Part:BBa_K2305004
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:
This biobrick is an improvement of the biobrick BBa_J37032 designed by iGEM2006_Imperial.
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.
DNA sequencing
The figure 2 is a comparison with the results of the sequencing. It verified the accuracy of the sequence further.
The OD value and the fluorescence value
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 64
Illegal BsaI.rc site found at 794
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