Team:Shanghai HS United/Result
Results
E. Coli Nissle 1917 expression system
(1) E. Coli Nissle 1917 fermentation system
pMTL83151-J23100-Butyrate and p15A-J23200-lacY were co-transformed into E. Coli Nissle 1917 for butyrate fermentation.
(2) The fermentation was carried out at 37℃, and we test several characters at different time. In our project, we also test the consumption rate when supplied with different carbon sources such as glucose and galactose.
Results and conclusions
1. measure the growth rate of the host strains
Fig1. the growth rate of host strains when supplied with different carbon sources
Note: each of the strains we have a repeat. Glu represents that we use glucose as the carbon source while fermentation; Gal represents that we use galactose as the carbon source while fermentation; GG represents that we use both glucose and galactose as the carbon source while fermentation; 1917 represents wild type strains; pButy represents that the host strain contained pMTL83151-J23100-butyrate plasmids and expressed butyrate synthesis gene cluster; placY represents that the host strain contained p15A-J23200-lacY plasmids and expressed lacY gene; 1 and 2 represent for sample repeats.
During fermentation, when we use galactose as the unique carbon source in the culture, the growth rate of the host bacteria is obviously slower than those strains cultured with glucose. After cultured for over 8 hours, most of the strains cultured with glucose entered stable phase. However, strains cultured with galactose stepped into log phase. When cultured with glucose or both of glucose and galactose, the difference of the growth rates of the host strains is insignificant. Strains expressed butyrate synthesis gene cluster did not grow in medium containing only galactose.
2. measure the consumption rate of glucose
Fig2. the concentration of glucose when supplied with glucose
Note: each of the strains we have a repeat. Glu represents that we use glucose as the carbon source while fermentation; GG represents that we use both glucose and galactose as the carbon source while fermentation; 1917 represents wild type strains; pButy represents that the host strain contained pMTL83151-J23100-butyrate plasmids and expressed butyrate synthesis gene cluster; placY represents that the host strain contained p15A-J23200-lacY plasmids and expressed lacY gene; 1 and 2 represent for sample repeats.
During fermentation, when cultured with glucose or both of glucose and galactose, the glucose was almost run out till cultured for 8 hours. The With the growth of the bacteria, the carbon source consumption rate increased. When the bacteria growth entered the logarithmic phase, the glucose consumption rate was the highest. However, when the culture medium contained glucose and galactose, the carbon source consumption rate was lower than that of the culture medium containing only glucose. We speculated that glucose, as the dominant carbon source, was more likely to be catabolized during fermentation. Different carbon sources were almost consumed when fermentation lasted for 8 hours, so we believe that the fermentation strain entered a stable period around 8 hours was related to carbon source consumption.
3. measure the consumption rate of galactose
Fig3. the concentration of glucose when supplied with galactose
Note: each of the strains we have a repeat. Gal represents that we use galactose as the carbon source while fermentation; GG represents that we use both glucose and galactose as the carbon source while fermentation; 1917 represents wild type strains; pButy represents that the host strain contained pMTL83151-J23100-butyrate plasmids and expressed butyrate synthesis gene cluster; placY represents that the host strain contained p15A-J23200-lacY plasmids and expressed lacY gene; 1 and 2 represent for sample repeats.
During fermentation, When the medium contained only galactose, the host bacteria expressing lacY gene began to consume lactose at 4 hours, indicating that lacY gene could promote the consumption of galactose by the host bacteria. However, the host bacteria expressing the butyric acid synthesis gene cluster and the wild-type strains did not grow, so that the galactose content in the culture medium did not change. In addition, when the medium contained two carbon sources, glucose and galactose, galactose was almost consumed after fermentation for about 8 hours. We speculated that the presence of glucose might contribute to the normal growth of the bacteria, thus promoting galactose consumption. In the experimental results, one wild-type strain grew normally in the medium containing only galactose and consumed galactose, which we speculated might be contaminated. The consumption rate of galactose in the medium also increased at about 4 hours, which was directly related to the host bacteria entering the logarithmic phase at this time.
4. measure the consumption rate of fermentation production--acetic acid
Table1. the concentration of acetic acid while fermentation
Note: each of the strains we have a repeat. Gal represents that we use galactose as the carbon source while fermentation; GG represents that we use both glucose and galactose as the carbon source while fermentation; 1917 represents wild type strains; pButy represents that the host strain contained pMTL83151-J23100-butyrate plasmids and expressed butyrate synthesis gene cluster; placY represents that the host strain contained p15A-J23200-lacY plasmids and expressed lacY gene; 1 and 2 represent for sample repeats. The numbers in red in the table is abnormal may be caused by some reasons we did not find out.
According to Table1, during fermentation, the content of acetic acid is slowly increased, verified that strains in our project can transfer carbon sources to other metabolites. Since acetic acid is a normal product of the metabolic cycle, we can detect an increase in the amount of acetic acid in the culture.
5. measure the consumption rate of fermentation production—butyrate
In our experimental, we did not detect butyric acid production. As previous studies have shown that butyric acid production can be detected when overexpressing butyric acid synthesis gene clusters in Clostridium paniculata, researchers had proved that this gene cluster can promote the host bacteria fermentation and transfer carbon source substances in the culture medium to butyric acid. Therefore, we speculated that butyric acid production was not detected in the fermentation process in E. coli, possibly because the butyric acid synthesis gene cluster was too long to fully expressed, or the expressed protein was not folded correctly. The reasons for this need to be explored further.
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