Team:Shanghai HS United/Engineering
Research -> Image -> Design
As a primary part of lactose, galactose is found in most galactose products and many baby formulas. But it is dangerous for galactosemia patients to eat galactose products. Galactosemia is an autosomal recessive disorder disease leading to abnormal in using galactose to produce energy. The patients have galactose 1-phosphate uridyltransferase (GALT) deficiency. There are three kinds of this disease: Type 1 galactosemia occurs in 1 out of every 6000-10000 babies, Type 2 galactosemia is less common than type 1 and occurs in 1 out of every 100,000 babies, Type 3 and Duarte variant galactosemia are very rare. Long-term outcome for older patients with galactosemia remains problematic although neonatal hepatotoxic syndrome is prevented by newborn screening and galactose restriction.
Based on the information presented above, we came up with the plan using engineering probiotics to degrade and digest the galactose in galactosemia patients. Our goal is to construct an engineering E. Coli Nissle 1917, which carries a butyrate synthesis path gene cluster with J23100 promoter (Part: BBa_K3585003). The engineering bacterial can transform galactose to butyrate, that is beneficial for galactosemia patients.
Build -> Test
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.
Learn -> Research -> Improved design
According to the articles, the presence of glucose inhibits the synthesis of many catabolic enzymes, a phenomenon called "glucose effect" or "glucose inhibition". Studies have shown that overexpression of LacY can eliminate the glucose effect. We design to overexpress lacY protein to achieve simultaneous metabolism of glucose and galactose, thereby increasing the efficiency of degrading galactose.
The second round of Build -> Test
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.
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.
The second round of Learn -> Research -> Improved design
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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