Difference between revisions of "Part:BBa K2553009"

Latest revision as of 21:53, 17 October 2018

pLuxlacO_superfolderGFP

It is used as the QS signal receptor/reporter module with yemGFP under the control of the synthetic lux promoter PluxlacO to evaluate the synthetic lux system. It can not be found in nature.

It can be used to detect the availability of upstream quorum sensing system. When activated by QS autoinducers, a green fluorescence can be detected as the transcription of GFP is initiated. Using the QS system to control transcription can improve both the efficiency and stability of the target protein's transcription. The autoinducing mechanism saves the effort of using artificial inducers such as IPTG, and reduced steps also mean fewer things can go wrong. Also, this system is self-regulating, which means that the colony will automatically adjust to ensure stable production without the need for interference from researchers.

For those aiming to work with QS system, this construction offers an easy way to evaluate the performance of the system.

Characterization by ultraviolet

1.Amplification DNA amplification it with BBa K2553007.

2. Double Enzyme Digestion (1) Conduct Double Enzyme Digestion on the fragments and original PET-28a, and operate electrophoresis. The target bands are collected and recovered. (2) The bands are mixed with the pEt 28-a plasmid as a ratio of 9:1, and the instruction were carried out using T4 ligase kit at 16 degrees for 1-4 hours. (3) Then, the ligated product can be directly transformed into a BL-21 expression strain.

3. Transfection The plasmid was diluted 1000-fold and 10000-fold, and the competent bacteria BL-21 was added for 30 minutes in an ice bath. Then we heated the bacteria at 42 degrees for one minute, and ice-bathed for five more minutes. After that, we added 400 ml of LB without Kana antibiotic and shook the bacterial fluid. Finally, the bacteria were sprayed on a petri dish with kana antibiotics.

4. Bacterial Fluid PCR to filter positive clones. To insure the plasmids were successfully transfected, we conducted bacterial fluid PCR. Our groupmates take ten 1.5 ml EP tubes, add 200 μl of kana resistant medium separately, directly draw a single clone with a small pipette tip and push the TIP directly into an EP tube. We covered the EP tube and shake the bacteria at 37 degrees for 2-3 hours. Then 1μl of the bacterial solution was used as a templte to identify the PCR positive clone. DNA gel electrophoresis was used to see if there was a target strip to judge.

5. Inducing Expression We added 1ml BL-21, whose OD600=1, into 15ml LB. Then the plasmids were induced for 2hrs until OD600=0.2, with 1 mM IPTG. 6. Test of induced protein. The induced bacterial liquids were disrupted by ultrasonic. Then the proteins were irradiated under ultraviolet light(waverlength250nm and 350nm). We found fluorecent green image.

Sequence and Features