Difference between revisions of "Part:BBa K539691:Experience"

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===Applications of BBa_K539691===
 
===Applications of BBa_K539691===
  
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== '''Experience by 2011 iGEM NCTU_FORMOSA''' ==
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== '''Experience by 2012 iGEM NCTU_FORMOSA''' ==
  
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[[Image:butano_figure1.jpg]]
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Our team modified [[Part:BBa_K539691]] from 2011 NCTU_ Formosa iGEM team by adding three kinds of zinc finger binding domain genes preceded in three protein<br>genes(alss, ilvC and ilvD) respectively. Using characteristic of zinc finger, we can improve the result without adding zinc finger binding domain.<br>More detail information, please refer to [[Part:BBa_K887000]]<br>
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[[image:picture.jpg|850px]]
  
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'''Figure.1 Firstly , we ligase B0030 with ilvC.'''
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== '''Experience by 2011 iGEM NCTU_FORMOSA''' ==
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'''Analysis of the isobutanol production in different temperature'''
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[[Image:butanol_figure2.jpg]]
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'''Figure.2 At the sametime , we ligase B0030 with ilvD.'''
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[[Image:butanol_figure3.jpg]]
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'''Figure.3 Meanwhile , we ligase B0032 with Alss.'''
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https://static.igem.org/mediawiki/2011/6/66/Butanol-12.png
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<br><b> Figure 1.</b><br>
  
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(A.)<b> Mean fluorescence intensity (MEFL) at 30℃,37℃,42℃</b>
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The vertical axis is mean fluorescence(MEFL), and the horizontal axis is time.
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We can regard GFP as our target protein,Kivd. Under different temperature, a tendency shows that the production of GFP proteins will increase in lower incubation temperature. The result shows that our low-temperature released device can work at these three temperatures. In this way, we can do the experiment as the same, but changing the genes of our butanol circuit.<br>
  
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[[Image:butanol_figure4.jpg]]
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(B.)<b> Production of isobutanol at 30℃,37℃,42℃</b>
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Cotransform [https://parts.igem.org/Part:BBa_K539691 BBa_K539691] and [https://parts.igem.org/Part:BBa_K539742 BBa_K539742] into DH5α, so the expression of alss, ilvC, ilvD and kivd are under the control of temperature. Coupling A. and B. diagram, the Kivd protein expression and the production of isobutanol are under temperature control, and both of their production will increase in lower incubation temperature. Therefore, we can conclude that the Kivd protein expression is related to the production of isobutanol.
  
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'''Figure.4 To make sure we obtain the correct part , colony PCR is needed.'''
 
  
  
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[[Image:butanol_figure5.jpg]]
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'''GC (Gas Chromatography) graph'''
  
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'''Figure.5 Secondly , we ligase Plac with B0032+Alss'''
 
  
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[[Image:GC13.jpg]]
  
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[[Image:butanol_figure6.jpg]]
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'''Figure 2. Cotransform two circuits, [[Part:BBa_K539691]] and [[Part:BBa_K539742]], into DH5α. Incubate DH5α at 37℃ until OD reaches 0.5. Before we switch the temperature to 30℃, we analyze the chemical composition of medium by GC.'''
  
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'''Figure.6 Thirdly , we ligase Plac+B0032+Alss with B0030+ilvC'''
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[[Image:GC14.jpg]]
  
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'''Figure 3. Incubate DH5α at 37℃ until OD reaches 0.5, then we switch the temperature to 30℃ incubating for 24hr and analyze the chemical composition of medium by GC.'''
  
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[[Image:butanol_figure7.jpg]]
 
  
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'''Figure.7 At the same time , we ligase B0030+ilvD with RNA thermometer+tetR+double terminator'''
 
  
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'''Comparison of isobutanol production under low-temperature released device or not'''
  
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[[Image:butanol_figure8.jpg]]
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[[Image:butanol5.jpg]]
  
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'''Figure.8 Finally , we ligase the whole circuit:promoter(lacI regulated)+Alss+ilvC+ilvD(each preceded by own RBS)and RNA thermometer+terminator'''
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'''Figure 4. Control group (non-temperature controlled device): BBa_K539671 and BBa_K539742 Experimental group(low-temperature released device): BBa_K539691 and BBa_K539742 GC graph in 30℃, 37℃, 42℃
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'''  
  
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We cotransformed [[Part:BBa_K539691]] and [[Part:BBa_K539742]] into DH5α, so the expression of ''Alss, ilvC, ilvD and kivd'' are under the control of temperature.
 
  
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And to realize whether ''Alss,ilvC and ilvD'' have any impact to the production of isobutanol, we compare the circuit which contain ''Alss,ilvC and ilvD'' genes with the one with only a single ''kivd'' gene in it.
 
  
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[[Image:Butanol-11.png]]
 
 
===User Reviews===
 
===User Reviews===
 
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Latest revision as of 13:57, 25 September 2012

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K539691

Experience by 2012 iGEM NCTU_FORMOSA

Our team modified Part:BBa_K539691 from 2011 NCTU_ Formosa iGEM team by adding three kinds of zinc finger binding domain genes preceded in three protein
genes(alss, ilvC and ilvD) respectively. Using characteristic of zinc finger, we can improve the result without adding zinc finger binding domain.
More detail information, please refer to Part:BBa_K887000
Picture.jpg

Experience by 2011 iGEM NCTU_FORMOSA

Analysis of the isobutanol production in different temperature

Butanol-12.png
Figure 1.

(A.) Mean fluorescence intensity (MEFL) at 30℃,37℃,42℃ The vertical axis is mean fluorescence(MEFL), and the horizontal axis is time. We can regard GFP as our target protein,Kivd. Under different temperature, a tendency shows that the production of GFP proteins will increase in lower incubation temperature. The result shows that our low-temperature released device can work at these three temperatures. In this way, we can do the experiment as the same, but changing the genes of our butanol circuit.

(B.) Production of isobutanol at 30℃,37℃,42℃ Cotransform BBa_K539691 and BBa_K539742 into DH5α, so the expression of alss, ilvC, ilvD and kivd are under the control of temperature. Coupling A. and B. diagram, the Kivd protein expression and the production of isobutanol are under temperature control, and both of their production will increase in lower incubation temperature. Therefore, we can conclude that the Kivd protein expression is related to the production of isobutanol.


GC (Gas Chromatography) graph


GC13.jpg

Figure 2. Cotransform two circuits, Part:BBa_K539691 and Part:BBa_K539742, into DH5α. Incubate DH5α at 37℃ until OD reaches 0.5. Before we switch the temperature to 30℃, we analyze the chemical composition of medium by GC.

GC14.jpg

Figure 3. Incubate DH5α at 37℃ until OD reaches 0.5, then we switch the temperature to 30℃ incubating for 24hr and analyze the chemical composition of medium by GC.


Comparison of isobutanol production under low-temperature released device or not

Butanol5.jpg

Figure 4. Control group (non-temperature controlled device): BBa_K539671 and BBa_K539742 Experimental group(low-temperature released device): BBa_K539691 and BBa_K539742 GC graph in 30℃, 37℃, 42℃


User Reviews

UNIQ7f12d312afad0dee-partinfo-00000000-QINU UNIQ7f12d312afad0dee-partinfo-00000001-QINU