Difference between revisions of "Part:BBa K887000"
 
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<partinfo>BBa_K887000 short</partinfo>
 
<partinfo>BBa_K887000 short</partinfo>
  
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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>
 
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We make a big change to improve the traditional gene.<br>
 
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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>
 
  
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[[Image:Butanol.5.jpg]]
 
  
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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>
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The following figure is isobutanol biosynthesis pathway.<br>                      
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But we are not satisfied.<br>
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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>
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[[image:picture.jpg|850px]]
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[[image:picture0.jpg|300px]]      [[image:picture8.jpg|150px]]      [[image:picture7.jpg|400px]]<br>
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As for this biobrick([[Part:BBa_K887000]]), we encoded three necessary protein genes, alsS, ilvC and ilvD, into our host(DH5a).
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If we ligate or cotransform this biobrick with another one([[Part:BBa_K539741]]), which encoded last protein gene, kivd, the synthesis pathway could be done.
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Theses enzymes are crucial for producing butanol.(Reference: Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.)<br>
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The figure below can proof that only when these two biobricks(means the four enzymes) were connected will isobutanol be produced.
 +
 
 +
[[image:picture1.jpg|400px]]<br>
 +
 
 +
It means that our modified E.coli will convert glucose(may from defoliation or kitchen waste) into isobutanol.
 +
However, according to many researches, we know that isobutanol is toxic to E.coli.
 +
This factor is really restrain the yield of isobutacol when using traditional genetic engineering method to produce isobutanol.<br>
 +
To solve this problem, we need to adjust the expression of the genes.
 +
In the method we designed last year, we control the pathway by stopping the mechanism when it reaches the step to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate, then under specific thermal control, the mechanism would continue to express. The advantage of our method is that the precursors are much less toxic for E.coli than our target product (isobutanol) is. We then Apply this method to our project. We first 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.
 +
 
 +
[[image:picture10.jpg|850px]]<br>
 +
 
 +
About specific thermal control, we use (37℃ induced RBS+tetR+double terminator) as our controller.
 +
When environmental temperature is higher than 37℃, tetR protein gene will be translated.
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And tetR protein will repress Ptet promoter.
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In this way, [[Part:BBa_K539741]] will not be expressed, and if we keep the temperature over 37℃, we can accumulate the intermediate, 2-Ketoisovalerate, which is non-toxic for E.Coli.
 +
Until having enough 2-Ketoisovalerate, we change temperature below 37℃.
 +
With this switch, we can gain our final product, isobutanol without killing E.coli at early stage.<br>
 +
 
 +
More details and results about our temperature control system, please refer to [[Part:BBa_K539691]] from 2011 NCTU_formosa iGEM team.<br>
 +
 
 +
 
 +
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([https://parts.igem.org/wiki/index.php?title=Part:BBa_K887000 Part:BBa_K887000]) with another one([[Part:BBa_K539741]]) 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.<br>
 +
 
 +
However, after sequencing, we found that there is a deletion of a base pair in the zif268 biding site.
 +
So, we designed a complementary primer to fix it by PCR point mutation technique.<br>
 +
Finally, we got correct DNA program([[Part:BBa_K887011]]).
  
  

Latest revision as of 16:41, 2 October 2012

Plac+B0034+zif268+alsS+B0034+PBSII+ilvC+B0034+HIVC+ilvD+37℃ induced RBS+tetR+double terminator


The following figure is isobutanol biosynthesis pathway.

Picture0.jpg Picture8.jpg Picture7.jpg


As for this biobrick(Part:BBa_K887000), we encoded three necessary protein genes, alsS, ilvC and ilvD, into our host(DH5a). If we ligate or cotransform this biobrick with another one(Part:BBa_K539741), which encoded last protein gene, kivd, the synthesis pathway could be done. Theses enzymes are crucial for producing butanol.(Reference: Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.)

The figure below can proof that only when these two biobricks(means the four enzymes) were connected will isobutanol be produced.

Picture1.jpg

It means that our modified E.coli will convert glucose(may from defoliation or kitchen waste) into isobutanol. However, according to many researches, we know that isobutanol is toxic to E.coli. This factor is really restrain the yield of isobutacol when using traditional genetic engineering method to produce isobutanol.
To solve this problem, we need to adjust the expression of the genes. In the method we designed last year, we control the pathway by stopping the mechanism when it reaches the step to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate, then under specific thermal control, the mechanism would continue to express. The advantage of our method is that the precursors are much less toxic for E.coli than our target product (isobutanol) is. We then Apply this method to our project. We first 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.

Picture10.jpg

About specific thermal control, we use (37℃ induced RBS+tetR+double terminator) as our controller. When environmental temperature is higher than 37℃, tetR protein gene will be translated. And tetR protein will repress Ptet promoter. In this way, Part:BBa_K539741 will not be expressed, and if we keep the temperature over 37℃, we can accumulate the intermediate, 2-Ketoisovalerate, which is non-toxic for E.Coli. Until having enough 2-Ketoisovalerate, we change temperature below 37℃. With this switch, we can gain our final product, isobutanol without killing E.coli at early stage.

More details and results about our temperature control system, please refer to Part:BBa_K539691 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.

Picture.jpg

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.

However, after sequencing, we found that there is a deletion of a base pair in the zif268 biding site. So, we designed a complementary primer to fix it by PCR point mutation technique.
Finally, we got correct DNA program(Part:BBa_K887011).


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