Difference between revisions of "Part:BBa K533006:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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===Applications of BBa_K533006===
 
===Applications of BBa_K533006===
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This is the expression device of mCherry.
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When IPTG is added, strong expression of mCherry can be detected. However, mCherry cannot be properly folded above 20 centigrade, as shown by our experimental results.
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{| align="center" style="border: none; background: none; border-spacing: 0;text-align:center;margin-left: 100px;"
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! style="border: 1px solid #39F;padding: 5px;" | Group
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| style="border: 1px solid #39F;padding: 5px;" | 1
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| style="border: 1px solid #39F;padding: 5px;" | 2
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|-
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| style="border: 1px solid #39F;padding: 5px;" |  Volume
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| style="border: 1px solid #39F;padding: 5px;" colspan="2" | 3ml
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|-
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| style="border: 1px solid #39F;padding: 5px;"  rowspan="3" | IPTG(mM)
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| style="border: 1px solid #39F;padding: 5px;"  colspan="2" | 0.1
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|-
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| style="border: 1px solid #39F;padding: 5px;"  colspan="2" | 0.5
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|-
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| style="border: 1px solid #39F;padding: 5px;"  colspan="2" | 1.0
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|-
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| style="border: 1px solid #39F;padding: 5px;" | Temperature
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| style="border: 1px solid #39F;padding: 5px;" |  30<html><sup>o</sup>C</html>
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| style="border: 1px solid #39F;padding: 5px;" |  18<html><sup>o</sup>C</html>
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|-
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| style="border: 1px solid #39F;padding: 5px;" |  Duration
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| style="border: 1px solid #39F;padding: 5px;" |  12h
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| style="border: 1px solid #39F;padding: 5px;" |  12h
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|-
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| style="border: 1px solid #39F;padding: 5px;" |  Result
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| style="border: 1px solid #39F;padding: 5px;" |  Media remained yellowish.<html><br /></html> E. coli white
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|  style="border: 1px solid #39F;padding: 5px;" | Media turned purple. <html><br /></html>E. coli cherry pink
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|}
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After Pro-rich mCherry expression, significant color change can be observed (bacteria cells turned cherry red as a result of mCherry expression)
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[[Image:Thuexp_cherryexp.png]]
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''From left to right, IPTG concentration: 0, 0.1mM, 0.5mM, 1mM. All induced at 18 centigrade.''
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We purified the protein by Ni column. After loading the cell lysate onto the column, we first washed the column with 5mM immidazole and then 20mM immidazole. The elute is a bit pink but the majority of protein is still on the column.
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The remaining protein is eluted by 200mM immidazole solution.
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[[Image:Thuexp_cherrysds2.png]]
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{| style="background:none; "
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| style="width: 30px;"|
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| style="width: 35px;"| 1
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| style="width: 35px;"|  2
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| style="width: 35px;"|    3
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| style="width: 35px;"|    4
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| style="width: 35px;"|      5
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| style="width: 20px;"|      6
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| style="width: 55px;"| Marker
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| style="width: 25px;"| 7
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| style="width: 35px;"| 8
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| style="width: 35px;"| 9
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| style="width: 20px;"| 10
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| style="width: 40px;"| Marker
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|}
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{| style="background:none;"
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| style="width: 180px;" | ''1. Sonicated ''
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| style="width: 180px;" | ''2. Supernatant''
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| style="width: 180px;" | ''3. Pellet''
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|-
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| ''4. Flowthrough ''
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| ''5. Buffer wash''
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| ''6. Resin''
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|-
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| ''7. 5mM Elution''
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| ''8. 20mM Elution''
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| ''9. Resin 2''
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|-
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| ''10. 200mM Elution''
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|}
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 16:14, 5 October 2011

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_K533006

This is the expression device of mCherry.

When IPTG is added, strong expression of mCherry can be detected. However, mCherry cannot be properly folded above 20 centigrade, as shown by our experimental results.

Group 1 2
Volume 3ml
IPTG(mM) 0.1
0.5
1.0
Temperature 30oC 18oC
Duration 12h 12h
Result Media remained yellowish.
E. coli white 		Media turned purple.
E. coli cherry pink

After Pro-rich mCherry expression, significant color change can be observed (bacteria cells turned cherry red as a result of mCherry expression)

Thuexp cherryexp.png

From left to right, IPTG concentration: 0, 0.1mM, 0.5mM, 1mM. All induced at 18 centigrade.

We purified the protein by Ni column. After loading the cell lysate onto the column, we first washed the column with 5mM immidazole and then 20mM immidazole. The elute is a bit pink but the majority of protein is still on the column.

The remaining protein is eluted by 200mM immidazole solution.

Thuexp cherrysds2.png

1 2 3 4 5 6 Marker 7 8 9 10 Marker
1. Sonicated 2. Supernatant 3. Pellet
4. Flowthrough 5. Buffer wash 6. Resin
7. 5mM Elution 8. 20mM Elution 9. Resin 2
10. 200mM Elution

User Reviews

UNIQ8ba4a123871ee8c7-partinfo-00000004-QINU UNIQ8ba4a123871ee8c7-partinfo-00000005-QINU