Today I’m writing a little shorter post on genetic manipulation, which somewhat falls in line with the previous antibiotics post, where I describe the use of antibiotic resistance markers for selection of genetically manipulated cells.
Last week I had a conversation with a housemate of mine (who doesn’t work in the science research field) and tried to explain to her what we actually do in the lab as part of ‘research’. And while I don’t think there is one, simple answer to that, as it really depends on the nature of the research area and subject (i.e. protein, RNA, DNA etc.), the thought flow and process usually goes like this:
Depending on what field of research one works in, the nature of experiments differs. Microbiology, for example, tends to look at the organism e.g. fungi, bacteria, algae and usually investigates their growth and characteristics relative to the organism as a whole and their environment. While a molecular biologist on the other hand usually looks at processes that occur inside the cell or intracellularly, such as DNA, RNA and protein expression, regulation and synthesis.
In molecular biology, a lot of times we want to answer what a gene does or how it is regulated and how this affects downstream pathways. For this we frequently “knock out” genes, meaning we remove or alter them, so they don’t carry out their natural function any longer. Subsequently we look at the phenotype or effects it has on downstream molecules to investigate whether the absence of this “knocked out” gene alters a) any downstream pathways/molecules, and if yes, b) how.
Currently, the “state of the art” tool for knock-outs and knock-ins is CRISPPR Cas9 – an endonuclease enzyme (an enzyme that catalyzes the ‘cutting’ of DNA) first discovered in bacteria that has a programmable guide RNA sequence (of choice), which can be used to alter almost any gene! This is done by designing the gRNA complementary to the sequence of the gene one intends to knock-out.
But how do we manage to get this cas9 and gRNA into our cells in first place? Short answer: a plasmid, which is a circular DNA construct. Long Answer: Let’s take E.Coli as our model organism for simplicity purposes, (widely used and easy to manipulate). As bacteria are prokaryotes, they do not have a nucleus, meaning the DNA floats around freely and the plasmid we want to integrate, only has to get through the outer cell membrane. This can be done in different ways but is commonly achieved by chemical treatment and abrupt temperature changes that make temporary holes in the cell membrane allowing us to introduce our plasmid of choice.
Source: Wikipedia (https://en.wikipedia.org/wiki/Plasmid)
The bacteria which integrated the plasmid(s) successfully, will then express the genes encoded on the plasmid such as proteins, like in this case our Cas9 (which in an enzyme -> type of protein). The rest, the bacteria basically does for us (the knock out), at the end we usually sequence the bacterial DNA to verify that we successfully knocked the target gene out.
Next week I’m going to do a ‘day in the life’ of a regular lab day of mine, because we have actually moved to a new lab space here at Yale. Since we’re all pretty excited about that I thought this would be a nice thing to do and introduce my second home :D. Otherwise, I’m slowly starting to really settle in, here in New haven, while the lab work is pretty challenging, yet very fun. I’m also going to try to post more often from now on!😊