1) Protein engineering

Molecular biologists often play with proteins, switching them on/off, fixing them to a support or just liberating them. In this project we are going to design a construct that can help with the rapid removal of residual RNAses from purified RNA sample. This is needed because it is hardly possible to work entirely RNAse free, and most RNAses are hyper-active.

We know that RNAses are proteins and we know that proteins can be cleaved by proteases. The best protease to 'clean up' proteins normally is trypsin (why?). Unfortunately, trypsin also degrades RNA very, very slowly. So we need to let trypsin do its work for a while, and then remove it from the solution.

To be able to remove it from the solution, we want to bind trypsin molecules to small (25 μm radius) coated plastic beads. We have a linker that rather easily connects arginines to the coating of the beads. Unfortunately, this compound will also bind arginines to other arginines, so it would be best if all but one arginine were removed from trypsin by mutagenesis.

The next problem is that the trypsins fall of the beads very slowly. So after removing the beads we will add a massive dose of BPTI to keep any left-over trypsin inactive. Consequently, no mutations can be made that make the trypsin-BPTI binding worse.

In the lab we have a very good trypsin vector that has over the years proven to be very resilient to mutations (i.e. if you make mutations it will still be produced, and no funny side effects are seen), and to produce lots of trypsin in E. coli, no matter which mutations are being made. Only the first three amino acids cannot be touched by mutagenesis because that is fatal to the vector.

The sequence of this nicely behaving trypsin is:

IVGGYTCQENSVPYQVSLNSGFHFCGGSLVNDQWVVSAAHCYKSRIQVRI
GEHNINVLEGNEQFVNAAKVIKDFYRKTLNNDIMLIKLSEPVKLNARVAT
VALPSSCAPAGTQCLISGWGNTLSSGVNQPDLLQCLDAPLLPQADCEA
SWPGKITDNMVCVGFLEGGKGSCQGDSGGPVVCNGELQGIVSWGYGCALP
DNPDVYTKVCNYVDWIQDTIAAN

Tell me how you would proceed. And I want you to hand in a shorter than 9 page article (in English) that follows the following line:

1. Title, authors, affiliation.
2. Abstract, About 200 words that highlights question, main steps and results.
3. Introduction of at least two pages. Explain the question, the engineering, the molecules involved, etc. Obviously trypsin and BPTI need to be introduced in detail.
4. Methods section of about 1 page. Describe just methods, no results or discussion here. Find the original references for all computer programs that you use. So explain how you extracted sequences from which database, etc. But don't write here which sequences you extracted.
5. Results as many pages as are needed. Give the results and discuss the details, potential errors at each step, etc. Keep a logical order in what you write. Alignments belong in this section.
6. Discussion 1-2 pages. Here you do not repeat the result section, but you discuss some overall aspects that weren't discussed yet in the result section. What are the known weaknesses of the methods you have used? Why do you nevertheless trust your results? Etc.
7. References. Anything copied from anywhere without proper reference costs you a full grade point! URLs (http://www.anywhere.org/) are also valid references, but it is always nicer to give the original reference.
8. List of appendices (if any).
9. Appendices. Try to be careful here. Much info is too uninteresting to mention, but a validation report of your model seems useful.
10. Throughout the article, use figures where useful. Make sure figures show what you want them to show and not much more than that. Use colours, but only where functional.
11. Number figures and references ordered throughout the article.
12. Number the pages.
13. Use paragraphs intelligently. You go to a new paragraph when you go to a new subject. So, for example, a paragraph cannot start with "However,...".
14. Make sure that sentences follow each other logically. I suggest you summarize each paragraph for yourself with a one-word title. If that is difficult, that paragraph is too messy. (Do not forget to remove those paragraph titles before handing in the article...).
15. Use a spell-checker.
16. I want you to work in groups of four. Be aware that if one of the four partners screws-up, you all get a low grade. The article must be handed in no later than 168 hours after the exam. After those 168 hours you loose one grade-point per hour.

Good luck
Gert

Ps

Ps, it might help your feeling with the whole problem to do a few simple calculations first:

1. What is the volume of a plastic bead?
2. How many beads can you get in one Eppendorf tube (volume 1 ml)?
3. The active groups at the surface of the bead tend to be 100 Ångström  away from each other. How many linkers can you bind to the bead?
4. If you can only use 5% of the volume of the Eppendorf tube for beads, what is then the maximal achievable 'concentration' of trypsin in the Eppendorf that holds your prcious RNA?
5. There is often up to 20 atogram of RNAse in any Eppendorf that has been open to the air in any normal lab for more than 10 seconds. Some RNAses cleave waith a speed of 10⁷ sec^-1. How much damage will this amount of RNAse do to your precious RNA in two weeks?
6. If the immobilized trypsin cleaves with a speed of 150 sec^-1, how long should you then keep the beads in the Eppendorf? And why, in practice should it actually be about 30 minutes?
7. Why dont we keep the beads in the RNA Eppendorf for 24 hours, just to be sure?

The radius of a bead typically is 25μ. Given that the volume of a sphere is 4/3.π.r³, and given that we are not interested in a couple factors of two here and there (so 4/3.π=4, 4.π=10, etc), the volume of one bead is about 5.10^-14Ångström³. So you can store about 2.10¹³  of those in a liter. The surface area of a sphere is 4.π.r². So the surface area of one bead is about 5*10^-9m². Which gives you 2.10¹³.5*10^-9m²=10^-3m²  surface area. Linearly, the trypsins will on average be 100 Ångström  separated, so you get 1 trypsin per 100.100 Ångström²  whic is about 10¹⁶t/m²  (in units trypsins / square meter), which with 10^-3m²  gives you 10¹³  t/L (trypsins/liter). Assuming we have 1 mL in an eppendorf, and 5% of the eppendorf will be beads, we need to divide that number by 2.10⁴  which gives with a bit of rounding 10⁹t/e. And with Avogadro's number rounded at 10²⁴ we have a trypsin concentration of 10^-13t/e or 10^-9M/L which is commonly known as a 1 nanomolar solution. in a liter.