Team:Illinois/Modelingteam

Modeling Team

Goals: Our team will be creating mathematical models of our bacterial decoder and of the various biological processes involved. More specifically, we will be modeling:

• simple sRNA regulation of one gene
• multiple sRNAs regulating one gene
• one sRNA regulating multiple genes
• promoter activity as a function of input concentration
• our bacterial decoder as a whole

We will be using Matlab and SimBiology often as tools to help us model various systems. Our team will also be taking measurements and recording data in order to compare our actual decoder with the models we have constructed.

For more information, please view the Modeling page.

June 23

We made a Matlab program that models simple sRNA regulation of a single gene. The program takes a vector containing times and numbers of sRNA, mRNA, and protein molecules and outputs a vector of solutions to the differential equations used for each time.

We will be measuring fluorescence of three E. coli cultures containing cells with pXG-0, pXG-1, and pXG-10 plasmids. These plasmids contain the gene for GFP under a constitutive promoter, so these measurements will act as controls for our experiments.

June 24

We made a program in SimBiology that models simple sRNA regulation of a single gene. The equations and constant values used were taken from a paper on sRNA regulation (please see the Research page).

We attempted to measure fluorescence of our cells today using a plate reader, but we ran into some technical problems. We may have to use a different plate reader, or we may be able to fix the problems on the current plate reader.

June 25

We created a working program in SimBiology that roughly models our decoder. In the program, each gene is regulated by two sRNAs. The sRNA synthesis rates are assumed to be constant for now, whereas in reality these rates will depend on input concentrations and promoter activity. We verified by changing sRNA synthesis rates that combinations of two sRNAs resulted in production of the correct fluorescent protein.

We cultured cells overnight in anticipation that we will be able to perform a fluorescence reading tomorrow.

June 26

We were able to use a plate reader on our cells today. Unfortunately, we learned that we did not properly culture our cells for accurate quantitative measurements, so we could only determine fluorescence to be positive or negative. Our pXG-1 cells tested positive for fluorescence, and our other two plasmids (pXG-0 and pXG-10) tested negative. We expected low fluorescence activity for pXG-10 but could not discern any.

June 29

We worked on making a model of simple transcriptional regulation by a repressor protein. However, we encountered some problems in making the model and using SimBiology. We will be fixing these problems soon.

July 3

We have begun work on characterizing various promoters from the Parts Registry. We transformed the Biobricks for GFP (BBa_E0240) and for our reference standard promoter that we will measure fluorescence against (BBa_J23101) into DH5α cells and cultured them overnight.

July 6

Transformations of GFP and the reference standard promoter were successful. We successfully miniprepped the DNA for both Biobricks.

July 10

Our team has begun work to characterize various promoters via fluorescence readings. We transformed two different arabinose promoters from the Parts Registry (BBa_I0500 and BBa_K113009) into competent DH5α cells and cultured them overnight.

July 13

The transformations of BBa_K113009 and BBa_I0500 yielded colonies. We grew one colony from each plate in 4mL of LB overnight.

We ran a PCR to amplify the pSB3K3 plasmid backbone for testing our promoters. For some reason, the gel we ran indicated a 6kbp band when it should have been 2.75kbp. We reran this PCR twice using the same settings.

July 14

We miniprepped our transformed colonies to obtain DNA for BBa_K113009 and BBa_I0500. We also ran a gel on our PCR reactions on the pSB3K3 plasmid backbone. Both reactions yielded ~2kbp bands, when they were expected to be at 2.75kbp. We have decided to use this DNA anyway, since this is the second time bands have shown up at 2kbp.

We planned on digesting our DNA overnight, but our DNA concentrations were too low: between 5 and 30 ng/μL per sample. We have to find out what we should do to compensate for low concentrations.

July 16

We set up digestion reactions for our plasmids, GFP, and promoters. The promoters required digestion with two noncompatible cutters (EcoRI and SpeI), so we are digesting first with EcoRI. We did not have enough DNA to run digestion reactions for the two arabinose promoters, however, so we may have to redo those transformations and minipreps.

After three and a half hours, we ran a gel on our digestions to gauge how much longer digestion should continue. We ended up running all our digestions for eight hours, then we heat-inactivated the restriction enzymes and performed PCR cleanups on the DNA.

July 17

Our digestion reactions were purified by gel electrophoresis. The plasmids formed very light bands and were extracted and purified. No GFP band could be detected, so that was unsuccessful.