The lab I found to be the most rewarding was our CRISPR/Cas9 lab. The complex is capable of completely disabling or “knocking out” an entire gene. In this specific lab, it was used to disable a gene in E.Coli bacteria. The complex is able to cut DNA when Cas9 binds to guid RNA, which will instruct Cas9 to cut the DNA when it comes across DNA that is complementary to it. In this experiment, Cas9 disabled the lacZ gene in the bacteria. A control group was established by transforming one set of the bacteria with plasmid containing the Cas9 gene and random guide RNA. In this case, the lacZ gene remained functional. Another set of bacteria was transformed with pKO plasmid. This plasmid had the Cas9 gene and RNA complementary to the lacZ gene. In this bacteria, the lacZ gene was successfully disabled. 

Because the lacZ gene codes for a protein which ends up creating blue pigment in the bacteria colonies, it was easy to visually see which bacteria had the gene disabled. My favorite part about this lab was the outside research it lead me to do. While in lab, we learned a lot about how CRISPR/Cas9 functions, which is detailed above. This lead me to wonder about its usage in potenitally editing or disabling genes and DNA linked to the developemnt of congenital heart disease (CHD). 

Research is still being conducted regarding how CRISPR/Cas9 might be used to edit genes and prevent development of CHD. It is my hope that in my lifetime, significant strides will be made in preventing the development of CHD. In the future,  I aim to become a pediatric cardiologist or cardiothoracic surgeon and work with kids who are living with this disease. I would also like to become involved in research surrounding technologies that may be able to help prevent this disease. Luckily enough, it sounds like CRISPR may have some promise. 

During this lab, we were able to continue practicing the use of PCR and Gel Electrophoresis. By amplifying the lacZ gene using PCR, we were able to tell if the lacZ gene was still functional in the bacteria or not. The image above is the result of the PCR completed. Gel Electrophoresis is another method we used to attempt to confirm or deny the functionality of the lacZ gene. Unfortunately, in this particular experiment, the bands smeared and the results were difficult to interpret. That image can be seen to the left. It was anticipated that white colonies would have the same bands as another lacZ control, and the blue colonies would have similar bands to another cas9 control. While the results were not adquate here, this was good continued practice with using Gel Electrophoresis. Finally, this lab provided continued practice of two skills essential to biological research: plating for tests and growth and presice pipetting skills.