The confirmation experiment of Sweet gene

Experiment Notebook.

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We get the Sweet gene groE which could help E-coli folding the protein correctly at high temperature from the genome of BL21. Caring this protein would make the host bacteria grow better at high temperature.
Experimental procedure:
Step 1: recover groE and Blank ( E.coli without sweet gene),and shake them at 37℃ for 12 to 16h at 150rpm.
Step 2: measure the OD600 of bacterial liquid .
Step 3: use 50ul bacterial liquid which has been diluted times to coat plates. Set temperature and induced factor (IPTG, 1 mM) control.
Step 4: add 5ul bacterial liquid which has been diluted times to liquid Luria-Bertani medium .Set temperature control. After cultivating for 12 to 16h, measure the OD600 of bacterial liquid.

Fig1. The experimental procedure of Sweet gene’s confirmatory experiment The data of Sweet gene’s confirmation experiment version 1.0
Cell OD600 groE Blank 0.038 0.018 0.023 0.022 0.026 0.040 0.016 0.014 0.042 0.023 0.032 0.018 0.030 0.031 0.036 0.030 0.018 0.027 Average value 0.029 0.025 Fig2. The initial OD600 of the groE and Blank Tem Cell 37℃ 42℃ groE （IPTG-） 0.071/0.065/0.057 0.034/0.041/0.037 groE (IPTG+) 0.054/0.079/0.065 0.041/0.050/0.049 Blank （IPTG-） 0.078/0.093/0.062 0.036/0.048/0.038 Blank (IPTG+) 0.056/0.070/0.072 0.034/0.040/0.040 Fig3. The OD600 of the E.coli after 16h Tem Cell 37℃ 42℃ groE （IPTG-） 0.064 0.037 groE (IPTG+) 0.066 0.047 Blank （IPTG-） 0.078 0.041 Blank (IPTG+) 0.066 0.038 Fig4. The average value of the OD600 of fig 2 The data of Sweet gene’s confirmation experiment version 2.0 The initial OD600 of the g+p ( groE on the backbone of pET32a) and p( plasmid pET32a) Cell OD600 g+p 0.123/0.138/0.135 p 0.270/0.262/0.256 After 4 h, we measure the OD600 of each kind of cell liquid:
Cell volume of bacterial liquid /ul 37℃ 42℃ g+p （IPTG-） 5 0.094/0.139/0.061 0.061/0.077/0.046/0.049 10 0.078/0.069/0.157 0.048/0.046/0.052 15 0.059/0.057/0.091 0.075/0.051/0.049 g+p (0.8mM) 5 0.020/0.024/0.009 0.017/0.029/0.020 10 0.015/0.035/0.016 0.021/0.027/0.029 15 0.032/0.044/0.029 0.025/0.026/0.033 g+p (1.6mM) 5 0.019/0.019/0.014 0.020/0.025/0.013/0.018 10 0.024/0.024/0.027 0.057/0.026/0.035 15 0.019/0.016/0.024 0.015/0.019/0.024/0.025 P (IPTG-) 5 0.064/0.080/0.064 0.030/0.042/0.038 10 0.069/0.055/0.064 0.034/0.039/0.040 15 0.067/0.053/0.053 0.039/0.035/0.051 P (0.8mM) 5 0.054/0.059/0.054 0.036/0.030/0.028/0.043 10 0.066/0.050/0.061 0.049/0.029/0.083/0.035 15 0.063/0.067/0.061 0.040/0.033/0.026 P (1.6mM) 5 0.082/0.055/0.048/0.057 0.044/0.052/0.052 10 0.051/0.077/0.065 0.068/0.057/0.047 15 0.061/0.053/0.062 0.063/0.053/0.055 Dealing with these data, we got the results as follows: Cell volume of bacterial liquid /ul 37℃ 42℃ 42℃/37℃ g+p （IPTG-） 5 0.098 0.058 59.18% 10 0.101 0.049 48.36% 15 0.069 0.058 84.06% g+p (0.8mM) 5 0.018 0.022 124.53% 10 0.022 0.026 118.18% 15 0.035 0.028 80.00% g+p (1.6mM) 5 0.017 0.019 109.62% 10 0.025 0.039 156.00% 15 0.020 0.021 106.78% P (IPTG-) 5 0.069 0.037 53.37% 10 0.063 0.038 60.64% 15 0.058 0.042 72.83% P (0.8mM) 5 0.056 0.034 61.08% 10 0.059 0.049 83.05% 15 0.064 0.033 51.83% P (1.6mM) 5 0.053 0.049 91.88% 10 0.064 0.057 88.60% 15 0.059 0.057 97.16% Averaging the different volume, we have a more visual figure. The Sweet gene plays a powerful role in the temperature struggle。 After 17 hours: Cell volume of bacterial liquid/ul 37℃ 42℃ g+p （IPTG-） 5 0.054/0.060/0.125 0.055/0.049/0.059 10 0.110/0.089/0.076 0.028/0.037/0.102/0.039 15 0.124/0.156/0.144 0.285/0.248.0.241/0.272 g+p (0.8mM) 5 0.048/0.053/0.100 0.028/0.061/0.047 10 0.045/0.043/0.038 0.066/0.074/0.035 15 0.070/0.086/0.065 0.043/0.043/0.074 g+p (1.6mM) 5 0.080/0.160/0.091 0.048/0.045/0.060 10 0.174/0.110/0.087 0.045/0.050/0.082/0.057 15 0.225/0.114/0.103 0.066/0.057/0.062 P (IPTG-) 5 0.086/0.126/0.092 0.125/0.059/0.061 10 0.095/0.079/0.081/0.078 0.068/0.061/0.074 15 0.098/0.092/0.076 0.072/0.133/0.079 P (0.8mM) 5 0.138/0.138/0.138 0.078/0.067/0.062 10 0.111/0.093/0.085 0.143/0.071/0.070 15 0.072/0.076/0.062 0.092/0.058/0.049 P (1.6mM) 5 0.126/0.082/0.078 0.062/0.065/0.079 10 0.118/0.094/0.103 0.061/0.072/0.073 15 0.086/0.059/0.043 0.065/0.097/0.061 The average of data after 17h: Cell volume of bacterial liquid/ul 37℃ 42℃ 42℃/37℃ g+p(IPTG-) 5 0.08 0.054 67.50% 10 0.092 0.035 38.04% 15 0.141 0.262 185.82% g+p(0.8mM) 5 0.067 0.045 67.16% 10 0.042 0.058 138.10% 15 0.074 0.053 71.62% g+p(1.6mM) 5 0.11 0.051 46.36% 10 0.124 0.051 41.13% 15 0.147 0.062 42.18% p(IPTG-) 5 0.101 0.082 81.19% 10 0.083 0.068 81.93% 15 0.089 0.095 106.74% p(0.8mM) 5 0.138 0.069 50.00% 10 0.096 0.095 98.96% 15 0.07 0.066 94.29% p(1.6mM) 5 0.095 0.069 72.63% 10 0.105 0.069 65.71% 15 0.063 0.074 117.46% After 17h, we found a strange phenomenon. The g+p lives better at 42℃ without IPTG, and we don’t know the reason. Besides, the Sweet gene still works in a temperature struggle. After 45h： Cell volume of bacterial liquid /ul 37℃ 42℃ g+p （ IPTG-） 5 0.036/0.070/0.048 0.086/0.078/0.083 10 0.072/0.049/0.047 0.039/0.067/0.050 15 0.061/0.055/0.060 0.061/0.052/0.060 g+p (0.8mM) 5 0.080/0.069/0.058 0.039/0.044/0.033 10 0.067/0.067/0.052 0.037/0.056/0.043 15 0.115/0.048/0.073 0.048/0.044/0.049 g+p (1.6mM) 5 0.072/0.061/0.070 0.041/0.059/0.039 10 0.048/0.053/0.059 0.071/0.051/0.046 15 0.104/0.032/0.042 0.038/0.037/0.036 P ( IPTG-) 5 0.068/0.097/0.082 0.034/0.051/0.045 10 0.059/0.131/0.106 0.042/0.023/0.032 15 0.078/0.075/0.144 0.046/0.043/0.046 P (0.8mM) 5 0.049/0.066/0.080 0.060/0.039/0.041 10 0.056/0.056/0.060 0.042/0.050/0.040 15 0.039/0.144/0.050 0.040/0.037/0.042 P (1.6mM) 5 0.050/0.102/0.085 0.051/0.032/0.035 10 0.050/0.097/0.173/0.051 0.030/0.027/0.035 15 0.066/0.066/0.065 0.033/0.061/0.042 The average of data after 45h: Cell volume of bacterial liquid/ul 37℃ 42℃ 42℃/37℃ g+p(IPTG-) 5 0.051 0.054 106.54% 10 0.056 0.052 91.96% 15 0.059 0.262 443.22% g+p(0.8mM) 5 0.069 0.045 65.70% 10 0.062 0.058 94.09% 15 0.079 0.053 67.51% g+p(1.6mM) 5 0.068 0.051 75.00% 10 0.053 0.059 110.38% 15 0.059 0.062 104.52% p(IPTG-) 5 0.082 0.082 99.59% 10 0.099 0.068 68.35% 15 0.099 0.095 95.62% p(0.8mM) 5 0.065 0.069 106.15% 10 0.057 0.095 166.08% 15 0.078 0.066 85.04% p(1.6mM) 5 0.079 0.069 86.92% 10 0.093 0.069 73.84% 15 0.066 0.074 112.63% After 45h, the strange phenomenon became stranger. There was no regularity anymore. The Sweet gene was no longer performing its function. We still compared the OD600 of one type of bacteria while time elapses. The volume of bacterial liquid is 5 ul. The volume of bacterial liquid is 10 ul. The volume of bacterial liquid is 15 ul. From these figures, we still got the same result as the data above.