2001 Annual Science Report

Scripps Research Institute Reporting  |  JUL 2000 – JUN 2001

Biochemistry Class Examination - Ellington's Laboratory

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339L Biochemistry Exam

Throughout this exam you are going to want to consult not only your textbook, but primary literature and the Web for answers. The best way I’ve found to find primary literature is to use the resource Entrez (http://www.ncbi.nlm.nih.gov/Entrez/) and the PubMed server therein. If you have a better resource, feel free to use it. Of course, many of the papers you’ll actually have to look up yourself in the library.

I have also set some papers aside for you to use, and these are on reserve in the Chemistry library. I have set aside six copies.

Please understand that this type of test is meant as a sign of respect for your capabilities. Please do not steal the references on reserve. Please do not cut articles out of journals. If either of these instances of disrespect comes to my attention, the test will be cancelled, and an in-class final of my own choosing (closed book) will be substituted. To determine if an in-class final has been instituted in place of this final, please continue to consult this Web site.

Research is a waste product of an economy (see, for example, “Guns, Germs and Steel,” by Jared Diamond, or just play the computer game Civilization II). Research potentiates technology which in turn potentiates human conquest of the environment. Accepting for the moment these simple precepts, it is likely that different planets in the Universe have different levels of â??economic’ activity (economics being broadly defined as the conversion of resources to real or symbolic capital). Planets with fewer resources have less waste and concomitantly less technology; planets with greater resources have more waste and therefore more technology (see, for example, the comparison of Micronesian cultures in “Guns, Germs, and Steel”). Obviously, this simple analysis can be leavened with any of a number of other assumptions, but it will do for now.

The conquest of an extraterrestrial (extraplanetary) environment is one of the greatest technological challenges facing a planet. Therefore, around the Universe, some peoples will leave their home planets, some will not, in part based upon how much economic waste (research) they produce.

Which category does Earth lie in?

While I have my own opinions on the matter, perhaps you can help to discern whether or not we have technologies suitable for leaving the surface of our home planet and venturing one baby step out towards the stars. This test is about your ability to terraform the planet Mars.

Below you will find a graph that neatly compares at least some of the parameters between Earth and Mars.
(1; 5 points) Based on this graph, some of the problems that confront terraforming the surface of Mars with mesophiles (organisms that live under ambient Earth conditions) are:

(a) It’s too cold.
(b) There is no liquid water.
(c) The atmosphere is too sparse.
(d) It’s too cold and the atmosphere is too sparse.
(e) It’s too cold, the atmosphere is too sparse, and there’s no liquid water.

To remedy the primary problems confronting terraforming, it is first necessary to change the Martian environment. A number of interesting solutions to this problem have been posed, including “Orbital transfer of very massive bodies (10 billion ton asteroids) from the outer solar system â?¦ using thermal nuclear rocket engines using the asteroid’s volatile material as propellant â?¦. If the asteroid is made of NH3 â?¦ as little as 10% of the asteroid will be required for propellant â?¦. About 4 such objects would be sufficient to greenhouse Mars” (provide an atmosphere that would have a significant greenhouse effect, retaining heat and even blocking harmful UV radiation).

This would have the practical effect of giving Mars a “temperate climate, and enough water â?¦ to cover a quarter of the planet with a layer â?¦ 1 m deep.” Surf’s up!

Of course, there is the cautionary problem that “As each object will hit Mars with an energy equal to about 70,000 1 megaton hydrogen bombs â?¦ such a program may be incompatible with the objective of making Mars suitable for human settlement.” Details, details.

The document continues, “A possible improvement to the ammonia asteroidal impact is â?¦ that bacteria exist which can metabolize nitrogen and water to produce ammonia. If an initial greenhouse condition were to be created by ammonia object importation, it may be possible that a bacterial ecology could be set up on the planet’s surface that would recycle the nitrogen resulting from ammonia photolysis [or extant nitrogen in the atmosphere, which stands at 0.12 mbar — AE] back into the atmosphere as ammonia, thereby maintaining the system without the need for further impacts” [beyond the initial four].

(2; 5 points) Name a bacteria that can metabolize nitrogen and water to produce ammonia. Cite your source.

(3; 20 points) Show the detailed pathway by which the bacteria from (2) uses nitrogen (N2) and water to create the greenhouse gas ammonia. Be sure to indicate where electrons come from and where they go. You can assume protons and energy (ATP).

(4; 20 points) One problem with an ammonia atmosphere is that the water on the surface will be, um, somewhat basic. Can some bacteria survive in alkali? Of course they can, they’re called alkalophiles. Draw a cartoon showing how alkalophiles maintain an interior pH lower than their exterior pH. Be sure you demonstrate an understanding of what your cartoon shows.

(5; 20 points) For an exterior pH of 9.5, an interior pH of 7.5, and an electrical gradient of 0.15 V (negative inside), calculate the delta G for the transport of a single proton from â??out’ to â??in.’

An alternative method for â??greenhousing’ Mars would be to send vast quantities of hairspray to the planet. No, but seriously, those CFC’s (halocarbon) gases that are so detrimental to the Earth’s ozone layer would be of great value for changing Mars’ atmosphere. “Greenhousing Mars via the manufacture of halocarbon gases on the planet’s surface may well be the most practical option. Total surface power requirements to drive planetary warming using this method are 1,000 MWe, and the required times scale for climate and atmosphere modification is on the order of 50 years â?¦. For purposes of comparison, a typical nuclear power plant used on Earth today has a power output of about 1,000 MWe, and provides enough energy for a medium-sized (Denver) American city.”

Thus, if we can just send Denver to Mars, we’re in good shape.

However, “The industrial effort associated with such a power level would be substantial, producing about a trainload of refined material every day and requiring the support of a work crew of several thousand people on the Martian surface. A total project budget of several hundred billion dollars might well be required. Nevertheless, all things considered, such an operation is hardly likely to be beyond the capabilities of the mid 21st Century.”

Without revealing too many of my own prejuidices, let me say: Ha! We can’t even build the Superconducting Supercollider.

(6; 25 points) What is not explored is whether or not biological processes might be used for the production of halocarbon gases. Assuming an adequate supply of halides on Mars (see, for example, http://www.spaceflightnow.com/news/n0006/23marsocean/), our best bet for biological incorporation of halides (primarily chlorine) into organics may be enzymes known variously as haloperoxidases, chloroperoxidases, or myeloperoxidases. Using tyrosine, chlorine ion, and any other necessary substrates, show the incorporation of chlorine via one of the above enzymes. You don’t necessarily need to show mechanism, but you do need to draw the structures of substrates and products, and to balance your equation. Cite any references that you use.

(7; 20 points) Based on the chlorotyrosine species that you derived, above, can you draw a pathway to the production of chloroethanol? Use your knowledge of amino acid degradation, the Krebs cycle, and gluconeogenesis.

While chlorinated organics will help with greenhousing, the introduction of fluorine would be even better, especially since “Fluorine in the bulk composition of Mars has been estimated at 32 ppm by mass versus 19.4 ppm for the Earth â?¦. Fluorine on Mars would have to be mined locally [likely as] fluorspar.”

(8; 5 bonus points) Cite an article which details the biological incorporation of fluorine into organic molecules. I couldn’t find one.

(9; 10 points) You decide to clone and express a chloroperoxidase, in part so that you can engineer it to work with fluorine, and in part so you can insert it into an alkalophile. Using the same amazing Entrez resource, go to the section marked â??Nucleotide’ (box in upper left hand corner). Identify, print out, and attach to this test the complete sequence of a chloroperoxidase of your choosing. Show the â??start’ and â??stop’ codons on the sequence.

(10; 20 points) Go to the site www.novagen.com and look up the sequence of the pET 30 vector series. Print out and attach to this test a map (not the sequence, a map) of the pET 30 vector. Below, re-draw a schematic of this map and explain what each feature of the vector does, especially if that feature has to do with protein production.

(11; 10 points) Why does the pET vector use a T7 RNA polymerase promoter? Where does the T7 RNA polymerase that will be used for chloroperoxidase production come from?

(a) From a T7 RNA polymerase gene encoded on the pET vector
(b) From T7 RNA polymerase added to the media during cell culture
(c) From a T7 RNA polymerase gene inserted into the genome of an E. coli in which the plasmid is grown
(d) From T7 RNA polymerase added to the plasmid during its extraction from the cell.

(12; 20 points) Design primers for the PCR amplification and insertion of the chloroperoxidase gene into pET30. The primers should overlap the reading from of the chloroperoxidase gene by at least twenty residues. The primers should be compatible with the cloning scheme you’ll describe below.

(13; 25 points) Using the primers from (11), draw a cartoon that shows how you would PCR amplify the chloroperoxidase gene and insert it into a pET30 vector so that functional chloroperoxidase protein would be produced.

(14; 5 bonus points) You belatedly wonder just why Nature hasn’t come up with a fluoroperoxidase enzyme yet, given that it would seem like a useful tool for natural product synthesis. Before initiating your hunt for chloroperoxidase mutants that can utilize fluorine, you examine the mechanism of chlroperoxidase again. Relying on your knowledge of organic chemistry, you slap your head and realize why there are no fluoroperoxidases. Explain.

You recall that in addition to having no atmosphere and limited water, Mars has other, related problems, such as UV light intensity that would sterilize virtually any organism sent to the planet’s surface. Now, one alternative is for organisms to hide under or within rocks, as they do in Antarctica (you can find green sheens of photosynthetic organisms several cm down in exposed Antarctic rocks). Still, the hellish UV irradiation is likely to be a problem. Except that there is a terrestrial bacterial champion for UV as well, Deinococcus radiodurans.

(15; 10 points) How much more resistant is Deinococcus radiodurans to radiation than your average organism? Cite a number and cite your source.

(16; 20 points) Deinococcus radiodurans keeps multiple copies of its chromosomes around, and is capable of stitching them back together even after having them torn into many pieces. Basically, D. radiodurans has an incredibly robust recombination machinery. Starting from the the five aligned chromosomal fragments shown below and using your own knowledge of recombination, show how recombinational repair could make one longer chromosomal fragment. Detail all necessary steps, including indicating what enzymes are participating.

(17; 5 points) You insert your vector that produces chloroperoxidase into Deinococcus, hire a Russian rocket to send it to Mars (hey, if a tourist costs $20 M to send to space, then a bug should be cheap, especially for a one-way trip), and await the evolution of sunny beaches. Use the cool Mars terraforming simulator [http://www.users.globalnet.co.uk/~mfogg/simul.htm] to figure out how much CFC gas you would need to raise Mars’ tropical termperature to a balmy 25 degrees C. Write the number below.

(18; 5 points) Which picture is frost on Mars? Which picture is frost on an Antarctic desert (hint: which atmosphere
(19; 0 points) Overall, I am of the opinion that the Earth does not have enough resources to â??waste’ on terraforming Mars. That is, the level of economic activity on the planet currently produces a level of technology that is not commensurate with a sustained effort to move off of the planet. Rather, we are likely nearing a period of resource limitation (not necessarily a resource crisis, but a limitation) that will necessitate turning our attentions to our own environment. In my opinion, by turning inward we will of necessity not be turning outward. Or, more starkly, when we cease to consume at a frenetic pace, we will also cease to look for new resources to maintain our level of consumption (and the level of â??waste’ technology that comes from such consumption). A larger, richer planet that had a longer period of time to wastefully develop technology would reach its environmentally critical phase later. Such a planet would therefore also have a longer period of time to develop wasteful space programs and wasteful technologies commensurate with terraforming, and thus would be more likely to make the jump to another planet’s surface.

Contrary or modifying opinions will be of interest.