Mammals vs Insects

The reason I have chosen to focus on the elimination of the plague in prairie dogs rather than in fleas is partially because of practicality and partially because of curiosity. After my project proposal I read a question from one of my class mates asking me why I chose not to focus on the initial host organism. Ideally you would think its best to eliminate an illness at its source because then it has no chance to spread. To be honest I had not even considered using CRISPR Cas9 on fleas instead of prairie dogs, mostly because of some tunnel vision. However, after thinking about it, I decided that it would be more beneficial to stay focused on my initial subject rather than altering my project.

This decision came down to a couple of factors. The main one being the lack of gene drive research on mammals. For the most part gene drive success has come through research on mosquitos and other insects, but not with mammals. It could go either way, but I felt that any research would benefit me and the group more if it was diverse and original. Any population modeling would be based on a slower gestation period and a different litter size. So, while slightly more complex, I thought that the best way to challenge myself and stay interested would be to tackle the prairie dog challenge.

Of course, my initial project and driving question had flaws, so through the development of my project I grew even more grateful that I didn’t alter the subject of my project. It turned out that dCas9 works great with mammals. There are a wide variety of activators and application examples in mammals. I would have focused on fertility reduction in flea populations, but instead I stuck with antibody production in prairie dogs which led me to dCas9. Now I feel like I have valuable new information to present to my classmates.

As I initially predicted, my decision to push through was actually very beneficial. While it challenged me at first, it also opened new doors to the world of gene regulation. I now know about many different tools to accomplish niche tasks. I think its very important to keep pushing the boundaries of CRISPR Cas9 technology because the components of the system each play different roles and have so much potential.

The Evolution of my Final

I began my final with the idea that CRISPR Cas9 gene drives could be the solution to the problem prairie dog species are having with the plague. As a widely exterminated and hated species, prairie dog populations have diminished by a large amount in recent years; because of that, they have little room to fight the sylvatic plague. The bacterium yersinia pestis is spread through fleas to prairie dog colonies and subsequently creates die offs of 70-90%. Currently vaccines given through bait are working, but not efficiently enough. The vaccine stimulates antibody production by introducing anti-V and anti-F1 antigens in small doses. However, the resistance can wear off within the year, and many prairie dogs aren’t even eating the bait.

So, my initial thought seemed fantastic, why make an inefficient vaccine when you can force genetic resistance through designed gene drives. However, there are big flaws within that train of thought. For example, the reason the Cas9 enzyme is useful is because of its ability to cut a specific gene of interest. The healthy gene will then fill the gap through HDR. The tool is useful when a gene is not performing the correct task, but in this case, our antibody producing gene is working just fine, however it is not producing enough antibodies. Because of that I decided to look into any way Cas9 could increase antibody production. With no luck I turned to the idea that another gene could be inserted to increase production. Once again, no luck. It took almost a whole week for me to give up on my trivial pursuit, but at that point I found out about dCas9. The reason it was not associated with my Cas9 searches is because its function is entirely different. I explain it in my previous blog post, but it works as a binding protein rather than molecular scissors, and can attach to promoters to increase gene expression. Problem number one solved.

I was still stuck on the idea that gene drives would be the best way to create population wide resistance. Gene drives have already shown their ability to alter populations by causing close to 100% inheritance rates; so why wouldn’t it be a good idea to use gene drive technology in combination with dCas9. Mainly because an engineered gene drive requires you to cut a gene in order to replicate the gene of interest. This system inherently uses the Cas9 enzymes main function to create preferable inheritance. It would require at least one extra step to attach the dCas9 to the gene drive and make it work in tandem with a normal Cas9 enzyme. This technique has also never been tested so it may be impossible in the first place. The solution I found was actually very quick and simple.

That solution being epigenetic editing. Epigenetic changes made with dCsas9 are heritable, and therefore there is no need to use a gene drive. Theoretically the over expression of immune response genes would automatically be passed down to prairie dog pups: increasing plague resistance. Of course the field of epigenetic editing is still rather obscure so inheritance patterns are not as predictable. In any case, I believe that through my research I have found the best way to  stimulate resistance to the sylvatic plague on a large scale.

The Use of dCas9 to Regulate Gene Expression

The goal of my final project is to identify a population wide cure for the sylvatic plague in prairie dogs. This solution must be efficient and cost effective, so I felt that CRISPR would be a fantastic tool to use. However, in order to use the tool properly, I need to understand what genes and antibodies fight the plague. After researching the sylvatic plague vaccine (SPV) that is laced in bait, I was able to identify the anti-V antibody that is produced. Since genes produce these antibodies, the most important piece to this project is the regulation of genes: specifically the activation of specific antibody producing genes. The Cas9 endonuclease normally functions as DNA scissors, but I don’t need to cut and/or replace a gene, I need to activate it in order to stimulate the production of anti-V antibodies. That path lead me to dCas9: an altered version of the Cas9 endonuclease that binds but does not cut.

dCas9 is created by mutating Cas9’s two nuclease domains. Each one of these domains cuts a single strand of DNA causing the double stranded break. The purposeful mutations   deactivate those nuclease domains and create a dCas9. The dCas9 is effectively a guided DNA binding protein now, however, it is highly specific and can be turned into an artificial transcription factor. By fusing transcriptional activators or repressors to the dCas9 endonuclease, it is possible to make dCas9 target promoters and increase gene expression. The p65 and VP64 activators have shown they are able to regulate genes when attached to dCas9.

Another use of dCas9 is for epigenetic gene regulation. This form of editing does not change the DNA but is does influence the expression of certain genes. These changes are heritable and therefore can be very useful when looking at population wide change. dCas9, when attached to a histone acetyltransferase, is able to acetylate histones. This opens up specific sequence for expression. The more tightly wound the DNA is around histones, and the distance between histones, can alter gene expression. The negative charges of these added acetyl groups force the histones to loosen up. The acetyltransferase p300 causes acetylation of histone H3 lysine 27 at the target gene.

In terms of my project to eliminate the plague in prairie dogs, epigenetic editing is the logical way to go. The alterations are heritable, so it would be relatively simple to spread resistance throughout colonies. In order to do this I would hypothetically use dCas9 to increase the expression of immune response genes associated with anti-V and anti-F1 antibody production. With more innate resistance, colonies would be much less likely to see large population drops when fighting the sylvatic plague.

Purification of Green Fluorescent Protein (Protocols 4-5)

Protocol 4: Bacterial Lysis

The purpose of protocol 4 was to remove more of the contents of the cell that we do not want. This is done by lysing the bacterial cell walls further and isolating small molecules from larger cell debris. The main tool used was a chromatography column, specifically a hydrophobic interaction chromatography column (HIC). This protocol requires preparation of the HIC column with equilibration buffer to maintain the environment previously supplied by our lysate.

Protocol 5: HIC Chromatography

The purpose of protocol 5 was to separate proteins based upon affinity to hydrophobic beads within the chromatography column. This protocol involved use of multiple buffers to separate our GFP from the rest of the the contents of the supernatant. After closely monitoring the column with UV light for both the wash buffer and the TE buffer, our purified GFP had been completely rinsed out to the last drop. The separation of our protein was therefore successful and we stored the collection tubes containing other concentrations of protein that was removed during HIC Chromatography. This is just one form of chromatography, and it will be a useful tool for other protein purifications within aqueous-based solutions.

 

Purification of Green Fluorescent Protein (Protocol 3)

Protocol 3: Concentration of Bacteria

The purpose of protocol 3 was to lyse bacterial cell walls in order to release soluble proteins from the cytoplasm of our E.coli. We are attempting to rupture the bacteria and release some of the proteins that we do want.

We were successful in this protocol; the microcentrifuge tubes with our more concentrated bacteria still glowed a bright green. By observation (as shown in the pictures) the bacteria seems to glow a brighter green. That may bed to a small lighting change, but because of the proteins we released from the cytoplasm it makes sense.

 

 

Purification of Green Fluorescent Protein (protocol 2)

Protocol 2: Inoculation of Liquid Media with Transformed Cells

The purpose of protocol 2 was to grow transformed cells in a liquid growth media. The liquid growth media allows for a higher yield of the GFP carrying cells that were grown on our LB/Amp/Ara plates in protocol 1.

After incubation of our culture tubes at 32 degrees celsius for 24 hours the transformed cells had clearly grown. Under UV light both tubes glowed a bright green color indicating the presence of GFP. The high concentration of transformed cells is shown in the pictures below. One tube has less liquid; This is most likely due to leakage while shaking the tubes. However, we still succeeded in getting a higher yield which we need for the following protocols.

 

 

 

 

 

 

Bacterial Transformation of E.coli Cells to Express GFP Gene

The purpose of this bacterial transformation lab was to create and subsequently grow colonies of E.coli that express the GFP gene. Through transformation we can introduce foreign DNA into a cell, and have that cell replicate the plasmid DNA that we want. The first step of this transformation was to submerge a single colony of bacteria and a loop full of pGLO plasmid DNA in our transformation solution (CaCl2). The transformation solution, or the cations specifically, create a shield over the negative charges by forming complexes with phosphate groups. This is called the adhesion zone; it allows our plasmid DNA to move into the cell. Heat shock opens the pores in the membrane of the bacteria and pushes our plasmid DNA into the cell. Another sudden decrease in temperature closes the membrane up to make sure the bacteria takes up our plasmid.

Our E.coli did take up the plasmid DNA. Six colonies showed significant growth and glowed. We know this means they have the GFP gene that we need for further protocols. Below are pictures of our results under UV light.

The lab protocol taught me how to effectively transform bacteria using heat shock. I now understand why heat shock is necessary in order to introduce foreign DNA into the bacteria, and how the process of heat shock works on a molecular level. It was good to practice aseptic technique and the overall process of transformation.

Chapter 24: Cells

Chapter 24 has a good mix of scientific history with explanations of the mechanics of cells. The author uses metaphor well in sections about the mechanics of cells. Comparing yeast cells to a Boeing 777 allowed me to visualize just how complex even the simplest cells are, but at the same time he acknowledges that the metaphor is limited. Imagining a human cell as the largest country on the planet is another visual tool that helps the reader to understand the intricacy of cells. He continuously pairs explanations of complex structures with the bigger picture of how each cell functions in the whole organism.

The background information on microscopes and Antoni van Leeuwenhoek feels a bit too long, and for the most part it provides no important information about cells. While the discoveries of protozoa and bacteria were important, the rest of the information -about microscopes and Leeuwenhoek – felt unimportant. The main value of this section is too highlight how relatively recent many biological discoveries are, and the value of advanced technology. It also helps the author connect the big picture to a zoomed in view of the human cell.

I had never really thought about how quickly interactions happen within our cells, and how many mistakes can occur in this process. For example, how every strand of DNA is damaged every 8.4 seconds. This fact highlights the healing power of our bodies and the frantic atmosphere of the human body at the molecular level. I did not know the scale of our electrical signals either, and just how relatively huge they are. However, my favorite new “discovery”, was how sponges make our cellular organization look unrefined. Their ability to recombine is rather remarkable and something to consider moving forward with synthetic biology.