Part:BBa_K3256439
Plux+mCherry
This composite part contains Lux promoter, RBS, and mCherry fluorescent protein. More detail about the toxin is shown below.
Introduction
Quorum sensing (QS) is a gene expression system responding to the fluctuation of cell density. This unique system enables bacteria to communicate with each other but in a different way. Unlike we humans communicate through talking, bacteria connect each other by coordinating gene expression. The existence of certain chemical signal such as Acylated Homoserine Lactone (AHL) will indirectly affect whether to express certain genes and thus serves as a way to represent the cell density. In our biobrick, LuxI and LuxR would express constitutively. LuxI would produce AHL, and AHL would bind to LuxR to form an operator complex. The AHL-LuxR complex will trigger the lux promoter, and mCherry fluorescent protein will express.
Result
Cloning
The Figure 1 is the electrophoresis results of the PCR products with 1Kb marker on the left side and target gene on the right side. The lengths are labeled beside each band.
Figure 1: DNA electrophoresis of part BBa_K3256437 with BBa_K3256439.
As the figure we show above, we successfully cloned QS gene into the E. coli.
Functional Test
Then, we tested the function by measuring the fluorescence intensity and the optical density of the transformed E. coli together with controlled group. We put our sample into the CLARIOstar® Plus plate reader. For each sample, we applied triple replication in order to get more precise data.
In the beginning, the fluorescence intensity remained relatively at low level. At about 150 minutes after IPTG induction, the fluorescence increased sharply and then remained in high intensity. The result was in accordance with our quorum sensing design. The sharp increase of fluorescence intensity occurred at O.D. value at approximately 0.2, which was expected to be too early. This phenomenon was observed in multiple tests. We expected that the promoter Plux was loose and expressed well with little concentration of AHL. For future work of integrating Quorum Sensing system in the bioassay, we may have to change the promoter or add operon for the tighter control.
Composite Parts Cloning
After we had done the function test of toxin gene and the QS gene circuit, we co-transformed the two vectors into E. coli BL21(DE3).
Figure 3 was the electrophoresis results of the PCR products with the marker on the left side and target gene on the right side. The lengths are labeled beside each band.
Figure 3: A. DNA electrophoresis of part BBa_K3256005 and BBa_K3256437 with BBa_K3256439. B. DNA electrophoresis of
part BBa_K3256008 and BBa_K3256437 with BBa_K3256439.
We successfully co-transformed two different toxin genes respectively with QS gene into the E. coli. So,
we will use them to do the following experiment.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 434
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 434
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 434
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 434
- 1000COMPATIBLE WITH RFC[1000]
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