A Blast From The Past
Annie c -
Hi everyone! Welcome (or welcome back) to week 6 of my blog!
As spring break wrapped up, things started to get busy in the lab again!
Regarding my mentor’s project, we started a new phase of testing this week! Previously, we had been testing the DNA inserts on the same backbone (remember all of the blog posts about plate reader tests?). This week, we began testing the inserts on separate but identical backbones. By doing this, we’ll be able to compare any variation in the expression levels and patterns in each environment. We started by digesting the inserts and the backbone, which is essentially where we cut at specific restriction sites to isolate each component. Then, we ligated the inserts and backbones together, plated the plasmids, and let them grow into colonies overnight. The next day, we performed colony PCR, a method to verify that the plasmid size was correct. After that, we ran a gel electrophoresis experiment to visually confirm the insert sizes.
I also had quite a bit of downtime to work on my project this week, which was mainly review!
I started by refreshing myself on the central dogma, where DNA gets transcribed into mRNA and then gets translated into amino acids since my project focuses on manipulating these processes to activate or repress certain genes. For the mathematical modeling component, I reviewed differential equations (a concept I haven’t touched since my AP Calculus days)—specifically ordinary differential equations since those are commonly used to model gene expression. So far, I’ve learned two different types of equations: the basic ODE (ordinary differential equation) used to model gene expression, and the Hill function, which is used to model the activation and repression of genes.
Another interesting term that I’ve stumbled upon is the steady state, which is exactly what it sounds like: the state of the circuit when no change is occurring. To find the concentration of proteins and mRNA at the steady state, all I have to do is set the ODE equal to zero! This can be useful in my modeling since I can use it as a baseline for comparison when I modify different parts of the circuit.
My main struggle right now is finding the different production and degradation rates for mRNA and protein to use for my equations. However, I plan on reading more literature to (hopefully) find their half-lives, which could potentially be used to determine the degradation and production rates.
Thanks for reading and see you next week!
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