Difference between revisions of "Part:BBa K737007"

Latest revision as of 02:06, 26 September 2012

Constitutive GVPC Generator

"It contains a constitutive promoter, GvpC protein , GFP protein and a terminator. This part can be used as a control of (J23106+P0440+R0040+B0034+GVPC+E0840). It can express GvpC protein and GFP constantly. When it transformed with (J23106+P0412+R0011+B0034+GVPA+E0840), the bacteria may can float with GFP!"

In order to quantitative analyze the expression of gvp gene inside the cell, we design a genetic circuit that could be realized by inserting the gvp gene alongside GFP as a polycistron[1]. Using this part, we can know the expression level of Gvp protein by the detecting the fluorescence of GFP. It will be transcribed into an mRNA and the two genes each have a same promoter (BBa_ J23106). So we can through the expression of GFP to detect gvp gene expression of roughly quantitative. GFP expression can through the determination expression of the specific excitation wavelength of fluorescence value to calculate. [1][2]

We made 2 polycistron circuits of gvpA/C and gfp genes.

Our devices are: Constitutive GvpA and GFP generator: Promoter (J23106) + RBS (B0034) + gvpA + RBS + GFP + terminator (E0840)

Constitutive GvpC and GFP generator: Promoter (J23106) + RBS (B0034) + gvpC + RBS + GFP + terminator (E0840)

Here is the result of SDS-polyacrylamide gel electrophoresis. We can see GFP and GvpC protein directly. Lane 4 is top10 control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper white boxes are in the position about 26KD are GFP protein (25.8KD). The lower white box in the position of about 20KD is GvpC-20 (20KD). Because the MW(molecular weight) of GvpA protein is too small, it’s very difficult to see it on the SDS-polyacrylamide gel. But the expression of GFP suggests it may be expressed. Some papers say that GvpA is difficult to see is that GvpA is too hydrophobic to run out of sample hole.

Figure1.The result of SDS-polyacrylamide gel electrophoresis. Lane 4 is top10 control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper white boxes are in the position about 26KD are GFP protein (25.8KD). The lower white box in the position of about 20KD is GvpC-20 (20KD). Because the GvpA protein can’t be seen on the SDS-polyacrylamide gel electrophoresis, we wanted to use GFP to detect the expression of GvpA. We use a polycistron of gvpA(gvpC) and gfp to detect the RFU. We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control. First, the RFU of these parts are rather high. Second, the E.Coli with these two plasmids can float. Third, we can see GvpC on the SDS-polyacrylamide gel. So, we can see the two parts can express Gvp Protein and work well.

Figure2.We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control.

After getting the plasmids of constitutive GvpA and GFP generator, constitutive GvpC and GFP generator, we transform the two plasmids into E.Coli to see if they can float. As expected, they float! It shows that the gvpA and gvpC genes work. In other word, only two genes (about 700bp) can make bacteria floating!

Reference

1. The gvpA/C cluster of Anabaena flos-aquae has multiple copies of a gene encoding GvpA P. K. Hayes. R. S. Powell Arch Microbiol (1995) 164:50-57

2. Engineered gene circuits Jeff Hasty, David McMillen, & J. J. Collins NATURE VOL 420 14 NOVEMBER 2002

Sequence and Features