Jul
24

Post #6: Bacteria, Transform!

Hi everyone! In my last post, I mentioned I would begin working with GFP-170 and GFP-250, well-characterized aggresome markers, to study the protein aggregation of my mutant receptor tc-TRa1. However, before I could begin those experiments, I had to create expression plasmids for tc-Tra1 that are not hybridized to GFP. After all, if both my receptor and the aggresome markers are GFP-tagged, then they will be indistinguishable under the microscope; they will both glow green! To solve this problem, I prepared mCherry tagged tc-TRa1 receptors.  Unlike GFP, which appears green under fluorescence, mCherry gives off a vibrant red color. This way I will be able to distinguish the aggresome marker from the receptor, and observe if there is any co-localization.

The process of preparing plasmids is one of my favorite parts of the research process! Though some might deem it tedious and time-consuming, I enjoy it because it is very systematic and I get to do a lot of hands-on work at the bench.

 

Figure 1. Hands-on work at the bench!

Figure 1. Hands-on work at the bench!

The very first step of DNA preparation is transforming E. coli bacteria. By mixing small quantities of our plasmid of interest with a small bacterial culture, and subjecting the bacteria to rapid temperature changes, we make the E. coli cells absorb the DNA we want to prepare. We then grow the culture over a period of 48 hours, allowing the bacteria to reproduce and produce large quantities of our DNA. During this time, we also introduce an antibiotic to the growth media. Our plasmid contains a resistance gene against this antibiotic, so only bacteria that are expressing our plasmid will be able to survive! Finally, we lyse the bacteria and use a specialized kit that allows us to isolate our plasmid from the rest of the bacterial waste.

We now have large quantities of mCherry tc-TRa1 to carry out several new experiments!

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