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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 time, the average swimming speed can be calculated.
Different strains were firstly introduced to suitable agar media(with specific resistance). Then the diameter of the colonies were 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
Among the stains that could swim the LPS completeness were examined one by one by searching information from the reference.
Conclusion
Combining the results of two parts, E. coli 2443 and Salmonella SL7207 were adopted in further experiment and entitled with PB (Propeller Bacteria) 001 and PB 002 correspondingly.
Polar Expression
AIDA 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.
Plasmid Construction
pET-GFP-Strp-AIDA is a DNA plasmid that can induce polar expression in E.coli. This transmembrane system is designed to ensure the expression of specific proteins on the outer membrane. The fragment of AIDA and Strepvadin is obtained from the lab. We use the restriction enzyme SacI to digest these two fragments to ensure the cohesive ends. Then a ligation reaction underwent between two digested parts to produce Strp-AIDA fragments. GFP is digested with restriction enzymes EcoRI and SacI. The end that was digested with SacI helped the ligation of GFP and Strp-AIDA. The signal peptide was added to the EcoRI digested end by PCR. To ensure the right ligation reactions, we double checked the direction of each fragment in this plasmid by enzyme digestions.
Transformation
The 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.
Lpp-OmpA system
Bacteria
The plasmid was built in the negative-gram strain salmonella SL7207.
Plasmid Construction
Lpp-OmpA-GFP is a DNA plasmid that can induce polar expression in Salmonella SL7207. This transmembrane system is designed to express specific proteins on the outer membrane of bacteria by using 9 amino acids of a primary membrane lipoprotein (lpp) with amino acids 46-159 of the outer membrane protein OmpA. In this construct, OmpA (residue 46-159) was synthesized by PCR with a template derivwhed from E.coli strain MG1655. The signal peptide and the first nine amino acids of one major lipoprotein were added to the upstream of OmpA peptide by PCR. The whole peptides were integrated to a T-vector, which contained a lacI- promoter. The fragment GFP was digested by restriction enzymes XhoI and NotI. Then the T-vector with Lpp-OmpA was digested the same two enzymes to ensure the same cohesive ends. A ligation reactions underwent between digested between GFP and Lpp-OmpA. The polar expression was examined by a fluorescent microscope.
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 AIDA system.
