Team:Shanghai HS United/Parts Collection
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Part: BBa_K3585003
BBa_K3585003 is a composite part constituting of butyrate synthesis pathway enzymes under constitutive promoter (basic parts BBa_J23100). The butyrate biosynthesis gene cluster can degrade galactose and synthesize butyrate.
Accelerate consumption rate of glucose
When cultured with glucose as the carbon source, the glucose was almost run out till cultured for 8 hours. When the culture medium contained glucose and galactose, the carbon source consumption rate was lower than that of the culture medium containing only glucose. However, the bacteria had butyrate synthesis gene, the consumption rate of glucose was accelerated.
Figure 1. 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.
Part: BBa_K3585006
BBa_K3585006 is a composite part constituting of constitutive promoter (basic parts BBa_J23100) and LacY gene. LacY codes for beta-galactoside permease which regulates the transport of lactose into the cell. BBa_K3585006 is designed to production of beta-galactoside permease on the cell membrane which enables the cell to use lactose as the carbon source.
Support growth of bacteria expressing lacY in galactose
Bacteria with lacY was able to grow using galactose as the unique carbon source in the culture, although the growth rate of the host bacteria is obviously slower than those strains cultured with glucose.
Figure 2. Growth rate of host strains when supplied with different carbon sources. 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.
Accelerate consumption rate of galactose
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.
Figure 3. Consumption rate of galactose. 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.
Part: BBa_K3585007
Based on the experiment information presented above and more related research articles, we came up with the new improved design engineering part BBa_K3585007, that has a butyric acid synthesis pathway along with a lac-Y gene that helps the probiotic to prioritize the ingestion of galactose.
We design a strong promoter J23200 to continuously overexpress lac-Y gene to achieve simultaneous metabolism of glucose and galactose. Continuous expression of butyrate synthesis pathway enzymes through a constitutive promoter, so the strain can degrade galactose and synthesize butyrate. Combining the butyrate synthesis pathway enzyme and lac-Y, the engineered strain can effectively degrade galactose in the presence of glucose.
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