Sequencing and troubleshooting

Our goal this summer was to create fusion proteins of thyroid hormone receptor alpha with GST and His tags, which I described more in depth in my last entry. In order to do this, we had to insert the gene encoding TRα or its domains into an entry vector (plasmid), before moving on to later steps in the project.

It is important that our inserts are in the correct orientation within our plasmid.  It is best to check if the insert is in the right orientation at this point in the project before proceeding; future steps are useless if the insert is in the wrong orientation, and checking here will save time and money. We checked the orientation of our insert by sequencing our entry clone.

As I mentioned in my last entry, some inserts had already been verified by the time I joined the project, but other inserts had proven difficult to sequence or had been confirmed to be in the incorrect orientation. Sequencing has been difficult and has rarely yielded usable results; a major problem we have been facing is troubleshooting our sequencing reaction.

A typical sequencing reaction includes both the DNA you want to sequence and sequencing primers, short sequences of RNA that will allow the sequencing reaction to continue. The primers must be complementary to DNA upstream of the region you wish to sequence. At first, we thought that the primers we were using, which were recommended by the manufacturer of the system we used, may not be optimal for the region we wanted to sequence, so we designed our own. Unfortunately, this did not improve our results; clearly the primers were not the issue.

After contacting the manufacturer, however, we learned that issues with sequencing were common, and our issues may have been due to secondary structure forming near the sites where our insert was located; at these recombination sites, the DNA folded in such a way as to interfere with the sequencing reaction. While they recommended some solutions, we are still having some issues with sequencing.  After a lot of trial and error, we are looking to other ways of confirming the orientation of the insert.

Despite problems with sequencing, we did manage to confirm the orientation of some of our inserts. The full TRα insert, the DNA binding domain insert, and the Hinge insert were confirmed to be in the correct orientation. This allowed us to move forward toward the second “phase” of the project.

Our eventual goal was to express our protein constructs with our peptide tags attached. In order to do this, we first had to perform a recombination reaction to get our insert into our destination vector. The destination vector is a plasmid, similar to the entry clone; however, the destination vector also encodes for the GST or His tags. The insert is cloned into the destination vector so that when the gene is expressed, it yields a fusion protein of GST and our insert.

The recombination reaction “moved” our insert out of our entry clone and into our destination vectors, in the same orientation. Then the destination vector was transformed into bacteria, which were grown overnight. As the bacteria grew and multiplied, so did our plasmid, yielding very high amounts of DNA. We could then extract that plasmid DNA for future use.

This high amount of plasmid DNA could then be transformed into a different strain of bacteria, BL21-A1 cells, which were more ideal for protein expression, which I’ll talk more about in my next entry.