Difference between revisions of "Part:BBa K887000"

Revision as of 17:20, 30 September 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.

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_K539742) 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 6049
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 6938
Illegal AgeI site found at 3158
Illegal AgeI site found at 4143
1000
INCOMPATIBLE 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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K887000 short</partinfo>
-In traditional genetic engineering method, we use strong promoter to initiate our gene expression, so that E.coli will overexpress the proteins we need in synthetic pathway. However, this overexpression of target proteins will cause E.coli wastes its limited growth resources, so the activity and performance of the enzymes may be too low. In this situation, the synthetic pathway is unbalanced, and the production of isobutanol will not be optimum. This is also a problem in the production of isobutanol which is poisonous to E.coli.<br>
+[[Image:Butanol.5.jpg]]<br>
-We make a big change to improve the traditional gene.<br>
-To solve the problem, we need to adjust the expression of the genes. We use the cellulose to produce glucose. Glucose can be catalyzed into isobutanol through afterward enzymes- Alss, Ilvc, Ilvd,and Kivd step by step.But isobutanol and isobutyraldehyde have biological toxicity and also can denature proteins,so we design a temperature control system to stop at the step that produce 2-Ketoisovalerate. We accumulate lots of the non-toxic intermediate as the precursor, 2-Ketoisovalerate, to a certain amount, and then convert the entire non-toxic precursor into the product, isobutanol, all at once. The toxic isobutanol and isobutyraldehyde will produce at the last minute and cause less harm.<br>
-[[Image:Butanol.5.jpg]]
+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.<br>
-In our design, we put E.coli in 37°C and control the pathway by stopping the pathway when it reaches 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 isobutyraldehyde at once. Then isobutyraldehyde will been produced into isobutanol with the enzyme of the widetype E.coli.<br>
-But we are not satisfied.<br>
-We encode four zinc fingers in front of each enzyme.Zinc fingers could tightly bind to specific DNA or RNA sequence. We replace the zinc fingers' functional domains with our enzymes to create fusion proteins to put the enzymes in order.  When the intermediates are produced, it could have the next reaction as quickly as possible. <br>
-[[image:picture.jpg|850px]]
+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.
+[[image:picture.jpg|850px]]<br>
+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_K539742]]) to finish the whole pathway, the productivity of isobutanol will be higher.<br>
+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.