Difference between revisions of "Part:BBa K4115017"

Revision as of 13:49, 10 October 2023

SacC (the extracellular sucrase gene)

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

Improved by Fudan iGEM 2023

In Fudan iGEM 2023, we aim to address the challenge of E.coli K12's limited ability to utilize sucrose directly, a common issue in symbiotic systems with cyanobacteria since cyanobacteria can synthesize sucrose by fixing carbon dioxide through photosynthesis. To overcome this, we adopted the approach from the ShanghaiTech 2022 project, wherein we introduced the fructofuranosidase enzyme (SacC) into E. coli. This enzyme promotes the hydrolysis of sucrose into fructose and glucose, enabling E. coli to efficiently utilize sucrose as a carbon source.

First Experiment

Methods/Protocols
1. Preparation of M9 Minimal Liquid Medium: Dissolve M9 solid dry powder (11.3 g/L) in DDH2O to create M9 minimal liquid medium. Autoclave the medium at 121°C for 15 minutes. After autoclaving, add a filtered sucrose solution (10 g/L) or glucose (4 g/L). (Note: Be aware that sucrose may hydrolyze into glucose and fructose during autoclaving.)
2. Medium Culture: Begin by culturing the transformed cells (J23101-SacC/J23107-SacC/J23109-SacC) in LB medium with the appropriate antibiotics at 37°C overnight. Dilute the initial LB culture of E. coli at a ratio of 1:100 with M9 minimal medium. Culture this diluted mixture at 37°C with the addition of sucrose (10g/L) or glucose (4g/L) while shaking at 220 rpm.
3. Measurement of growth curve: At specified time intervals (0, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, and 38 hours), take a 1 mL sample of the bacterial solution. Determine the concentration of the bacterial solution by measuring the optical density at a wavelength of 600 nm (OD600) using a spectrophotometer. Create a growth curve based on these measurements.

Analysis
In the initial experiment, our objective was to compare the growth curves between sucrose-utilizing E. coli (SacC) and empty E. coli strains. This aimed to determine whether the introduction of the fructofuranosidase enzyme (SacC) into E. coli facilitates the hydrolysis and utilization of sucrose as a carbon source.
We conducted a comparative analysis of the growth curves of SacC (J23101/J23107/J23109-SacC) and empty E. coli in M9 medium supplemented with sucrose. Additionally, we cultured empty E. coli in M9 medium with glucose as a reference. Our experimental results revealed that all bacterial groups reached a growth plateau around 7.5 hours. Remarkably, E. coli expressing SacC displayed growth patterns similar to empty E. coli utilizing glucose, indicating their efficient utilization of sucrose (with no specific differences observed among different promoters J23101/J23107/J23109). In contrast, empty E. coli relying on sucrose as the carbon source exhibited slower growth and attained lower bacterial concentrations. This disparity highlights the challenge faced by empty E. coli in effectively utilizing sucrose as a carbon source. Introducing the fructofuranosidase enzyme (SacC) into E. coli facilitated the hydrolysis and utilization of sucrose, leading to enhanced growth and higher bacterial concentrations.

Figure1 Growth Curves of Sucrose-Utilizing E. coli (SacC) and Empty E. coli Bacterial growth curves in M9 minimal medium with sucrose (suc) as the default carbon source, unless otherwise stated as glucose (glu).

Second Experiment

Methods/Protocols
1. Preparation of M9 Minimal Liquid Medium: Dissolve M9 solid dry powder (11.3 g/L) in DDH2O to create M9 minimal liquid medium. Autoclave the medium at 121°C for 15 minutes. After autoclaving, add a filtered sucrose solution (10 g/L) or glucose (4 g/L). (Note: Be aware that sucrose may hydrolyze into glucose and fructose during autoclaving.)
2. Medium Culture: Begin by culturing the transformed cells (J23101-SacC/J23107-SacC/J23109-SacC) in LB medium with the appropriate antibiotics at 37°C overnight. Dilute the initial LB culture of E. coli at a ratio of 1:100 with M9 minimal medium. Culture this diluted mixture at 37°C with the addition of sucrose (10g/L) or glucose (4g/L) while shaking at 220 rpm.
3. Measurement of growth curve: At specified time intervals (0, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, and 38 hours), take a 1 mL sample of the bacterial solution. Determine the concentration of the bacterial solution by measuring the optical density at a wavelength of 600 nm (OD600) using a spectrophotometer. Create a growth curve based on these measurements.
4.Provide extra factors: After 25 hours of culture, separate the bacterial cultures and add Sucrose, Glucose, MgSO4, or CaCl2.

Analysis
As our experiment progressed, we observed a slight decline in growth curves for all groups after reaching their peak during the growth plateau. To delve into this phenomenon, we hypothesized that E. coli might be deficient in specific growth factors, even when provided with carbon sources in the M9 minimal medium. Our objective was to investigate this hypothesis.To test this hypothesis, we conducted a comparative analysis of the components in M9 medium and M9 minimal medium. We identified variations, including an additional 4 g/L glucose, 0.241 g/L MgSO4, and 0.011 g/L CaCl2 in M9 medium.

To pinpoint the limiting factor affecting E. coli's continued growth, we repeat the experiments with empty E. coli the second time, while isolating several tubes from empty E. coli cultures at the 25-hour mark, and supplemented with carbon source, CaCl~2~ or MgSO~4~. Our results indicated that the growth curve notably improved upon the addition of MgSO~4~, suggesting that MgSO~4~ was the limiting growth factor.but the growth curve is not inhibited by carbon source and CaCl~2~.

To pinpoint the factor limiting the continued growth of E. coli, we repeated the experiments with empty E. coli. We isolated several tubes from the empty E. coli cultures at the 25-hour mark and supplemented them with carbon sources, CaCl2, or MgSO4. Our results clearly indicated that the growth curve notably improved upon the addition of MgSO4, suggesting that MgSO4 was the limiting growth factor. Interestingly, the growth curve was not affected by the addition of carbon sources or CaCl2.

These second experiments enabled us to identify the critical growth factor affecting E. coli's growth, emphasizing the significance of considering all factors, particularly inorganic salts, when culturing E. coli.

Figure2 Growth Curves of Empty E. coli Bacterial growth curves in M9 minimal medium with sucrose (suc) or glucose (glu) as the default and supplemented (at 25 h) carbon source.

Figure3 Growth Curves of Empty E. coli Bacterial growth curves in M9 minimal medium with sucrose (suc) or glucose (glu) as the default carbon source, supplemented with CaCl2, or MgSO4 (at 25 h).

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.^[1]
At the same time, the introduction of SacC will cause E. coli to produce a large amount of organic acids, which is exactly the organic carbon source preferred by nitrogen-fixing bacteria (A.caul). The intersection of nutritional structures will help the entire symbiotic system to be more stable and efficient.
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.
[1] Lee, J.W., Choi, S., Park, J.H., Vickers, C.E., Nielsen, L.K., and Lee, S.Y. (2010). Development of sucrose-utilizing Escherichia coli K-12 strain by cloning beta-fructofuranosidases and its application for L-threonine production. Appl Microbiol Biotechnol 88, 905-913. 10.1007/s00253-010-2825-7.

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. Before experiments, we will check the pH value of M9 medium. Also, we want to try a higher sucrose concentration(20 g/L) to see if there will be a more significant difference.
It is likely that the increase in monosaccharide concentration accelerates the carbohydrate metabolism of E. coli due to the rapid decomposition of extracellular sucrose by SacC. Escherichia coli will then quickly produce a large amount of organic acids causing the pH to drop.