Field of Science

Probably more a 'graphic novelette'

I was reading through all the bioephemera archives that I missed through being hideously busy and was motivated to find something pretty and artistic connected with Synthetic biology (if my lovely gene designs weren't pretty enough...) Somewhere at the beginning of our course we were sent a link to a quick comic explaining synthetic biology, which I didn't bother with but which the engineers in our course were incredibly pleased with, and found really useful.

I went back and took a look at it last night. And actually, it's really good:The full story can be found here.

And it's actually in a nature paper! Which means it is most definitely not just a childish 'comic' and deserves to be taken seriously, like any other graphic novel.

The Lab Rat guide to DNA Synthesis

The structure of DNA is a double helix of two sugar-phosphate backbones joined by hydrogen-bonds between nitrogenous bases, as shown below:

Image from Bioinformaticsweb.org


The letters of the DNA code come from the bases; adenine (A), thymine (T), guanine (G) and cytosine (C). They code for amino-acids, which make up proteins, in groups of threes, i.e GCC codes for alanine, GGA codes for glycine etc.

Each base, along with the associated sugar and phosphate, forms its own little subunit. Joining these together in the correct order can code for any protein you want. As a Lab Rat I don't know very much about this process, except that I send the sequence off and get back a little vial full of DNA (or a stab containing the bacteria that have my DNA held on a separate plasmid). So what I'm writing here is just what I've managed to find out about the process - it might not reflect the most up-to-date method used in the top sequencing companies, but it's a plausible way to make DNA.

There are two main types of DNA synthesis. Firstly there's small sequence oligonucleotide (aka small-bit-of-DNA) synthesis, to make primers and things. Secondly there's whole gene synthesis, which deals with larger sections of DNA. As whole gene synthesis mostly involves sticking together little bits of DNA, I'm mostly going to focus on small oligonucleotide synthesis.

The basic process involves sticking the growing DNA strand to a solid support and then just washing the next DNA base through, over and over again. This is pretty much automated nowadays, so you just program a robot to do it. The supports used are mostly either Controlled Pore Glass or macroporous polystyrene (plastic with small holes to select for size, allowing the salts and bases to be washed away before the larger DNA molecule is eluted). Both of them covalently attach to the end of the DNA chain, holding it in place as the nucleotides are washed through.

In their natural state, however, nucleotides are not very reactive, so special modified versions are used. Large bulky groups such as DMT and cyanoethyl are used to block the ends of the bases and the phosphorous linkages, to stop them reacting or participating in reactions.

The first base is then attached to the support and the DMT group (attached to the bottom of the base - the five sided ring) cleaved off with an alkaline wash. The next subunit is then activated before being added to the support. This involves adding tetrazole, which cleaves off the three big rings shown on the left, making the subunit more reactive. The activated subunit is then washed through the column, where it can react with, and bind too, the preceding base.

Once all the bases have been added in the correct order the mixture is purified, to isolate the required sequence. This is done by desalting, usually with chromatography, to produce the final product.

In case anyone was wondering, the robot/machine/computer used for synthesis looks like this:
Image taken from monash university website.

Cell wall under attack - bacterial response to antibiotics

ResearchBlogging.orgI took a quick break away from synthetic biology and DNA synthesis research the other day, to dive back into my happy little world of antibiotic research, in preparation for my new project in October. I'll be working with Streptomyces bacteria again, which after a whole summer of E. coli I'm quite looking forward to. What I'll be doing with them is examining the response of the cell wall to antibiotics.

The bacterial cell wall is made up of glycopeptide molecules (sugars and proteins joined together) and surrounds the whole cell. Without it, bacteria swiftly loose their integrity and salt-balance across the membrane, which is why many antibiotics target the cell wall in order to kill bacteria. Both for antibiotic resistance, and for surviving conditions that could damage the cell wall, bacteria have a system of monitoring the state of the cell membrane and responding quickly to any changes.

The system that was discovered in Streptomyces coelicolor (which I'll be working on) was named the sigE system, and consisted of an operon (string of genes) encoding four genes:

SigE encodes for a sigma-factor, a protein used in bacteria to switch on certain sets of genes. The cseA codes for a cell membrane lipoprotein, possibly used in a sensor system, while cseB and C are a two-component signalling system (very common in bacteria). CseC is a sensor (a histadine protein-kinase sensor for those who are interested) while cseB is the response regulator, acting out a response when it receives a signal from cseC.

And now...the science :)

In order to test that this operon was involved in cell membrane responses to antibiotics the lab carried out a variety of experiments, all producing evidence that lead towards this conclusion. The main experiments were as follows:
  1. Removing the sigE operon and placing it on a separate plasmid, that activated resistance to Kanamycin. The bacteria were then plated on agar containing antibiotics and challenged with a kanamycin disk. Cell wall attacking antibiotics induced kanamycin, whereas antibiotics that attacked (say) the ribosome didn't.
  2. Keeping the sigE in its original chromosomal context, the group then challenged it with different concentrations of vancomycin (an antibiotic which attacks bacterial cell walls). They then measured the level of the sigE operon proteins being produced in the cell. Higher concentrations of vancomycin, lead to more proteins.
  3. Going back to the sigE-kanamycin resistant protein, they tried knocking out the sigE promoter, effectively switching all these genes off. The effect seen previously disappeared.
  4. Leaving the lab, they then did some computational work, scanning the database to see what genes the sigE sigma-factor actually switched on. They found a group of 12 genes, all of which coded for cell-wall synthesis enzymes.
All of this leads up to some pretty conclusive evidence - in case of cell wall damage, the sigE operon is switched on. The interesting thing is, is that this isn't just a response to antibiotics either. It is highly unlikely that the system is able to respond to every different cell-wall destroying antibiotic, instead, the response is triggered by cell-wall intermediates, or degradation products that signal "Help - cell wall is being destroyed!" and switch on the sigE response, which produces proteins to mend it again.

But there are still a lot of unanswered questions. What is the cseC actually sensing? What is the exact purpose of the cseA? Why produce both a sigma-factor and a heafty response pathway? Which intermediates are used for activating? And, most importantly, can we hijack this somehow to kill bacteria?

I can't wait to get to work with it :D

---

Hutchings, M., Hong, H., Leibovitz, E., Sutcliffe, I., & Buttner, M. (2006). The E Cell Envelope Stress Response of Streptomyces coelicolor Is Influenced by a Novel Lipoprotein, CseA Journal of Bacteriology, 188 (20), 7222-7229 DOI: 10.1128/JB.00818-06

Hong, H., Paget, M., & Buttner, M. (2002). A signal transduction system in Streptomyces coelicolor that activates the expression of a putative cell wall glycan operon in response to vancomycin and other cell wall-specific antibiotics Molecular Microbiology, 44 (5), 1199-1211 DOI: 10.1046/j.1365-2958.2002.02960.x

Jacobs C, Frère JM, & Normark S (1997). Cytosolic intermediates for cell wall biosynthesis and degradation control inducible beta-lactam resistance in gram-negative bacteria. Cell, 88 (6), 823-32 PMID: 9118225

Sequencing vs. synthesis

Sequencing = the process of finding out what base-pairs a piece of DNA consists of (AAAGGGAAA etc)

Synthesis = the process of actually making the DNA from separate base pairs.

In my last post I got the two processes chronically mixed up. The links lead to a description of DNA sequencing, whereas the process I describe doing with my DNA is preparing them for DNA synthesis.

I was going to edit the post but I couldn't find any meaningful way to do so, without either deleting the information or deleting the links, both of which contain (hopefully) quite interesting science.

I will write a post about synthesis at some point.

Sequencing

A friend of mine, who actually reads my blog occasionally, was very interested in the idea of DNA sequencing, fascinated by the thought that DNA could just be created in companies and then shipped out when needed. He mentioned I should write a post on DNA sequencing.

I thought about it, and realised, with a daunting sense of dread, that actually I would have to do quite a bit of research before being able to write coherently about DNA sequencing. I know the general idea, but not enough to explain to someone who doesn't already have quite a good idea of whats going on. Luckily there already is a very clear and comprehensive explanation of it over at Genetic Interference:

Part One
Part Two

So I'll just add to the story a little by describing things from the point of view of me...the scientist actually ordering the DNA.

First I need to find out what I want. This requires a literature search. For example, when I started looking for my pigment colours, I went on a quick trawl through PubMed, looking for any genes that had been shown to produce colour in E. coli. The vio gene shown in this post is just one example, at the moment I also have a brown pigment, and (hopefully soon) two genes that make green and red as well.

The next stage is to find the actual DNA sequence. Usually it's in the PubMed paper, or in NCBI - which has a huge database of all proteins that people have registered. If it's very new research, you might have to email or phone the researchers. Once you have the DNA it's a good idea to double-check it as well...compare to homologous proteins or ones with similar domains. I used the MUSCLE comparison tool for this, simply because it's the one I'm most used to.

Once you're certain you have the right sequence for what you want, you contact a DNA synthesis company. Prices vary... as far as I can work out it varies from 20 (if you're REALLY lucky) to 50p per base pair. A smallish gene is usually about 1kilo-base pair(kb) just to give an idea of the scale of things. And the price tends to just up once you get over 1kb as well. The vio gene which we are getting for free is about 6kb long.

Then you send your sequence off to get made! You can have various options for synthesis (as I am just discovering). The codons (AAA, GGG etc) can be optimised for your organism - in the case of more than one codon (the three bases) coding for one amino acid, different organisms will prefer to use different codons. You can get restriction sites removed and added (for cutting and pasting DNA parts), and extra parts added to the gene, such as an area for the beginning of protein coding, or a degradation tag, which will cause the end product protein to break down (very useful if it's a long-living protein you want to get rid of quickly).

Larger genes get sent in bacteria, on little loops of DNA called plasmids to keep them replicating inside the bacteria. You grow your culture up, and then can extract your precious DNA from them. Small bits of DNA, like primers, just arrive as naked DNA, inside a little plastic vial, and can be made up to solution with water.

After synthesis, it's a good idea to get them sequenced as well... just to check you have the right stuff.

Stages of Design

At the moment, as I mentioned in my last post. I'm trying to design a gene to get synthesised. I'm using GeneDesigner from 2.0, and I now have about 100 saved copies of my gene in various incarnations.

My first design was simple enough...the operon (a set of genes one after the other) surrounded by the biobrick prefix and suffix
For this design I used the whole vio operon, including all the bits at the beginning and end that weren't part of the gene. I was scared of cutting anything out, in case we got our nice designed gene back and it didn't make any product at all.

However, when I looked at the sequence I found that the genes within the operon were out of line. Each amino acid (the blocks that make up proteins) is coded for by three bases, for example the sequence AAAGGG will make two amino acids. AAA = lysine and GGG = glysine. However on my operon this was out of alignment; instead of getting XXX / AAA / GGG / XXX, I was getting XXA / AAG / GGX

To counter this I re-designed it, putting all the genes as separate blocks and double checking that they all made the correct proteins:
The end result was a little cramped, but it meant all the proteins were being produced as displayed, along with the prefix, suffix, and a ribosome binding site at the start of the gene.

So I showed it to my supervisor. Who looked at it, and then looked at me, and then said in a very kind voice, "Why do you *need* all that stuff around the genes?"

The thing is I'm a little afraid of taking it out. Just in case there's some sort of importance to it. But in terms of genetic engineering, and further gene manipulation, it's more useful to have each gene smartly laid out, with it's own ribosome binding site. We also want to add a promoter (at the suggestion of the ever-wonderful DNA2.0) which is a kind of START site for the gene, and allows it to be turned into mRNA (which is then turned into proteins). As the operon product has some antibiotic properties (if they're expressed at too high a level they can kill the bacteria), we want it under an inducible promoter as well, so we can turn this operon on and off by adding different chemicals to the bacteria.

So here is my plan at the moment:
Each gene is preceded by a ribosome binding site (rbs and B0034 are the same thing, I just don't know how to relabel things in GeneDesigner). The operon is preceded by a promoter and the whole thing is enclosed within the suffix and prefix.

I'm showing it to my supervisor today. I really hope it's good enough, I want to get this sequenced.

(as a point of interest, these are only a subset of the different versions currently on my computer. I also have variations on all of the above with codon optimisations, restriction sites removed and added, and different/differently placed ribosome binding sites)

EDIT (added after meeting)

I showed him the design, and this time it was pointed out that I was missing a gene...

How embarrassing.

But the rest of the design was good! So it looks like this will be the final product, bar a little fiddling about with the actual sequence:

When corporations are AWESOME

A very small miracle happened last night.

In order to get on of our pigments, we need to synthesise a piece of DNA. A very large piece of DNA, that would normally cost around £3000 to get synthesised, effectively blowing our synthesis budget for this project. I've been spending most of last week agonising about how much of the actual gene we wanted to synthesise, I didn't want to cut too much out, in case it stopped working.

I got an email from my supervisor last night: DNA2.0 have agreed to synthesise is for us.

For free.

Free!

They also have awesome free software you can use to send them the bits you want synthesised. It puts every bit of DNA on a separate arrow, which can be looked at in sequence form or just an image (as shown on the right). In the sequence form it shows you what protein sequence it makes, where the restriction sites are and which codons can be optimised for different organisms. You can also take some of the more standard parts (i.e promoters, ribosome binding sites etc) from a list down the side, and drag them into your construct.

You can move the little DNA arrows around into different orders as well. And colour code them if you want. It works a little slow, but I think that just might be my computer.

I'm still in a little happy daze from the offer to be honest. It is going to make the whole project so much easier and quicker.

Carnival Time!

The ninth edition of Scientia Pro Publica is up on Pleiotropy. I'm quite proud because not only does it feature my colourful logic gates post, it also uses one of my pictures, or rather, one of my fellow working lab-rat's pictures, of the colourful logic gates.

It's also got loads of other great and well-explained science stuff on it, so definitely worth a look.

How to (almost) damage expensive equipment

Around a week ago, a couple of men came into the lab and installed a new plate reader. This is a nifty little device that can measure the both the optical density and the fluorescence of a sample over long periods of time, essentially saving you from having to take little mini-samples out and measuring them every half-hour. It's about the size of a large printer and you put the samples into little plastic wells about half a centimeter wide and a centimeter deep then load them all up and let the machine take the readings for you.

One of the things we're working on at the moment is pigments, which we want to characterise by measuring the fluorescent spectra they give off. Very easy in a plate reader. Characterising one of them yesterday, we took the cell-samples, lysed the cells, extracted the pigment with acetone and then loaded them into the wells for the plate reader.

Luckily, someone left the wells out on the bench for a while before loading them. Because when we came to load them we immediately noticed one vital and unforgotten fact...

Acetone melts plastic.

The acetone from the extraction had melted right through the bottom of the wells. If that had been in the machine the sample would have dripped through onto the lens, which is apparently *very* expensive, and put the whole machine out of commission.

ooops...