Difference between revisions of "Part:BBa K887000"
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In our design, we put E.coli in 37°C and control the pathway by stopping it when reaching the step to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate. Then under specific thermal control that moves E.coli to 30°C, the mechanism would continue to express another intermediate(isobutyraldehyde) at once. Then isobutyraldehyde will been produced into isobutanol with the enzyme of the widetype E.coli.<br> | In our design, we put E.coli in 37°C and control the pathway by stopping it when reaching the step to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate. Then under specific thermal control that moves E.coli to 30°C, the mechanism would continue to express another intermediate(isobutyraldehyde) at once. Then isobutyraldehyde will been produced into isobutanol with the enzyme of the widetype E.coli.<br> | ||
| − | More details and results, please refer to [ | + | More details and results, please refer to [https://parts.igem.org/Part:BBa_K539691:Experience BBa_K539691 Experience] from 2011 NCTU_formosa iGEM team. |
Revision as of 12:43, 1 October 2012
Plac+B0034+zif268+alsS+B0034+PBSII+ilvC+B0034+HIVC+ilvD+37℃ induced RBS+tetR+double terminator
This is the Biobrick we made last year. We designed a temperature control system so that we can prevent E.coli from dying early because of rising concentration of isobutanol.
In our design, we put E.coli in 37°C and control the pathway by stopping it when reaching the step to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate. Then under specific thermal control that moves E.coli to 30°C, the mechanism would continue to express another intermediate(isobutyraldehyde) at once. Then isobutyraldehyde will been produced into isobutanol with the enzyme of the widetype E.coli.
More details and results, please refer to BBa_K539691 Experience from 2011 NCTU_formosa iGEM team.
In 2012, we aimed to promote the yield of isobutanol production with our modified E.coli.So, we encode three kinds of zinc finger binding domain genes in front of each enzyme.
Zinc finger proteins contain a DNA binding domain and a functional domain. DNA binding domain could recognize specific DNA sequence, which called DNA program. Zinc fingers could tightly bind to specific DNA or RNA sequence. Thus, we connect the zinc fingers' functional domains with our enzymes to create fusion proteins for aligning the enzymes in order when the pathway is in progress. By doing so, the enzymes would no longer disperse around the cell. It means when the intermediates are produced, they could have the next reaction as quickly as possible.
As we ligate this Biobrick(Part:BBa_K887000) with another one(Part:BBa_K539741) to finish the whole pathway, the productivity of isobutanol will be higher.
Besides, in order to assemble a production line in E.coli, we have to add DNA program as well as our fusion protein genes in to E.coli. Fortunately, the design of our biobricks has the same order of zinc finger as 2010 Slovenia iGEM team, so we decided to use their DNA program (Part:BBa_K323066) instead of synthesizing one.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 6049
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 6938
Illegal AgeI site found at 3158
Illegal AgeI site found at 4143 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2744
Illegal BsaI site found at 5835
Illegal BsaI site found at 6092
Illegal BsaI.rc site found at 836
Illegal BsaI.rc site found at 1430
Illegal BsaI.rc site found at 3563