Difference between revisions of "Part:BBa M36776:Experience"

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== Stanford Location ==

All glycerol stocks are made out of E. Coli K12 strain and stocked in the BioE44 F13 box. They are made for the testing of our pH sensor.

Matt's Glycerol Stock of the mutant from DNA 2.0: 0133009642

Sylvia's Glycerol Stock of the mutant: 0133009642

(Order ID from DNA 2.0 for the mutant sequence: 135227.)

(These two samples serve as replicant groups for the mutant samples for both out first and second tests.)

Glycerol stock of negative control (K12 cell from the teaching lab) : 0133011408

Glycerol stock of the positive control (E.coli with p82 plasmid made before): 0133010301

Glycerol stock of the pH sensor sequence without mutation: 0133010286

(Gene ID from DNA 2.0: 133530)

== Experiment Steps ==

We restarted the K12 strain in the fridge on Nov 10th, planning on having our first round of test before thanksgiving break. We made competent cell using it the second day and then stocked it in the -80 freezer.

Nov 15th: received the sequence with mutation that might cause reduction in RBS binding.

Nov 17th: transfecting the sequence into the competent cells and plated them on LB+amp plates, including the media with transfected cell included by DNA 2.0.

Nov 18th: discovered that only the pre-made media grew because instead of competent cell, we mistakenly took the unmodified cells and tries to transfect the construct into them. In order to get replicant of cells with mutant sequences, we transfected the DNA into competent cell again and plated it on LB+amp plate. We also picked single colony from the plate with growth and grew the colony in LB media.

Nov 19th: Picked single colony from the new plate. The plate showed a sign of over growth, but not to the extent that we could not pick out any colony. We grew the colony in the LB media. Restarted the cells with P82 plasmid that we transfected before, as well as the unmodified cells.

Nov 21st: made buffers. We used HCl, LB media(with amp for positive control and testing group, without amp for negative control) and Sodium Acetate to make the buffers of pH 4.5, 5 and 5.5; HCl, LB media(with amp for positive control and testing group, without amp for negative control) and phosphate for the solution of pH 6, 6.5, 7. The calculation of how much buffer to put was made beforehand. However, since we did the calculation assuming we would be using water, the calculation was not precise and using it as just reference. The pH values were measured by the pH meter in the teaching lab. The final pH of the solutions were: LB without amp: 4.51, 4.90, 5.43, 6.08, 6.54, 7.1; LB with amp: 4.56, 4.93, 5.42, 6.01, 6.48, 7.05. We inoculated the cells into corresponding tubes and made glycerol stocks of the rest. After 2 hours of growth, we tried to make the first measurement of GFP and beta-gal signals using the fluorometer and spectrometer in the teaching lab. However, the short growth time limited the signal and we didn't see anything significant.

Nov 22nd: did second test of GFP and beta-Gal signal after 18 hours of growth. However, because of limited lab hours, we only separate protein from cells for beta-gal and left them in the -20 freezer.

(Thanksgiving break, during which we got our correct DNA sequence from DNA 2.0)

Dec 1st: restarted the K12 strain for transfection of the correct DNA.

Dec 2nd: did the rest of the beta-gal test for the samples collected before. However, the result was not significant, possibly because of the protein degradation during the time. We then realized that we didn't test the cell density of the sample and therefore could not normalize the results. Since pH changes highly affected cell survival, data without normalization would not be meaningful. We therefore decided to abandon data from before thanksgiving. We transfected the DNA into the competent cells and plated them on LB+amp plates. Restarted the glycerol stocks of the mutant cells and P82 cells.

Dec 3rd: we found nothing grew on the plates and latter realized that they were mislabeled and were actually LB+kan+amp plates. Therefore we had to plate them again. We inoculated the cells with P82 plasmids, unmodified cells and mutant groups in the media with different pH to start our testing. Based on the data and growth time from last time, we decided to make the measurements after 24 hours and 46 hours.

Dec 4th: picked single colony of the newly plated cell in the morning. Did the first measurement of the cells already inoculated in the evening after 24 hours of cell growth. (data shown in the next subsection) Put the cells with the correct DNA in the media and made glycerol stocks of the rest, and started separate positive control, negative control with it.

Dec 5th: did the second measurement after 46 hours of cell growth for the mutant cells and first measurement for the designed ones after 24 hours of growth.

Dec 6th: did the second measurement after 46 hours of cell growth for the cells with correct DNA, along with the positive and negative controls.

(All the transfections, glycerol stocks, GFP measurements, Beta-gal measurements and cell density measurements were done using standard procedure. GFP measurements were done using the fluorometer in the teaching lab, while cell density and beta-gal measurements were done using the spectrometer in the teaching lab.)