Team:ZJUT-China/Contribution
Human Practices
- ZJUT-iGEM team's contribution in this year is to add quantitative experimental characterization data to part BBa K2556051 . and part BBa K2728001 .. Then, I will explain our team’s contribution to the two parts separately.
Firstly, We added further measurement data to the part BBa K2556051 .. In 2017, ZJUT-iGEM team established a cell autolysis system, which was induced by arabinose (Fig. 1 ). However, they only measured the expression of this system in E. coli DH5ɑ host cells (Fig. 2).
- ZJUT-iGEM team's contribution in this year is to add quantitative experimental characterization data to part BBa K2556051 . and part BBa K2728001 .. Then, I will explain our team’s contribution to the two parts separately.
Firstly, We added further measurement data to the part BBa K2556051 .. In 2017, ZJUT-iGEM team established a cell autolysis system, which was induced by arabinose (Fig. 1 ). However, they only measured the expression of this system in E. coli DH5ɑ host cells (Fig. 2).
In this year, we transferred the plasmid in different host cells and measured the expression of this system. 1.We transferred the plasmid containing lysis gene into E. coli DH5ɑ host cells and plotted the corresponding growth curves (Fig. 3).
2.We transferred the plasmid containing lysis gene into E. coli BL21 host cells and plotted the corresponding growth curve (Fig 4).
3.We used the coated plate method to verify the expression of cleavage gene. The bacteria was inoculated into medium containing arabinose and medium without arabinose (Fig. 5 and Fig. 6).
- From our data, it can be found that the lysis gene is stably expressed in the E. coli DH5ɑ host cells, but occurs leakage expression in E. coli BL21 host cells. Our results show that when the arabinose operon was added to induce lysis gene expression, the growth of both E. coli DH5α and BL21 were significantly inhibited, with their OD600 values decreased significantly. In E. coli DH5α host cells, the OD600 decreased from 0.232 after 4 hour cultivation to 0.088 after 6 hour cultivation, and the difference was 0.144 (Fig. 7). The OD600 in E. coli BL21 host cells decreased from 0.278 after 4 hour cultivation to 0.093 after 6 hour cultivation, and the difference was 0.185. However, when the host cell was E. coli BL21, the OD600 increased significantly after 4-5 hours of lysis gene expression. The OD600 increased from 0.071 in the 8th hour to 0.214 in the 10th hour, the difference was 0.143 (Fig. 8). The results of the coating plate experiment also showed that the lysis genes are very effective. These results suggest that in E. coli BL21 host cells, leakage of expression occurs after 4-5 hours of lysis gene expression. Therefore, we should pay attention to the time period which the lysis gene works and complete the discarding of the lysis gene during this time. By changing the growth environment, overacidity and overheating would be adopted to make the bacteria no longer have reproductive capacity.
- Then, we added further measurement data to part BBa K2728001 .. In 2018, BGIC-GOBAL team registered pFrmR, a engineered promoter which was induced by formaldehyde. From they Experimental Characterization, they found it was hard to count the promoter due to the leakage. In this year, we use the catalytic ability of catalase to add quantitative experimental characterization data by counting the A650 of the reaction solution of the bacterial culture and 3, 3’ ,5, 5’ -Tetramethylbenzidine (TMB)
- The qualitative experiment:
(1)Culture E. coli harboring pFrmR - Cat gene in LB medium with different concentrations of formaldehyde (0 mg/ml, 10 mg/ml, 20 mg/ml, 40 mg/ml, 80 mg/ml, 100 mg/ml, 120 mg/ml) for 12 h .
(2)Measure the absorbance of the bacteria liquid at the wavelength of 600 nm and dilute the bacteria solution until the OD600 reached 0.5.
(3)Take 40 μl diluted bacteria liquid out in 96 - well plates .
(4)Add 160 μl TMB reagent in the bacterial liquid containing 96-well plate and incubate it at 37℃ for half an hour.
(5)Measure the absorban>ce of the reaction solution 96-well plate at the wavelength of 650 nm using ultraviolet spectrophotometer.
Related conditions：
Data: The measured absorbance is shown below:
- Analysis: When the E. coli were cultured with or without formaldehyde, they could react with TMB and the solution turned blue, which indicated that both of them produced catalase, and the absorbance at 650 nm was similar. The results indicated that FrmR promoter leakaged. There is no difference between the two situation because of the long culture time and the excessive amount of expressed enzymes, shortening the culture time can reduce the expression of enzymes, and whether the start-up of pFrmR can be seen.
- The quantitative experiment:
Considering to make the induction phenomenon more obvious by shortening the test culture time, we decided to take the bacterial liquid at specific times during the growth process and react with TMB. To avoid the effect on cell growth caused by sampling. We performed 32 culture batches and each group of measurements was repeated three times in parallel. We took E. coli containing pet-28b-cat plasmid as the control group, andE. coli with pet-28b-pfrmr-cat as the experimental group.
(1)Pick one colony of the strains into 5 ml LB medium and cultivate it overnight.
(2)Take 10 μl seed culture in 3 ml LB medim with 0 mg/ml, 80 mg/ml, and 120 mg/ml of formaldehyde.
(3)Take 200 μl cell culture at 3, 4.5, 5, 6, 7, 7.5, 8 and 8.5 hours respectively and stored at 4℃.
(4)Measure the absorbance of the bacteria liquid at the wavelength of 600 nm and dilute the bacteria solution until the OD600 reached 0.5.
(5)Take 40 μl diluted bacteria liquid out in 96 - well plates .
(6)Add 160 μl TMB reagent in the bacterial liquid containing 96-well plate and incubate it at 37℃ for half an hour.
(7)Measure the absorbance of the reaction solution 96-well plate at the wavelength of 650 nm using ultraviolet spectrophotometer.
Related conditions：
Data: The measured absorbance is shown below:
- Analysis: It can be seen from figure 1.3 that after cultivation for 4.5 h and 5 h in LB medium containing 80 mg/ml and 120 mg/ml formaldehyde, reaction of the bacterial liquid with TMB, the value of A650 are very similar to the value of positive control group. This could indicate that although pFrmR exists promoter leakage phenomenon at the early stage of culture, the FrmR promoter was abled to induce by formaldehyde between 4h and 5h. Besides, the reason why the difference between the experimental groups was not obvious after 6h was that catalase might had been overexpressed and the detection agent TMB was extremely sensitive to Cat.
