Talk:BE.109:Systems engineering/RT-PCR data analysis
Post a summary of your results and analysis of the bacterial photography system here. This should include all information that you think would be useful to biological engineers who might want to use this system or its constituent parts in the future. It should be the kind of information you would find on the Registry page associated with the system.
Our situation is special because we did two standard curves instead of one standard and data. One standard was done with DNA from lys-n-go reactions. One curve from standard conditions, and one curve from our variable condition. The condition we varied was the amount of air-flow allowed the cells. The standard culture was given air-flow, but the variable condition was completely sealed off in order to stress the cells. The curve from the standard air-flow condition had an r squared value of 0.8219, and equation of y = -0.35x + 12.44 The curve from the standard no air-flow condition had an r squared value of 1.000, and equation of y = -.37x + 13.22 Each curve was made with three values (the 1000, 100, 10 copies of LacZ)
Because of outside factors, our group tested light and dark cells versus each other. Our standard was done with DNA from the lys-n-go reaction. The curve had an equation of y = -0.63x + 21.29, with an R^2 value of 0.633. The poor corelation can be attributed to the first measurement of the undiluted DNA.
We varied cell density and culture volume by increasing cell density and lowering volume - the two factors worked against each other, which we did not plan well. This condition lowered beta-gal activity and lowered copies of lac Z in the cells, so the net effect of our varied conditions was to reduce the total number of cells in the sample. The curve from the standard DNA had an r squared value of 1 and an equation of y = -0.33x+13.03. This curve was made with two values, the undiluted sample and 1:10 dilution, which corresponded to 10,000 and 1,000 copies of lac z gene, respectively.
One standard was done with DNA from lys-n-go reactions. One curve from standard conditions, and one curve from our variable condition. The condition we varied was the amount of air-flow allowed the cells. The standard culture was given air-flow, but the variable condition was completely sealed off in order to stress the cells. The curve from the standard air-flow condition had an r squared value of 0.8219, and equation of y = -0.35x + 12.44 The curve from the standard no air-flow condition had an r squared value of 1.000, and equation of y = -.37x + 13.22 Each curve was made with three values (the 1000, 100, 10 copies of LacZ)
We compared cells growing in LB medium vs. SOC medium. The raw qPCR results had 2869 copies in the sample (average) for the LB triplicates and 6211 copies (average) for the SOC triplicates, while the LB no RT failed to reach the threshold (ie had <100 copies) and SOC no RT had 875 copies. The primers only and blank wells also had <100 copies. The melting temperature data was narrow and consistent, showing that contamination with other DNA was probably minimal.
The best fit line yielded y = -0.24x + 9.82 with r^2 = 0.92.
Since we added the same amount of RNA (in terms of mass), the results indicate that a greater proportion of the mRNA in the SOC cells was devoted to producing LacZ. However, the better measure is copies of mRNA/cell. After calculations, we found that, assuming 100% RT efficiency, that the LB samples had 4.2 million cells' worth of mRNA while the SOC samples had 14.4 million cells' worth. Therefore, there were 0.68 copies of the LacZ mRNA per 1000 LB cells and 0.43 copies of the LacZ mRNA per 1000 SOC cells. Overall, the SOC cells produced less LacZ mRNA, but they also produced less mRNA overall. This makes sense given that SOC medium has nutrients that the E. coli no longer have to produce mRNA in order to produce those nutrients.
We compared samples +Chl and -Chl antibiotic. The Bgal activity units for these showed approximately equivalent activity in protein.
The standard curve for q-PCR was y= -0.34x + 13.87 and r^2= .986
The No RT line was as expected and approximately overlapping the x-axis.
The triplicates for both reactions were approximately identical.
The +Chl showed greater activity than the -Chl in RNA.
The +Chl sample has more copies/cell.
Love always, Sonal and Jon
We setup a comparison between a culture with glucose and a culture without glucose, both growing in M9 media. The goal was to silence the background activity in one plate of cells by depriving the cells of glucose and to then observe the change in beta-gal activity. The OD600 of these cells after they had incubated for two days was 0.00 for standard growth conditions and 0.0145 for growth in the absence of glucose. This indicated that our experimental growth conditions were not suitable for our cell types. We believe that either the media (M9) contained a substance toxic to the cells, or that the media lacked a nutrient that the cells required. To analyze this problem we could create M9 media cultures with cell types known to use M9 media (this would tell us whether or not our media was contaminated), or we could perform the same experiment but with cultures with varied M9 compositions (MgSO4, M9, glucose). I don't think I needed to include all this, but whatever dude
We were given a new set of cells which were both grown under the same conditions. The RNA that we isolated from these cells was also lost probably due to contamination with RNase.
For the q-PCR, we were given new RNA samples, one from cells grown in the light and one from cells grown in the dark.
We're not done posting yet.
Team Lavender = Team Amazing :)
We tested two different cell samples that were grown under the same conditions to determine if the system for detecting light was stable in all the cells. The standard equation was y = -.25x + 11.93 with r^2 = .977, though we had to set our threshold value to .02. We found that there was a difference in the number of copies of mRNA in the cell samples, which does not correlate with the enzymatic activity we found. There may have been some contamination in one of the samples because the -RT control had DNA amplification.
Our experimental variable was light intensity/reflection. We were thinking that the reflection from whatever surfaces might have had an affect on the angle with which light entered out cameras (I guess the optimal situation is to have light comig from one direction). So, we placed a piece of aluminum foil beneath the experimental plate (a black surface was beneath the control plate). Even though the cell density seemed to differ (OD600,Var: 0.2835, Con: 0.3539, 1 OD600~ 10^9 cells/mL); maybe the difference is due to the extra heat of the aluminum foil), there was no significant difference in neither the beta-gal activity (Var: 222 units Con: 204 units; ~10% difference) nor the LacZ mRNA levels.
Our experimental variable was temperature. Our standard cells were incubated at 37 degrees C and our variable cells were left to grow at room temperature. The variable cells did not grow well at room temp and consequently had zero B-Gal activity compared to the 3,500 units of activity for our standard cells. To measure the occurence of transcription, we conducted q-PCR with the RNA obtained from both conditions. We compared the data from the conditions to a standard curve we constructed with different dilutions of DNA. We found our standard cells to express approximately 3 Lac Z copies per cell and our variable cells to express 0.09 Lac Z copies per cell. This data was in accordance to the B-gal activity we observed. Basically, it seems that temperature can have a huge effect on the rate of transcription, translation, and protein activity in our cell. - From a healthy Jess and a sick Irene and Scottie :(
Our experimental variable was temperature. We detected the activity of two samples, both in the dark. One of these samples was placed in the incubator at 37 degrees Celsius; the other sample was placed at room temperature. There was very little cell growth in the room temperature sample as seen in the OD600 readings. The B-galactosidase activity in the incubator sample was determined to be 1061.45 Units, while the B-galactosidase activity in the room temperature sample was 43.77 Units. The mRNA copies of LacZ correlated with the B-galactosidase activity. The total number of copies from the RT-PCR experiments were found to be in the 1000s for the standard samples and in the 10s for the variable sample. In addition, while the mRNA transcipts per cell were determined to be extremely low (7.26*10^-4 and 3.81*10^-6 copies per cell for the standard and variable samples, respectively), the variation between the two samples again correlates with the B-galactosidase activity found earlier. Thus, the bacterial photography system is not optimal at lower temperatures and should be run at the standard 37 degrees Celsius.
Our experimental variable was light intensity. We varied the light intensities by placing one culture close to the light in the incubator. The B-gal activities for the high light intensity, 5214 units, was higher than the one for the low light intensity, 4653 units. However, the number of copies of LacZ measured by qPCR for the high and low light intensity samples are about the same. This can be explained by our bad standard curve, which has a R^2 of 0.0008. The equation is y = -0.03x + 3.58. Judging from just the B-gal activities, higher light intensity induces higher gene expression.