Part:BBa_S04123
J23100:S04107
Construction intermediate
Improvement made by Shanghai_HS_United 2020
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 in 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 the parts mentioned above. We aim to construct an engineering strain to degrade galactose and hence provide galactosemia patients with a 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).
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).
Sequence and Features
- 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]
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