacteria that have taken up the plasmid. To insert the synthesized DNA in the plasmid, we add the two types of DNA to a tube with a buffer that contains ions and molecules needed to carry out the reaction. Then we add some glue – an enzyme, ‘ligase,’ which connects the ends of the DNA together.

Dr. Braun at the bench setting up a DNA ligation

Dr. Braun at the bench setting up a DNA ligation

How does the ligase know whether it’s connecting the plasmid to the synthesized DNA or to itself? It doesn’t! So we need to develop ways to tell whether or not our synthesized DNA is in the right spot. The pJET kit we’re using has a neat way to do this. The plasmid is already cut right in the middle of a gene that generates a toxic product. If you don’t insert a piece of DNA, the ligase will re-connect the two ends of the plasmid, completing the toxic gene sequence. The toxic gene will be expressed, and those bacteria will die. However, if the synthesized DNA was inserted into the plasmid, it will interrupt the toxic gene, preventing it from killing the bacterial cell. The cell will live, replicate the DNA, and now you have a self-maintaining stock of your DNA part.

This is only the first step. Once this process is complete we’ll have to ‘read’ the plasmids to make sure that the DNA we have is what we think it is. Even if they’re correct, these plasmids don’t have all the necessary information yet so we’ll have to do some additional steps to add the rest of the information. There’s a lot of science to share – ask questions in the comments below!