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Team:HKU-HKBU/polar expression protocols
From 2009.igem.org
Contents |
Strain selection
Swimming plate assay
The fastest-swimming bacteria among our candidates were determined by this assay. By measuring the diameter on the swimming plate at differnt Different strains were first introduced to suitable agar media, and the diameter of the colonies are measured every hour in the first 8 hours in order to obtain approximated data of their swimming speed. The data are also recorded overnight for confirmation. No or inconspicuous increase in diameter is classified as negative result..
| Strain | BL21 | NCM3722 | MG1655 | MG3 | 2443 | SL7207 |
|---|---|---|---|---|---|---|
| Swim plate assay result | - | - | - | / | ++ | + |
LPS completeness
Conclusion
Combining the results of two parts, E. coli 2443 and Salmonella SL7207 are adopted in further experiment and entitled with PB (Propeller Bacteria) 001 and PB 002 correspondingly.
E. coli polar expression system
Bacteria
Two negative-gram E. coli strain candidates were tried in our experiment—BL21 and 2443. They are both LPS complete strains, which is a necessity of polar expression of the plasmid which contains AIDA.
Transformation
This plasmid was transformed to the competent cell BL21 and 2443 respectively by electroporation and the T7 polymerase was co-transformed at the same time to promote the expression of this promoter. After recovering with SOC solution for 30 minutes, they were spread to Agar plates with Ampicillin resistance. Then two single colonies on the two plates were picked up for pre-culture.
Fluorescent microscope
After 16 hours’ pre-culture of E. coli BL21 and E. coli 2443 with plasmid GFP-strp-AIDA in LB broth, they were transferred to two slides and exposed under fluorescent microscope using oil immersion lens with a magnification of 600 times.
Western Blotting
Preparation of membrane protein samples
Firstly, the bacteria samples were centrifuged with a speed of 3,500g for ten minutes to harvest the cells. Cell lyses were done by sonic waves at 40% 10’’ for 6 times. Ultracentrifuge was used to achieve the total membrane pellet with a speed of 100,000g for one hour. Re-suspend the pellet with PBS to get total membrane solution. Then the inner membrane was solublized by PBS containing 0.05M MgCl2, 2% TritonX-100. Then the outer membrane proteins were obtained by a new round of ultracentrifuge with the speed of 100.000g for 1h. Via this method, two layers of membranes were separated to collect two protein samples from inner membrane and outer membrane respectively.
Equal loading of protein samples
The quantification of these three protein sample solutions is done by BCA analysis. After analyzing the concentrations of each protein samples, we used the formula: total amount of protein = sample concentration * sample volume to adjust the sample volume to the same loading volume by adding the mixture buffer. Equal loading of protein samples were ensured in western blotting after these adjustments.
Salmonella polar expression system
Bacteria
The plasmid was built in the negative-gram strain salmonella SL7207.
Transformation
The plasmid was transformed to the competent cell salmonella SL7207. After recovering for 30 minutes with SOC solution, the bacteria were spread to an agar plate with ampicillian resistance. Then one single colony was pick up for pre-culture.
The following steps are the same with those in the E. coli polar expression system.
