Cultures, colonies, proteins and ducks

Never, I repeat, NEVER go on a night out when you know that you are working on E. coli cultures the next day! The smell is terrible!

But never mind the smell, I got colonies!! That means I must have done something right! Or terribly wrong… But I prefer to believe the former! Hopefully, these colonies¹ are bacteria expressing the plasmids² I want them to express, either that or they are bacteria which have somehow gained themselves Kanamycin and Ampicillin³ resistance and have re-ligated⁴ without taking up my plasmid... However, I am optimistic that my experiment has worked!

Anyway, so these next two paragraphs are for science geeks only, explaining, in more scientific terms, what it is that I’m actually doing and how I’ve done it. If you're not a science geek and you actually have a life, please feel free to skip the next two paragraphs!

KIBRA is an important protein in a number of different pathways, including in the kidney and the brain (KI-dney-BRA-in). As well as being linked to memory, KIBRA is associated with the Hippo pathway⁵ and with the migration of podocytes⁶ in the kidney. It is seen to interact with a number of different components from various signalling pathways. In the case of memory, KIBRA reacts with proteins and lipids from other signalling proteins. One way in which they do this is via a C2 domain, the section of the protein that I am working on. Previous studies have shown that the KIBRA C2 domain is calcium-dependent, but a naturally-occurring mutant has recently been discovered in the human KIBRA gene, where two amino acids⁷ in the C2 domain have been substituted (SNPs⁸). My job is to discover whether this C2 variant is still calcium-dependent, as well as comparing the structures of both the normal wild type⁹ and the mutant C2 domains using NMR¹⁰ and X-ray crystallography¹¹. This will help us to understand the calcium affinity¹² of KIBRA and whether it aids the function of the protein. If you want to find out any more about this then I recommend this review article: KIBRA: A new gateway to learning and memory? and for those who are really interested, check out this review: Membrane binding and subcellular targeting of C2 domains .

I’ll bring you up to speed on what I have done so far. Firstly, I amplified two different lengths of DNA coding for C2 variants using PCR and picked the shorter length as that was the only one that gave a positive result on the gel. Then I used restriction digests, ligated the DNA inserts with four different plasmid vectors with complementary sticky ends and then transformed the plasmids into dh5α cells (E. coli). Afterwards, I plated them up onto the relevant antibiotic-selection plates and left them to incubate overnight, which brings me to the colonies I mentioned earlier! Big, fat, juicy colonies! I almost danced around the lab whilst setting up for the next experiment. Who knew that bacteria could make someone so happy?! To make sure that the colonies were all positive for the plasmid inserts, I used PCR of the transformed DNA. As you will see below, I got positive results for all of the colonies I tested! All that was left was to get the transformed DNA into expression vectors.

So what all the science gobbledegook above is trying to tell you is that I am working on part of a protein called KIBRA to determine its structure and function, which will help us to understand the role KIBRA plays in memory. This is quite a slow process as I essentially have to make my own KIBRA proteins to experiment with. At the moment, I have almost reached the stage of protein expression¹², meaning that I have nearly made my very own proteins! This is probably the most difficult part of the process so far because bacteria can be temperamental and can decide not to give you any purified protein (basically, it is difficult to get stuff you can use).

All of these incubation periods allow for a bit of free time, when you can often find me sitting with the ducks by the lake or on my laptop playing Tetris... I mean doing extra research! Oh, and updating my blog of course! Therefore, I should be able to update you on how my protein expression is coming along very soon. But first, I have a science conference to go to!

Science Glossary

1. Cluster of identical/cloned cells on a plate made to promote the growth of the bacteria you wish to grow
2. A section of DNA (in this case circular) used to transport foreign DNA (our DNA inserts) into the chromosomal DNA of a bacterial cell.
3. Types of antibiotic. We use plasmids coding for antibiotic resistance to later help us to select the bacteria which have taken up the plasmids.
4. The joining of two DNA strands, sticking them together.
5. Involved in the control of organ size, growth and apoptosis (death).
6. Cells in the Bowman’s capsule in the kidneys.
7. Simple organic compounds, the 20 building blocks of protein.
8. Single-nucleotide polymorphism = a mutation where a single nucleotide within the genome varies between members of a species.
9. The naturally occurring protein.
10. Nuclear magnetic resonance (the same technique used in MRI scans).
11. A method to discover the arrangement of atoms in a crystal using X-rays.
12. The stages after DNA has been translated into polypeptide chains, which are then folded into proteins