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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 Schedules and 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.)

== Data Tables ==

Cell Density(OD600)

Mutant Plasmid PH 4.5 5 5.5 6 6.5 7 after 24 hours K12 (negative control) -0.011 -0.013 -0.012 1.294 1.141 1.331 P82(positive control) -0.005 0.047 -0.01 1.155 0.873 1.079 Transformed E.coli 1 -0.006 -0.009 -0.008 1.012 1.193 1.318 Transformed E.coli 2 -0.004 -0.01 -0.01 1.142 1.078 1.258 Average of Transfored E.coli -0.005 -0.0095 -0.009 1.077 1.1355 1.288 PH 4.5 5 5.5 6 6.5 7 after 46 hours K12 (negative control) 0.005 0.003 -0.001 1.262 1.212 1.359 P82(positive control) 0.023 0.036 0.003 1.289 1.068 1.067 Transformed E.coli 1 0.012 0.003 -0.002 1.309 1.318 1.144 Transformed E.coli 2 0.006 0.006 0.0004 1.6 1.416 1.272 Average of Transfored E.coli 0.009 0.0045 -0.0008 1.4545 1.367 1.208

Correct Plasmid PH 4.5 5 5.5 6 6.5 7 after 24 hours K12 (negative control) 0.012 0 0.002 1.198 1.375 1.235 P82(positive control) 0.02 0.007 0.206 0.925 1.003 1.205 Transformed E.coli 1 0.01 0.015 0 1.167 1.141 1.165 Transformed E.coli 2 0.02 0.006 0.005 1.04 1.107 1.218 Average of Transfored E.coli 0.015 0.0105 0.0025 1.1035 1.124 1.1915 PH 4.5 5 5.5 6 6.5 7 after 46 hours K12 (negative control)0.01 0.005 0 1.096 1.268 1.467 P82(positive control) 0.018 0.012 0.038 1.067 0.984 1.25 Transformed E.coli 1 0.017 0.026 0.001 0.952 1.16 1.287 Transformed E.coli 2 0.017 0.011 -0.005 0.908 1.346 1.235 Average of Transfored E.coli 0.017 0.0185 -0.002 0.93 1.253 1.261

GFP normalized by cell Density

Mutant PH 4.5 5 5.5 6 6.5 7

after 24 hours K12 (negative control) -909.0909091 -2615.384615 -583.3333333 63.36939722 43.82120947 57.09992487 P82(positive control) -2800 170.212766 -1400 8907.359307 11796.10538 9545.875811 Transformed E.coli 1 -1166.666667 -2222.222222 -875 800.3952569 904.4425817 1341.426404 Transformed E.coli 2 -2750 -668000 -67300 1146.234676 637.2912801 1023.052464 Average of Transfored E.coli -1800 -352631.5789 -37777.77778 983.7511606 777.6309996 1185.947205

                                                          PH        4.5                    5                     5.5                6                      6.5                  7

after 46 hours K12 (negative control) 3000 2666.666667 -11000 3257.527734 75.08250825 84.62104489 P82(positive control) 782.6086957 472.2222222 7333.333333 7968.192397 9638.576779 9653.233365 Transformed E.coli 1 1416.666667 3666.666667 -6000 1940.412529 2189.681335 2743.006993 Transformed E.coli 2 2166.666667 3833.333333 607500 2785.625 2060.734463 2147.798742 Average of Transfored E.coli 1666.666667 3777.777778 -159375 2405.293915 2122.896854 2429.635762

Correct plasmids pH 4.5 5 5.5 6 6.5 7 after 24 hours K12 (negative control) 3666.666667 16500 53.42237062 77.81818182 63.15789474 P82(positive control) 1650 2428.571429 242.7184466 11022.7027 10155.5334 8499.585062 Transformed E.coli 1 2600 5000 8742.073693 9001.752848 7531.330472 Transformed E.coli 2 1650 3000 5400 9810.576923 9304.426378 8200.328407 Average of Transfored E.coli 1966.666667 4428.571429 17000 9245.582238 9150.800712 7873.268989 pH 4.5 5 5.5 6 6.5 7 after 46 hours K12 (negative control) 450 1000 5.474452555 5.126182965 4.771642808 P82(positive control) 3722.222222 2583.333333 342.1052632 85.28584817 81.30081301 87.2 Transformed E.coli 1 2235.294118 2307.692308 23000 10777.31092 8850.862069 7976.689977 Transformed E.coli 2 3000 3181.818182 -13400 11308.37004 7652.30312 8302.834008 Average of Transfored E.coli 1176.470588 810.8108108 -12000 11041.93548 8217.07901 8167.327518

Beta-gal 

time = 15 minutes miller units = 1000*Abs420/(Abs600 * t * v) volume of cells = .030 Beta-gal Mutant PH 4.5 5 5.5 6 6.5 7 K12 (negative control) 1616.161616 -7521.367521 1851.851852 829.4693457 128.5422144 156.941314 P82(positive control) -10666.66667 756.501182 1555.555556 675.3246753 750.922744 1938.008444 Transformed E.coli 1 1111.111111 493.8271605 2222.222222 228.3706632 132.2529571 136.5705615 Transformed E.coli 2 -2222.222222 0 -444.4444444 301.6151002 156.6687281 150.1501502 Average of Transfored E.coli -222.2222222 233.9181287 740.7407407 267.2031363 143.8426538 143.2022084 PH 4.5 5 5.5 6 6.5 7 K12 (negative control) 2222.222222 888.8888889 72.99270073 369.78619 1048.246611 P82(positive control) -1234.567901 -14814.81481 -1345.02924 4373.63324 5426.829268 4240 Transformed E.coli 1 1437.908497 -854.7008547 -71111.11111 128.3846872 86.20689655 1053.26772 Transformed E.coli 2 -522.875817 #VALUE! 13333.33333 1590.797846 1073.138517 133.1533963 Average of Transfored E.coli 457.5163399 #VALUE! 34444.44444 842.2939068 616.298661 602.6962728

Correct PH 4.5 5 5.5 6 6.5 7 K12 (negative control) 1296.296296 #DIV/0! 71111.11111 159.5251345 140.6060606 41.38551507 P82(positive control) -333.3333333 -952.3809524 -43.14994606 720.7207207 562.7561759 1003.227294 Transformed E.coli 1 2000 -444.4444444 854.9938113 1244.522349 972.8183119 Transformed E.coli 2 111.1111111 1111.111111 9333.333333 6.41025641 1240.590184 1247.947455 Average of Transfored E.coli 740.7407407 0 9333.333333 455.1175553 1242.586002 1113.44244 PH 4.5 5 5.5 6 6.5 7 K12 (negative control) 1777.777778 5777.777778 N/A 137.8751014 177.0066597 204.4989775 P82(positive control) 21234.5679 3148.148148 -935.6725146 5090.076018 4993.224932 4440.888889 Transformed E.coli 1 2091.503268 769.2307692 -31111.11111 5807.656396 879.3103448 4282.137615 Transformed E.coli 2 261.4379085 6262.626263 5777.777778 866.3729809 543.173188 4379.667117 /Users/ysy/Desktop/Screen Shot 2013-10-26 at 2.01.53 PM.png

Average of Transfored E.coli 0 0 0 0 0 0