Difference between revisions of "Part:BBa K3081053"

Revision as of 02:45, 21 October 2019

AHL receiver device to expression of dCas9 and sgRNA

Quorum sensing system is widely used in gene circuit design as a sensor of cell density. As a transcription factor, LuxR senses AHL and then activate the transcript of plux. In our project, we want dCas9 to express when bacteria density is high. So we put the dCas9 gene under the control of plux, and get this composite part. And we can use golden-gate assembly methods to change the sequence of sgRNA.

Figure1 We transfer this part in E. coli ,BL21(DE3), and culture them in M9 medium containing a series of AHL concentration for 6 hours. Receiver cells show remarkable dose-dependent change in length with addition of AHL.

Figure2 The Pattern diagram of Quorum sensing system.

Figure3 We transferred this part into E. coli, BL21(DE3) as receptor cells, and drop or coat them on the solid medium like A. Then drop 5 μl M9 containing 0.5 Mm IPTG. Solid medium were incubated about 16h. The bacteria density in one drop showed great difference with different distances from IPTG.

A time-scale quorum sensing system can be transformed to spatial-level through a donor/receiver system. The donor cells, which merely express and release AHL, would activate the GFP expression of receiver cells through AHL diffusion. This is validated on the agar plate, by dropping the donor cells in the center and receiver cells around them with different distances. We found a progressive decrease in fluorescence as the receiver cells locate farther from central AHL donor. GFP gene is exchanged for dCas9 with companion of constantly-expressed single guide RNA to establish the qs-CRISPRri system. Through the donor-receiver system, we realized spatial-level control of cell growth. Receiver colonies located nearer to the donor grow much slower than farther ones, which is observed in either dropping or smearing plate.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1321
Illegal XhoI site found at 5538
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 5728
Illegal BsaI.rc site found at 1004
Illegal BsaI.rc site found at 5324

@@ Line 17: / Line 17: @@
 Figure3 We transferred this part into <i>E. coli</i>, BL21(DE3) as receptor cells, and drop or coat them on the solid medium like A. Then drop 5 μl M9 containing 0.5 Mm IPTG. Solid medium were incubated about 16h. The bacteria density in one drop showed great difference with different distances from IPTG.
+A time-scale quorum sensing system can be transformed to spatial-level through a donor/receiver system. The donor cells, which merely express and release AHL, would activate the GFP expression of receiver cells through AHL diffusion. This is validated on the agar plate, by dropping the donor cells in the center and receiver cells around them with different distances. We found a progressive decrease in fluorescence as the receiver cells locate farther from central AHL donor.
+GFP gene is exchanged for dCas9 with companion of constantly-expressed single guide RNA to establish the qs-CRISPRri system. Through the donor-receiver system, we realized spatial-level control of cell growth. Receiver colonies located nearer to the donor grow much slower than farther ones, which is observed in either dropping or smearing plate.