Molecular biology update
I’ve been busy in lab all summer doing a lot of cloning/mutation experiments, and just wanted to post a quick update on what I’ve been up to.
Cloning, Cloning, and more cloning
Two-electrode voltage clamp electrophysiology (TEVC) can be a powerful tool, but in my studies, a huge weakness has been receptor expression. Simply put: no receptors, no current.
The central dogma (IMO) of two electrode voltage clamp electrophysiology. In my case, the problem was no protein => no currents, but just because you have no currents =/=> no protein; verify with another method like a western blot (particularly: check for surface expression with biotinylation experiments!)
An RNA gel used to analyze the quality of RNA. Distinct, clean bands indicate high-quality RNA, streaks suggest RNA degradation. Note that the ladder is hard to see (lane 1 and 14). We have a hard-time preventing the RNA ladder from getting degraded. Usually the 3,000 BP band appears, which lets us approximate RNA sizes pretty well.
We get receptors (protein) to express by injecting RNA that codes for the into the frog eggs, so quality is important. Furthermore, RNA is sensitive to RNAses; enzymes that destroy RNA. They’re found everywhere in the environment and pinpointing the source of contamination in a lab can be a grueling task. I’m meticulous when it comes to contamination, and I’ve been confident that I don’t introduce RNAses or contaminate samples, but for some reason, one of our glutamate receptor expression system would fail 70% of the time. The RNA would look high quality but fail to produce any results. After various cloning side-projects, I started looking more into the glutamate receptor DNA, and found that, unlike our P2X4 receptor system, the glutamate receptors didn’t have a PolyA tail at the end of the gene.
PolyA tail present in our P2X4 receptor genes, but not in any of our glutamate receptor genes.
A PolyA tail is a repetitive series of Adenosine sequences that protect RNA from degradation. Therefore, I decided to make a new vector that would protect all genes and promote protein synthesis (translation). If you recall, a vector is a circular piece of DNA that commonly helps bacteria become resistant to antibiotics. We exploit this by putting genes into the vector, so that as the bacteria replicate, they make copies of the vector, and therefore, our gene. What I did was add small pieces of a frog gene to the begining (5’) and end (3’) of our receptor genes. The frog gene pieces themselves are untranslated, meaning they don’t become proteins, they just promote protein synthesis. I also added a PolyA tail after the end of the frog gene, to protect the RNA once its made.
A plasmid map for one of the vectors I designed. Image generated using Geneious version 10.2, created by Biomatters.
This took a while because I didn’t have any of the frog gene pieces, or the polyA. To assemble these pieces, I designed a 4-piece slic experiment: vector + receptor + frog gene end + polyA tail. The vector was cloned using PCR, where I added the beginning (5’) of the frog gene. SLIC is powerful and easy to design on paper, but when you start to combine multiple pieces, things get exponentially more difficult. One big challenge was assembling things of different sizes: the vector and receptor were several thousand base pairs long, but the end (3’) of the frog gene and the PolyA tail were only 100 BP long. That means that they were too big to add during a PCR experiment. On the other hand, compared to the receptor and vector, they would be tricky to assemble in the right ratio (usually 2.5-5X the vector).
One of the first PCRs I ran when I was optimizing the cloning conditions. PCR was performed at different Tm and DNA concentrations to minimize off-target products.
In the end, I got a vector that had the genes I was interested, but things were getting so complicated and time consuming, that I ordered another pre-made vector from addgene, just in case I couldn’t get things to work in time. I didn’t go straight to addgene because, on paper, cloning it myself would have been faster. I technically only had to wait on the PolyA and 3’ frog gene to be synthesized, whereas ordering from add gene would take a week for the bacteria to arrive, then I’d have to grow, sequence, and then clone my receptors into their vector (using SLIC). Me attending a conference for a week and a holiday also delayed things further. Plus, you can’t get sequencing results during the weekend, so that time is basically lost.
I started to get a little impatient and found a short-cut to getting the vector: clone a polyA tail on to the end of the receptor using PCR, but don’t worry about putting it into a vector or bacteria, just use the PCR product for RNA synthesis. In theory that should work, but this strategy also required optimizing, not only to get the PCR to work, but because circular DNA messes up RNA synthesis. My suggestions
1) Use linearized DNA
2) Use the minimal amount of DNA possible (50-500pg) for PCR, since linearization isn’t 100% efficient
3) Use 1-2ug of PCR product for your RNA synthesis
DNA gel of PCR products (3uL of a 50 uL reaction) using Platinum Superfi. Concentrations are in picograms.
I use ThermoFischer’s Platinum SuperFi 2X green master mix for everything cloning related in lab (15 second/kb extension time!!) and easily get >1.5ug per reaction using 50 pg of linearized DNA. If you do the math, a 25uL miniprep of a plasmid yielding 200ng/uL plasmid could technically last you several years, if you linearize a few micrograms, then dilute that for PCR (1 microgram = 1000 nanograms = 1,000,000 picograms, or 2000 PCRs where you use 500pg per reaction)
In any case, the vector I ordered is amazing: you can use it in both frog eggs and in mammalian cells. A downside of my original plasmids and the plasmid I designed was that doing studies in one system and then moving to another system would be difficult and require more cloning if the vectors weren’t compatible/ideal. For more information, read the original paper here.
A ridiculous amount of effort on my part (70+ hours a week all summer) but with the help of my undergraduate and high school students, we were able to solve the problems that came up in these experiments while still moving the project forward. With these new tools, we’re going to accelerate progress and take everything to the next level.
Here are some good resources on SLIC if you’re interested:
In my next post, I’ll describe some of the new equipment I’m introducing/building in lab.
**Spoiler**: It’s rasberry pi related!
