Difference between revisions of "Part:BBa K4115017"

Revision as of 08:39, 3 October 2022

SacC (the extracellular sucrase gene)

The sequence of Zymomonas mobilis gene sacC that encodes the extracellular sucrase. Sequence and Features

Usage and Biology

The SacC gene was found in Zymomonas mobilis, and its expression product is extracellular sucrase, which is exhibited sucrase but not levansucrase activity, behaving like a true sucrase. Heterologous expression of SacC aims to improve the utilization of sucrose in the medium by E. coli responsible for the synthetic production of biologics.
If it is necessary to cultivate engineered E. coli in a culture environment with sucrose as a single carbon source, the transfer of the SacC gene is very beneficial to its growth and adaptation to a single carbon source environment. In addition to helping E. coli to more efficiently utilize the sucrose produced by photosynthetic organisms in the system, the SacC gene may play a very important role in assisting the fermentation industry, purification of carbohydrate products, and other processes that require sucrose decomposition.

Cultivation, Experiment and Improvement Process

100 mL Fermentation E. coli DH5α with BBa_K4115017

Figure 1: Fermentation of E. coli with BBa_K4115017 in 300ml conical flask in biological shaker, scale: 100 mL M9 medium (with 20% sucrose), 37 °C, 220 rpm, initial pH 7.5, OD₆₀₀ measured every 2 hours.

Methods/Protocols
1. Before culturing, E.coli is stored at 4 degrees. Culture experiment group(pUC-SacC, The plasmids carried by the engineering bacteria in this experiment have been registered, numbered BBa_K4115036) and control group(pUC-Empty) in LB medium for 2.5h(OD600~0.6). Use these E.coli as the seeds for the following steps.
2. 1:100 add seeds to 100 ml M9 medium: 1.13g M9 salts, dissolved by 95ml ddH2O; 1M MgSO4 200ul; 1M CaCl2 10ul; 20%(m/v) sucrose solution 5ml (10 g/L as the final concentration). M9 salts solution, 1M MgSO4 and 1M CaCl2 are sterilized by autoclaving. 20% sucrose solution is sterilized by a filter. After autoclaving and cool at room temperature, add kanamycin. The final concentration of kanamycin is 30 mg/L.
3. Culture at 37 degrees, 220rpm. Use Nanodrop 100 to measure OD600 every 2h. If the OD600 is higher than 1.5, measure the OD600 after dilution. Every time take out 80ul liquids for tests.

Analysis of the First Experiment

The experimental group(pUC-SacC, shown by the blue line on the figure) grows faster at the beginning(8~18h). After that, the experimental group reached the stationary phase at OD600~2.0, while the control group was still growing slowly until OD600~3.0.
In conclusion, the expression of SacC accelerates the growth of E.coli, but makes E.coli reach the stationary phase at a lower OD600.
We are confused about the differences in the stationary phase. One hypothesis is that the expression of SacC causes some metabolism burden. Considering J23101 used in BBa_K4115036 is a relatively strong constitutive promoter in the Anderson library, I decided to construct two more plasmids, using J23107 and J23109 to express SacC. The plasmids construction process is similar to that of pUC-Empty and pUC-SacC(full length).

Replacements of J23101

Since constitutive promoters in Anderson library only have several base pairs differences from each other, we directly use PCR to introduce the site-specific mutations to the promoter region.

Second-time experiment for SacC

Figure 2: Fermentation of E. coli with BBa_K4115017 in 300ml conical flask in biological shaker, scale: 100 mL M9 medium (with 20% sucrose), 37 °C, 220 rpm, initial pH 7.5, OD₆₀₀ measured every 2 hours.

Methods/Protocols
1. Before culturing, E.coli is stored at 4 degrees. Culture experiment groups[J23101-SacC(BBa_K4115036), J23107-SacC(BBa_K4115037), J23109-SacC(BBa_K4115038)] and control group(pUC-Empty) in LB medium for 2.5h(OD600~0.6). Use these E.coli as the seeds for the following steps.
2. 1:100 add seeds to 100 ml M9 medium: 1.13g M9 salts, dissolved by 95ml ddH2O; 1M MgSO4 200ul; 1M CaCl2 10ul; 20%(m/v) sucrose solution 5ml (10 g/L as the final concentration). M9 salts solution, 1M MgSO4 and 1M CaCl2 are sterilized by autoclaving. 20% sucrose solution is sterilized by a filter. After autoclaving and cool at room temperature, add kanamycin. The final concentration of kanamycin is 30 mg/L.
3. Culture at 37 degrees, 220rpm. Use Nanodrop 100 to measure OD600 every 2h. If the OD600 is higher than 1.5, measure the OD600 after dilution. Every time take out 40ul liquids for tests.

The phenomenon of fermentation bottles

Figure 3: The color of the culture medium after the experiment changed differently. The corresponding experimental groups from left to right are:J23101-SacC(BBa_K4115036), pUC-Empty, J23109-SacC(BBa_K4115038), J23107-SacC(BBa_K4115037)

Figure 4: The result of rough measurement of pH of the culture medium after the experiment. The corresponding experimental groups from left to right are:pUC-Empty, J23101-SacC(BBa_K4115036), J23107-SacC(BBa_K4115037), J23109-SacC(BBa_K4115038)

Analysis of the Second Experiment

J23101-SacC and J23107-SacC show similar growth tendencies. J23109-SacC grows a little bit faster than the control group. But all of them reach a similar stationary phase(OD600~2.1). The results are similar to last time. These results show that the burden of SacC expression does not cause the lower OD600 of the stationary phase. Also, we find the color and pH values of the culture medium are different between the four groups. Maybe their metabolism pathways are different. So next time, we intend to measure the sucrose concentration in their culture medium to see if there are some differences.