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.

July 21

We may have determined why our plasmid backbone pSB3K3 has been turning up in gels as 2kbp rather than 2.75kbp. There is a [http://partsregistry.org/Help:Plasmid_Backbone_Availability_2009 Parts Registry link] that seems to indicate that the pSB3K3 backbone with the two Biobricks we have been using has problems. It also suggested an alternative Biobrick in the kit. We ran PCRs on the old Biobrick with this backbone, the Biobrick suggested by the Parts Registry, and a third Biobrick with the backbone from the 2008 Parts Distribution. Unfortunately, our gel indicated that the PCRs were unsuccessful. We will have to try again tomorrow.

July 22

We retried PCRs for the pSB3K3 plasmid backbone, using two samples of template: Spring 2009 Plate 2 Well 15L and Spring 2008 Plate 1014 Well 1F. We ran four different PCRs, two for each under two different PCR programs: the program for amplifying PJU-334 and the program suggested for amplifying pSB3K3. Our gel indicated that none of the four PCRs worked.

July 23

Once again, we attempted to PCR the pSB3K3 backbone. We ran a PCR on the 2008 and 2009 DNA each under the suggested program for amplifying pSB3K3. Again, our gel indicated no results.

July 24

We tried the PCR on the pSB3K3 backbone again, using the suggested program and two different Biobricks from the Spring 2009 kit with the backbone. The gel we ran indicated no DNA.

July 27

We attempted to PCR the pSB3K3 backbone once again, using the PCR program for PJU-334 and running it on three Biobricks from the Spring 2009 kit: Plate 1 Well 7E, Plate 1 Well 7M, and Plate 2 Well 15L. Our gel indicated that only 7E worked, though it looks like the DNA is still less than the expected 2.75kbp.

July 28

The successful pSB3K3 PCR was cleaned up and the DNA was eluted. We ran digestions of GFP (with XbaI) and pSB3K3 (with EcoRI and PstI) overnight. We also grew up one colony from each of four plates of cells transformed with four Biobrick promoters: K113009, R0040, K0911011, and J23101.

July 29

The cell cultures of our four Biobrick promoters were miniprepped and digested, first with EcoRI and then with SpeI. The GFP digestion we did yesterday was cleaned up, then digested with PstI. The double digestion of pSB3K3 was cleaned up, and 5μL of the reaction was analyzed on a gel. The gel showed that nothing went noticeably wrong in the digestion.

We then ran two gels on our digestions using TAE buffer rather than TBE. The gels indicated that none of the promoters had been sufficiently digested, or that the small promoter DNA (between 30 and 80 bp) had possibly run off the gel. GFP was digested successfully, for the band shown corresponds to the plasmid that it was in, but there is no band for GFP itself, which is 876bp long.

We set up single digestions with EcoRI for the promoters again to run overnight.

July 30

We cleaned up the EcoRI digestions from last night and started the second round of digestion with SpeI to run overnight. We also inoculated a colony with the GFP Biobrick to grow overnight.

July 31

We accidentally ran our SpeI digestions using pure, undigested DNA of each promoter rather than using the DNA previously digested with EcoRI. Therefore, we added more Tango buffer to inactivate SpeI on these samples and added EcoRI, whereas we added SpeI to the samples we meant to digest last night. Both groups of samples were allowed to digest for 2.5 hours before we ran an analysis gel on 5μL, which indicated that we needed to digest more. We then continued the digest for another 2.5 hours, then ran a gel with the intention of extraction. The gel did not show fragments corresponding to our promoters (between 30bp and 80bp for all but K113009, which should have been 1210bp). Since we did not see a promoter band for K113009, it probably means that our samples have not been allowed to digest for long enough, or that there is something wrong with our restriction enzymes.

We also restreaked the plate with cells containing BBa_E0240 (GFP).

August 3

We inoculated colonies containing Biobricks BBa_K113009, BBa_R0040, BBa_K0911011, BBa_J23101, and BBa_E0240 (GFP) each into 5mL of LB/ampicillin and grew them overnight.

August 4

We miniprepped the DNA from the overnight cultures and set up digestions for overnight. We meant to digest the promoters first with SpeI and GFP first with PstI. However, we ran out of SpeI, so we were only able to do one of our promoters and GFP.

August 5

Our two digestions were accidentally left in the 37°C room for too long, so the digestion ran for eighteen hours. We will have to redo these digestions and all the others when we get more restriction enzymes in.

August 6

Since the Hybrid Promoter group successfully obtained the pSB3K3 backbone via miniprep and digestion using a different Biobrick with the plasmid backbone, we attempted a PCR on this new Biobrick to obtain the backbone that way. The PCR was successful, as shown by the ~2.75kbp band.

August 7

We ran a double digestion on pSB3K3, a single digestion with PstI on GFP, and single digestions with a new SpeI enzyme from NEB on our four promoter Biobricks BBa_R0040, BBa_J23101, BBa_K113009, and BBa_K091101. We also ran four of the same PCR as yesterday to produce more of the pSB3K3 backbone. We ran all our reactions on two gels, and according to the pictures it looks like we were successful so far. There is actually a very faint second band on two of our PCR samples, but we believe that this will not be an issue since, in the worst case scenario, we can gel extract the proper band after future digestion.

August 10

After looking at our sources again, we found some very important things out. We found that the promoters could be synthesized by annealing oligonucleotides designed to include EcoRI and SpeI overhangs rather than by digestion/gel extraction. We also found that the J23101 standard promoter-GFP construct exists as BBa_I20260 in Well 17F of Kit Plate 2. We digested GFP with XbaI and promoters K113009 and K091101 with EcoRI for 4 hours, then heat-inactivated the restriction enzymes.

August 11

Today we analyzed the digestions we performed yesterday on K113009, K091101, and GFP, which indicated a successful band for GFP but no band for the promoters.

We decided to try adding another 2mu;L of EcoRI to our promoter digestions and digest for longer. We ended up digesting for five and a half hours then heat-inactivating the restriction enzymes. Our analysis gel on these digestions accidentally ran for too long, so the promoter bands (if they were present) ran off the gel. We decided to toss the tubes rather than run another gel.

We performed a gel extraction on GFP and began four more digestions of GFP. These new digestions were done first with PstI for three hours.

In addition, we found a Biobrick with the low-copy plasmid pSB4K5 and a lactose promoter that we hope to use with the decoder design. We transformed the Biobrick into competent DH5α cells and grew them overnight. We also restreaked the plate containing BBa_R0040.

August 12

We ran several digestions today: BBa_K091101 with SpeI, GFP with XbaI, and a double digest on pSB3K3 samples with EcoRI and PstI. The digestions were run for four hours, then an analysis gel was run with the pSB3K3 digests. For some reason, the gel indicated two bands when only one was expected (since this plasmid was made by PCR), meaning that we will have to gel extract the proper band.