Selection for the monomeric FliC protein has taken place using the streptavidin-biotin complex that can be stabilized on magnetic beads. The reactions take place in 1X PBS buffer and at room temperature, the standard conditions for the protein of Burkholderia pseudomallei. The streptavidin beads were bound to 200pmol of the biotinylated protein that makes the initial complex. Then, following denaturation, 200pmol of R0 N59 RNA nucleic acid were incubated at room temperature for 30 minutes. The respective RNA washes are removed from the solution, allowing the bound FliC ligands to be eluted out of the target-pool complex.
An ethanol precipitation was carried out to purify and visualize the eluted RNA samples, since the RNA is transparent and often has beads and other contaminants in solution beforehand. Reverse transcription is proceeded next, transcribing the RNA into and amplifiable DNA pool. This was done with the initial removal of solution from the beads (W0), the third wash (W3), and the elution (E1). Then a cycle course reaction was run in order to test the proper amount of amplification to be done in large scale PCR. Without the proper amount of nucleic acid, one either may not have enough to continue on with the ext binding and selection round, or will have extraneous strands of varying lengths that will skew data. Gel electrophoresis using 3.8% agarose gel at 100V for 30 minutes was run in order to see the amplification of the DNA. The Figure 1 shows the gel for the second cycle course reaction run.
The cycle course PCR shown was the second attempt at cycle course for R1. The initial gel only showed the base pair ladders. Since only the ladders were showing, either the cycle course did not amplify the DNA, or the RT failed and there was no DNA to be amplified. In order to save from possibly having to do three first round cycle course reactions, RT was rerun. The second cycle course (as shown in Figure 1) had limited amplification of W1 as compared to W3. This could be due to variability in incubation time and movement of the beads between the two washes. A light band can be seen in the E1 that signifies overamplification in cycle 15. The proper amplification would reside at approximately 12 rounds, since this is the darkest band without a wide band that shows the DNA polymerase to be acting on too many strands of DNA. However, a no template negative control showed a band, which indicates a problem. Since the NTC did not show any species in the initial cycle course reaction, there are two possibilities. One possibility is the contaminant not being present in the first cycle course due to the PCR not being run properly (yet RT was run correctly). The second possibility is that RT did not run properly the first time, yet on the second it was run correctly, and the species was somehow picked up during the transition or during contamination of the cycle course. Since the second could have been a possibility, the binding and selection was rerun in order to eliminate the contamination from the picture.
The problems encountered mainly arose with the NTC of the second cycle course reaction. This presents a huge problem, since it is impossible to tell exactly what this species is. Since it is about the same length as the pool (59 base pairs) it can be assumed that some species was transferred into the PCR tube when addition of reagents (i.e. not switching a tip, touching a tip to the lab table, etc.). Since these species may or may not be potential binding ligands, they must be completely limited from the reactions. Another problem was my initial cycle course not showing any species. Attention to detail and additions and not leaving enzymes out of ice in reactions before commencing the reactions could help eliminate these errors.
Conclusions and Future Work:
The future steps of the reaction will be to continue on with the preliminary steps of R1 reaction in order to proceed into the later steps and R2. Currently the rerun of the round has gotten to RT. After cycle course, lsPCR will amplify the DNA in order to proceed into transcription (TNX) to obtain RNA to be used in R2. A PAGE gel is used in order to purify the RNA from the TNX, with than is ethanol precipitated out to visualize the RNA and isolate it completely from the gel.
So far the reaction has carried out fairly well, considering that at the moment, besides the positive NTC, the selection is possible and can produce viable results. Continuing on with the rounds should be able to produce an applicable ligand. The conditions for the first two rounds of selection can be seen in Figure 2. The rounds needed for PCR could possibly be less than 12 if the ideal amount of RNA is recovered from TNX. This would mean that the binding species would be fairly complementary to the target FliC protein. Given this, much of the pool should be able to bind, and thus will have less washed away by the protein, since most of that was removed in the R1 washes.