Part:BBa_K3585006
J23200-lacY
In E. coli, 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 use the strong promoter J23200 to continuously overexpress the lacY protein to achieve simultaneous metabolism of glucose and galactose, thereby improving the efficiency of degrading galactose.
Sequence and Feature
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 820
- 1000COMPATIBLE WITH RFC[1000]
Contribution
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.
Engineering success
The BBa_K3585006 was designed to produce beta-galactoside permease on the cell membrane which enables the cell to use lactose as the carbon source.
To test our design, we made the following experiments and presented the results as follows:
Supporting 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 1. 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.
Accelerating 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 2. 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.
According to the experiments results and figures, the BBa_K3585006 is functional in producing beta-galactoside permease.
Improvement
Previously, team iGEM08_Caltech designed a composite part BBa_S04123, which contained a constitutive expression promoter J23100 and LacY coding sequence. Besides this part, they designed many similar parts, such as BBa_S04108, BBa_S04110, BBa_S04111, BBa_S04112, BBa_S04122, BBa_S03971, BBa_S0455, and etc. They developed an engineered bacteria to convert lactose into glucose and galactose, on the basis of which they aimed at curing lactose intolerance. However, they failed to get transformants. They attributed their unsuccessful experiment results to overexpression of LacY or failed ligation.
After 7 years, group CityU_HK 2015 also aimed to cure lactose intolerance. They used one of the parts designed by group iGEM08_Caltech, BBa_S0455 and made a contribution by adding the transcription data of both LacY and LacZ. However, they only analyzed the function of LacZ without mentioning the corresponding results of LacY. In 2020, our group iGEM20_ Shanghai_HS_United has improved the design and granted our composite part BBa_K3585006 an enhanced function compared with those of parts mentioned above. We aim to construct an engineering strain to degrade galactose and hence provide galactosemia patients with new possible treatment. The first ingenious design is our promoter. We exchange the old part’s promoter J23100 with J23200, whose sequence has been optimized (Fig1).
Fig1 The blast result of part BBa_S04123 and BBa_K3585006.
The second design is our strain. We choose a probiotic E.coli Nissle 1917 as the host strain so that our engineering strain will be edible by galactosemia patients.
The third design is our butyrate synthesis gene cluster. We design a new plasmid that contained the butyrate synthesis gene cluster (our part number: BBa_K3585003). Through transformed BBa_K3585006 and BBa_K3585003 into E.coli Nissle 1917. We successfully degrade galactose. The LacY protein is responsible for helping galactose pass through the cell membrane and the butyrate synthesis cluster is responsible for accelerating the degradation of galactose (Fig2).
Fig2. The difference of design between the old group and our group.
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