Difference between revisions of "Part:BBa K2305004"

 
 
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pcat promtor controlled plux and GFP
 
pcat promtor controlled plux and GFP
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===Function===
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Pcat+RBS+GFP+T
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The purpose of this biobrick is to produce as much green fluorescence as possible, and ensure a strong GFP expression in E.coli.
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This biobrick was made from 2 parts:
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1.[https://parts.igem.org/Part:BBa_K081005 BBa_K081005]
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2.[https://parts.igem.org/Part:BBa_J37032 BBa_J37032]
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'''This biobrick is an improvement of the biobrick [https://parts.igem.org/Part:BBa_J37032 BBa_J37032] designed by iGEM2006_Imperial.'''
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[[Image: BIT_Figure_gold1.png |thumb|center|500px|BBa_J37032 and BBa_K2305004's comparison chart]]
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To test the correct functioning of this biobrick, we have performed three different types of experiment：
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1.Determination of plasmid molecular weight by agarose gel electrophoresis；
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2.DNA sequencing ensures its accuracy；
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3.Measured the OD value and the green fluorescence value by microplate reader.
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===Agarose gel electrophoresis===
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We calculated the new part’s base number to be 1007bp. The figure1 is the result of agarose gel electrophoresis. We can make a preliminary judgment that the sequences are correct.
 +
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[[Image: BIT Figure gold 1-1 ehp agar.png|thumb|center|200px|BBa_K2305004's result of agarose gel electrophoresis]]
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===DNA sequencing===
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The figure 2 is a comparison with the results of the sequencing. It verified the accuracy of the sequence further.
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[[Image: BIT Figure gold 1-2 cexu.jpeg|thumb|center|500px|BBa_K2305004's result of DNA sequencing]]
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===The OD value and the fluorescence value===
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[[Image: BIT Figure 2305004 1wwj.png|thumb|left|500px|Fig.3]]
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[[Image: BIT Figure 2305004 2wwj22.png|thumb|none|500px|Fig.4]]<br clear="all">
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The iGEM_BIT2017 added a strong promoter on the basis of the previous part. We used the microplate reader to compare their OD value and the fluorescence value under the same experimental conditions (Fig.3;Fig.4). As the figure demonstrates, in the first eight hours the OD600 value increased gradually, the BBa_K2305004 expressed high level expression in E.coli than the previous part.We can see that under the same control variable, the new part of the fluorescence is about 80 times than the old part. It can be an expression part is improved over the initial design.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 11:33, 29 October 2017


pcat controlled plux and GFP

pcat promtor controlled plux and GFP

Function

Pcat+RBS+GFP+T

The purpose of this biobrick is to produce as much green fluorescence as possible, and ensure a strong GFP expression in E.coli. This biobrick was made from 2 parts:

1.BBa_K081005

2.BBa_J37032

This biobrick is an improvement of the biobrick BBa_J37032 designed by iGEM2006_Imperial.

BBa_J37032 and BBa_K2305004's comparison chart

To test the correct functioning of this biobrick, we have performed three different types of experiment：

1.Determination of plasmid molecular weight by agarose gel electrophoresis；

2.DNA sequencing ensures its accuracy；

3.Measured the OD value and the green fluorescence value by microplate reader.

Agarose gel electrophoresis

We calculated the new part’s base number to be 1007bp. The figure1 is the result of agarose gel electrophoresis. We can make a preliminary judgment that the sequences are correct.

BBa_K2305004's result of agarose gel electrophoresis

DNA sequencing

The figure 2 is a comparison with the results of the sequencing. It verified the accuracy of the sequence further.

BBa_K2305004's result of DNA sequencing

The OD value and the fluorescence value

Fig.3
Fig.4

The iGEM_BIT2017 added a strong promoter on the basis of the previous part. We used the microplate reader to compare their OD value and the fluorescence value under the same experimental conditions (Fig.3;Fig.4). As the figure demonstrates, in the first eight hours the OD600 value increased gradually, the BBa_K2305004 expressed high level expression in E.coli than the previous part.We can see that under the same control variable, the new part of the fluorescence is about 80 times than the old part. It can be an expression part is improved over the initial design.




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 64
    Illegal BsaI.rc site found at 794