"

From 2014.igem.org

Our project is divided into four parts:

Part 1: The Construction of the Worker System
Step 1: Gene Cloning
To find the optimal temperature for oprF and RTS operon amplification, we set a gradient in temperature. Then we amplified oprF and RTS sequence by PCR in 58℃. The sequence was stored in -20℃. The PCR conditions were listed as table 1-1.

Table 1-1 ： Gradient PCR System

Components(50μl)	Volume(ml)
PrimerStar Buffer	10
dNTPs(2.5mM)	5
Primer-F(10μM)	1.5
Primer-R(10μM)	1.5
Template	1.5
PrimerStar	0.5
ddH2O	30

Step 2: Improvement of oprF
We choose the 188th and 196th amino acid of OprF as anchor points. Designing primers (Table 2-1）to clone the target fragment, and then add the GS linker and the gene fragment coding CBP at the end of the target fragment.
The protein regarding the 188th amino acid as anchor point and added GS linker was named as OprF-1 and the protein regarding the 196th amino acid as anchor point and not added GS linker was named as OprF-2.

Table 1-2  Primers of Improving oprF

Primer	Sequence(5’→3’)
oprF-CBP-F	CCGGAATTCAACTGAAGAACACCTT
oprF-CBP-R1	CCAGCCGCCATGATGCGGGGAAACCGGTTCCGGAGCCGGAGCGGC
oprF-CBP-R2	ATAGTTTAGCGGCCGCCGGCCAGCCGCCATGATGCGGGGA
oprF-GS-F	CCGGAATTCAACTGAAGAACACCTT
oprF-GS-R1	TGAACCTCCGCCACCTTTCGAACCACCGAAGTTGAAG
oprF-GS-R2	CCAGCCGCCATGATGCGGGGATGAACCTCCGCCACC
oprF-GS-R3	ATAGTTTAGCGGCCGCCGGCCAGCCGCCATGATGCGGGGA

 

Step 3: Construction of Vectors

Digest pET28a vector to donate a skeleton with EcoRI and NotI. Retrieve and purify the target genes with kits produced by Omega.

Afterwards, pET28a(+) vector, gene oprF-1/oprF-2/RTS operon were linked together to recombine a new vector: pET28a(+)-oprF-1/oprF-2/RTS. The reaction system for digestion and conjunction were listed in the table 1-3, 1-4.

Table 1-3  Reaction System for Digestion

Components(50μl)	Volume(μl)
10×H Buffer	5
BSA	5
EcoRI	2.5
NotI	2.5
Fragment	25
ddH2O	10
Conditions	37℃ 1h

Table 1-4  for Gene Conjunction

Components	System components(10μl)
Solution I	5
Insert	4.5
Vector	0.5
Conditions	16℃ 1h

Fig 1-1 Digested gene and plasmid

 

Step 3: Transferring the Plasmids into E. coli BL21 Strain 

The recombined plasmids were transferred into BL21 strain, which later was grew in LB culture medium with 100μg/ml Kanamycin, 37℃ for one night. To ensure the conjunctions were correct, we confirmed the vectors by colonial PCR and gel electrophoresis, following the digestion of EcoRI and NotI to see the possibilities of reverse connection. The conditions for colonial PCR and dual-enzyme digestion reaction were listed in the table 1-5, 1-6.

Table 1-5  Reaction System Colonial PCR

Components(10μl)	Volume(ml)
2×Ex Taq Mix	5
Primer-F(10μmol/L)	0.3
Primer-R(10μmol/L)	0.3
Template	0.3
H2O	4.1

Table 1-6  Conditions for dual-enzyme digestion reaction (37 ℃ 1h)

Components(10μl)	Volume(μl)
10×H Buffer	1
BSA	1
EcoRI	0.5
NotI	0.5
ddH2O	7
Condition	37 ℃ 1h

The E. coli BL21(DE3) strain transformed with pET28a(+)-flA was kindly donated by Prof. David O'Hagan and Prof. James H. Naismith from University of St Andrews, Saint Andrews, Scotland, United Kingdom. 

Step 4: Expression of Protein
Plasmid pET28a(+)-oprF-1/oprF-2/RTS/flA was transformed into E. coli BL21(DE3) and we get for protein expression analysis. The strains were grown in Luria broth containing 100ug/ml kanamycin at 37℃, 250rpm until an absorbance of 0.4–0.6 at 600 nm was reached. We then added IPTG to 0.5mM and continued the incubation at 28℃ overnight to induce the overexpression of OprF-CBP/OprF-GS-CBP. The cells were collected, suspended with 10mM imidazole containing 0.1mM protease inhibitor PMSF and then disrupted using Selecta Sonopuls. After centrifugation, the sediment was treated with 1*SDS gel loading buffer and kept in boiling water for 5 minutes and applied to SDS-PAGE.

Step 5: Surface Displaying Copper Ions
To identify that whether our OprF has anchored on the cell membrane of E. coli, we performed immunofluorescence assay. HA tag was added to the N-terminal of OprF-CBP so that the recombinant protein OprF-CBP-HA can be specifically recognized by anti-HA antibody. When FITC labeled anti-IgG antibody was used as the secondary antibody and interacted with the primary antibody, green fluorescence could be observed in the cell membrane of E. coli under the fluorescent microscope.