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Spidermonkey's Sporadic Lab Stuff

by Spidermonkey

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Hello World from the lab!

Part spider - part lab monkey, will do science for cash, strawberries, or for a place to be that's out of the rain (but preferably for cash).

The rest of what I get up to goes into the Tunnel of Goats :)

Urgent Science Stuff:

Please help reform English libel law
Current UK libel laws are very bad for science and free speech.

Cool science stuff:

RCSB Protein data bank: Molecule of the Month in alphabetical order

The PCR Song It's the little things that keep you going...

NCBI's Entrez Gene My favourite starting point for finding out what is known about any gene of interest.

NCBI's PubMed Where to go to find pretty much all research published in the last 30 years or so (may go back a lot further now). All newly published research is quickly added to the site.

The Genetic Code - table of the DNA/RNA triplet codes for amino acids. This is how DNA codes for protein.

Another PCR song Disco frenzy :)

Recent visitors

Back to the drawing board

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What I really need is for the plate on the left to look like the plate on the right (i.e. have bacterial colonies carrying the plasmid I am so lovingly trying to make that will make my favourite new protein and give them enough antibiotic resistance to grow on these agar plates).
Something is not happy. Probably the ligation step... Tomorrow will bring more troubleshooting.
15th Nov 2009, 20:30  


Caine says:

Well, damn. Good luck tomorrow!

15th Nov 2009, 22:13

Factotum says:

What's involved in the ligation step?

16th Nov 2009, 00:13

Spidermonkey says:

Thanks Caine.

Facto - here follows a brief summary of ligation (with a song!) which I hope helps explain.

Ligation reactions join together pieces of DNA using the ligase enzyme from T4 bacteriophage (we buy it from Invitrogen and it is one of the standard molecular biology tool kit components). I want to join together 2 pieces of DNA to make a circular plasmid I can transfect into human liver cancer cells I have going in culture. Plasmids are small circles of DNA that naturally occur in bacteria, extra to their main chromosome, and contain genes for things like antibiotic resistance. Unlike their main chromosome, plasmids can be exchanged between different species and strains of bacteria which is why the spread of antibiotic resistance occurs so easily.

The larger piece of DNA (the vector) is the basic plasmid that contains the gene sequence to make green fluorescent protein, + useful bits of sequence that give resistance to an antibiotic and enable the genes to be functional in bacterial and mammalian cells.

The vector has been cut open (linearized) using restriction enzymes (more from the tool kit) which cut DNA at specific sites.

The smaller piece of DNA is my gene of interest that was copied out of another plasmid using PCR and mutated slightly by adding an extra nucleotide at a specific point.

Combining the 2 pieces of DNA in a single tube, along with pH buffers and ligase enzyme and incubating over night at 16C should have joined them together into 1 circle again. The whole ligation mix is then added to a tube of specially treated E. coli cells, which should take up the plasmid DNA. The cells are then spread out onto the agar plates and incubated overnight. If it has worked I should see bacterial colonies in the morning (only bacteria that have taken up the plasmid can survive on the antibiotic-laced agar plate).

As you can see above, something has gone wrong :(

Here's one more PCR song
It's the little things that keep you going :)

16th Nov 2009, 03:19

Caine says:

How are you hoping these will work on the liver cancer cells?

16th Nov 2009, 03:47

Spidermonkey says:

The liver cells are just a tool at this point, a mammalian cell with all the cellular machinery (e.g. ribosomes) necessary for transcribing the DNA on my plasmids into messenger RNA and then translating that into protein. The protein will be Constitutive Androstane Receptor (CAR) joined on to green flourescent protein (GFP). This protein is a receptor for all sorts of things from hormones to products of metabolism to environmental toxicants and pharmaceutical drugs (it is the reason why too much acetaminophen/paracetamol is so very toxic, for instance). When it encounters a molecule it can bind (e.g. hormone or toxicant) in the cytoplasm of the cell it then travels into the nucleus where it recruits various partner proteins (these vary depending on what is bound). This protein complex then binds to the DNA upstream of genes that deal with the metabolism of whatever molecule has been encountered. Depending on which partner proteins are recruited, target genes are activated or repressed (switched on or off) with consequences for the metabolism/detoxification/activation of the compound. Tagging this protein with GFP should enable me to see where the protein is in the cells. I can then use the cells to screen for compounds that bind to CAR. This is important for screening compounds for toxic downstream effects, be they drugs or environmental pollutants. There is also emerging strong data that CAR is involved in energy metabolism, diabetes and obesity so tools to help understand it's function will be useful there also.

I could use any immortalized cell line, it's just I happen to have some of these growing nicely at the moment and they take up plasmids pretty well. Most cell lines commonly used in labs are some form of cancer as cancers have undergone the necessary changes to grow and divide indefinitely, whereas normal cells have a finite number of cell divisions (this is more RAB and Tilia americana's area - telomere biology).

16th Nov 2009, 04:17

Caine says:

Ah. After reading that twice, I think I have it!

16th Nov 2009, 04:23

Spidermonkey says:

I feel I have only a tenuous grasp on this at the best of times but I'm getting better (I hope)!
Thanks very much for your patience, it's hard to put into words without going off on tangents about every sentence. I will have to draw some diagrams sometime. It's much easier with diagrams :)

16th Nov 2009, 04:31

Caine says:

:) I'm good with diagrams, that's language that's more intuitive to me.

16th Nov 2009, 04:42

Spidermonkey says:

Words and pictures. I need them both to make a good diagram. Have to work out the words to know what to put in the diagram and need the diagram to visualise what I need words for. Then take the words away, like pulling out a table cloth from under a vase of flowers, and hope the diagram speaks for itself. :)

16th Nov 2009, 05:03

Spidermonkey says:

Time for bed. Goodnight!

16th Nov 2009, 05:04

Factotum says:

Thanks, Spidermonkey. Fascinating process!

16th Nov 2009, 12:16

crickson says:

Are you ready to dump the ligase yet and recombine your way to success?

17th Nov 2009, 00:51

Spidermonkey says:

I couldn't possibly comment. We have a metric tonne of ligase in the freezer and all the little restriction enzymes... I know, this is the old-fashioned way. Hardly anyone clones like this any more, but if it works I'll be happy.

17th Nov 2009, 22:59