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can this project work out within 8 menths? - (Dec/04/2007 )

We are a group of undergraduates trying work on a project about molecular evolution,and we would like to know whether our project discribed below can be done within 8 months.
And here's our primary experimental design: We use a bacteria Rhodococcus sp., a local strain of Taiwan, to carry out the whole experiment. Since the bacterial strain can degrade toluene, a kind of polutant that is toxic to organisms, as carbonsource, and having a relatively fast growth rate and simple developmental cycle. We colured the bacteria using toluene as the sole carbon source for a few thousand generations, during which raising the concentration of toluene starting from their upper limit of toluene degrading capacity.
Meanwhile, we use some computer programs to analysis the population dynamics of the bacterial population, and repeatedly check the bacteria's genome and the amino acid sequences of some proteins after a certain time span, which in order to find any spontaneous mutation that makes the bacteria more resistant to toluene. PS. Rhodococcus sp.'s genome has already been fully sequenced, and surpricingly it can be obtained from the net, and so does the whole metabolic network of the bacteria.
But in order to make sure that the toluene resistant phenotype is resaulted directly, or at least mostly, from genetic mutation(s), we have to limit the mutation(s) occuring spontaneously on a specific gene or group of genes. And this is the main barrier that we've met.
So, is there any method that can limit the mutations to accumulate on a certain gene(s)? And can this project be done within 8 months? Two months is enough for 1500 generations, and population dynamic analysis by computer programes could be done at fast in a month.
Please give us some advices for improvement. And if our primary experimental design can't be done in 8 months, please give us some guides to change our project content.

THANkS A LOT! ///////////////////////// Orz


You must be a very talented undergrad. This is almost a comprehensive exam proposal for someone to be evaluated for the qualification for PhD.

My general impression is this is a pretty big project, good enough for an entire course of postgraduate for an Msc, maybe more, depending how deep you want to dig.

For a groups of unskilled students I would say you guys may be able to accomplish a partial success at the most, to complete everything proposed to do is clearly too much. In a worst case scenario, you may get nothing accomplished in the eight month period.

Tell us how much $$$ can you spend and how many people will be involved. Will there be any active involvement from the faculty side?

Are you going to go through the process of multiple cycles of mutagenesis--> selection stages with a gradual increase in the selection pressure? Unless you are doing this with a robotic system, you are not going to be able to do 1,500 cycles. Each selection step will kill a lot of bacteria, you need to grow them up, save for future use, etc, etc.

Is the pathway understood at the molecular level? Do you know many key enzymes/proteins maybe involved? How are you going to narrow it down to a few targets should you discovered some interesting phenotypes? or are you going to do this randomly?

Other issues to consider: Bacteria transmit genetic materials horizontally among themselves in the form of plasmid. Many drug resistance phenotypes are carried by these plasmids. This is a very important pathway for bacteria to evolve new phenotypes, something it is very difficult to duplicate in your proposed study.


Could you clone the gene(s) and run them off plasmids to limit the mutation, i.e. add fresh plasmid to each 100 generations or something similar?


The objective of this experiment appears to be to make a super toluene consuming Rhodococcus. (via degradation or carbon assimilation?)

Firstly, your bacteria must be able to explore the genetic landscape to finally reach a ultimate peak of fitness (aka mountain. The Rhodococcus. is currently (we hope) on a small hill of fitness and not on the ultimate peak.

I don't know much about Rhodococcus biology, however to explore a genetic landscape, your bacteria must first leave their 'hill' of fitness. Leaving the hill of fitness can be bad. In the best case senario, the drop in fitness is mild, and the protein can withstand a few changes with no change in activity, then a few more mutations later, the protein has move to a higher hill of fitness (aka protein has become better), and the project works. In the worse case senario, it takes a few consorted changes (amino acid 1, 3 and 34 must both change at the same time) for the protein to move to taller 'hill'. As a result you won't see any evolution, the intial drop of fitness is too strong, and forces the protein to return to its original 'hill'.

This is why having more then one copy of the gene under positive selection is good, as one gene is allow to travel/mutate while the other copy keeps things working.

Second is the mutation rate. The mutation rate in most organism including bacteria is slow by project standards. You need something to mutagenise the genome to force the mutation rate to rise to acceptable speed. So you throw in a mutagnic chemical or two, however you will find that mutation rate may still be too slow, as the bacteria's DNA repair machinery are moping up all the genetic mistakes. You will probably need a strain which has mutations in its DNA repair machinery... note such strains are usually rather ill and not the best bugs to work with.

Third is the accumulation of benefial mutations. If your bacteria must be able to mate, so that several different beneficial mutations all the better. If they can't exchange DNA, you will have several different strains with different mutations that each makes them slightly better., those will compete until one strain wins, then you will have to wait for a second benefial mutation to occur again, before you see an improvement again.

Selection pressure, be careful here, using toluene as a sole carbon source may not work, since the organism needs to be able to break down toluene into some smaller carbon molecule product and then utilise said carbon product as building block to make more cells. Utilisation of toluene is not the same thing as toluene tolerance or even toluene degradation. I don't know anything about Rhodococcus. Can it live on (eat) solely toluene or does it simply degrade toluene (break it down to bit and then discard those bits). Living on toluene is more difficult that simply breaking it down.

And if it is not possible to get the bug to live only on toluene, how can you differentiate bugs that have better tolerence to toluene and one which can degrade toluene. Because if the bugs can not live only on toluene you will have to feed them something like glucose. And a bug may live on said 'glucose' while ignoring the toluene, or it can live on glucose and break down toluene at the same time.

The selection screen become also more difficult as you are also running into the 'pool of commons' problem. A few bugs degrade toluene well but most don't, the pool of toluene is rather large and thus no change in toluene concentration is detected. The usually thing most people do is to spread the cells over many wells so that each well will contain only a few cells and then check each well. THe work just spirals out of control.

QUOTE (uglycell @ Dec 4 2007, 05:27 PM)
Meanwhile, we use some computer programs to analysis the population dynamics of the bacterial population

Could you clarify by what you exactly mean by this? How are you going to analyse the population dynamics of the growing bacteria population? Do you have some kind of devise that automatically counts the cells in the population?

QUOTE (uglycell @ Dec 4 2007, 05:27 PM)
we have to limit the mutation(s) occuring spontaneously on a specific gene or group of genes. And this is the main barrier that we've met.

It is not possible to limit mutations to a single gene or area in vivo.

(Unless you are using proteins such as Activation induced cytidine deaminase, unfortunately expression of this protein is problematic. The only cells that I know who can express functional AID in any useful quantity is B lymphoma. But that is another story)

The best I can think off is to stick your genes of interest into a plasmid. (Perhap all the genes that are involved in toluene degradation). Mutagenise said plasmid invitro with incoperation of inosine triphosphate (ITP) into the DNA by PCR, could also use mutagenic DNA polymerase, or other strange things. Transform of the many different types of mutate plasmids into the cells. Grow said cells abit in toluene, allow cells to exhange plasmids (you will have to check the cell biology to see if that can happen. Maybe there is an F1 equivalent plasmid.) Add some mutagens to the media to increase mutation rates futher. Use the all important selection screen to pick the best mutant bug.

After selection with increasing toluene for a while, re-extract the plasmids, analyse by sequencing, mutagenise the plasmids again and retransform. Repeat.

Can the experiment be done in 8 month?

If you are making a plasmid containing all the toluene resistence genes... well it depends how many gene are there? Can you transform your bug with plasmids. (either by electroporation- the best method or chemical transformation)

Do you have help to make plasmid. If you have to make the plasmid on your own or the technical help you have is not good enough to make a poly gene plasmid, then it is difficult

Can your bug use toluene as a carbon source. It need not be the sole carbon source, we can work on it if it actually use toluene as a carbon sourse. If it merely degrades toluene, then there is much thinking required to design a selection screen. If you can not apply enough selection pressure, the project will fail. The current selection screen is dependent on the bug being able to use toluene as a carbon source. A fact that must be confirmed.

In closing, the project is difficult and requires more planing. More undertanding of the biology and molecular techniques of Rhodococcus is required. If you started the project right now, I have a feeling you will not see much evolution as experience with my PhD project has thought me, if there is even the slightest possibility of something going wrong it will most certainly go wrong. It is a law.


I personally dont like this type of exploration project for people just about to learn how to do science. For undergrads, it is more important to show them how to do science right and how fun it can be. The goal is to demonstrate a person how to initiate a project through hypothesis driven process. The project itself should be more toward conservative side so the students should not get discouraged at the end should something fail.
This project is high risk one. If you can not get the strains with the intended phenotype, no further sequencing and molecular characterization works can be done. It is clearly wrong type of project to pursue for you guys.


blink.gif, my..I myself cant really think that this is an undergrad project. Just like some forumer here had mentioned, this looks liks a phd project.