Team:TJU China/Results
Results
1.1 Standard curves
Our aim is to test the accurate amount of BV using smURFP we produced and purified. Then we get a series of standard curves of different concentration of BV and smURFP, as shown in the text-center below. Then we can use these curves to calculate how much BV is there in birds’ blood, or in other solutions.
Form the results, we found that we can get good standard curves if we use more than 15μM (15μM included) protein for detection. And from these four curves, we got a final standard curve, as shown in the figure below.
Figure. Standard curves between concentration of BV and RFU. (concentration of smURFP should be more than 15μM)
1.2 BV production
The result after induction (with plasmid pET22a of HO-1 gene). The upper one is the control group, and the inferior one is the experimental group.
2.1 molecular cloning
Escherichia coli BL21 & EHEC O157: H7:
a) Surface display - LPP
PCR results for LPP. M is DNA marker. Lane 1 is the result
for LPP anchoring protein. Lane 2 is the result for smURFP
protein. Lane 3 is the result for overlap PCR.
b) Surface display – INPNC
PCR results for INPNC. M is DNA marker. Lane 1 is the result for INPNC
anchoring protein. Lane 2 is the result for smURFP protein. Lane 3 is the
result for overlap PCR.
c) Co-expression
PCR results for co-expression system. M is DNA marker. Lane 1 2 3 is the result for sumURFP protein. Lane 4 5 6 is the result for HO-1 protein. Lane 7 8 9 is the result for overlap PCR.
Citrobacter rodentium:
a) Surface display – INPNC
PCR results for INPNC. M is DNA marker. Lane 1 2 3 is the result for smRUFP protein. Lane 4 5 6 is the result for INPNC anchoring protein. Lane 7 8 9 is the result for overlap PCR.
b) Surface display – Brka
PCR results Brka. M is DNA marker. Lane 1 2 3 is the result for leading peptide and smURFP protein. Lane 4 5 6 is the result for Brka protein. Lane 7 8 is the result for overlap PCR.
c) Co-expression
Figure. Result of PCR for coexpression with promoter P2. M is marker. Lane 1 to lane3 are results for P2. Lane 4 to lane 7 are the results for smURFP. Lane 8 to lane 11 are the results for HO-1. Lane 12 to lane 14 are the results for P2-smURFP.
Bifidobacterium longum:
a) Surface display –GLBP
M is DNA marker. Lane is promoter HU, lane 2 is a meaningless sequence, lane 3 is smURFP, lane 4 is hu-meaningless sequence, lane 5 is Hu-meaningless sequence-smURFP, lane 6 is glbp
b) Surface display –AcmA
M is DNA marker. Lane 1 is the PCR result for ACMA, Lane 2 is the plasmid of HU-meaningless sequence-smURFP, lane 3 is the digestion result of plasmid HU-ACMA-smURFP
c) Co-expression
M is DNA marker. Lane 1 is the PCR result of HU, Lane 2 is the PCR result of smURFP-histag, lane 3 is the PCR result HU-smURPF-histag, lane 4 is the PCR result of RBS-HO-1, lane 5 is the PCR result of HU-smURFP-histag-RBS-HO-1
2.2. Protein expression
Escherichia coli BL21 :
a) Surface display – INPNC
The western blot result of INPNC in E.coli BL21. M is marker, Lane 1 is negative control. Lane 2 is INPNC-smURFP-His, which is about 40.4 kDa. Lane 3 is positive control.
b) Co-expression
| Tube number | 1 | 2 | 3 | |||
|---|---|---|---|---|---|---|
| Well number | 1' | 2' | 3' | 4' | 5' | 6' |
| RFU | 4315 | 4644 | 4783 | 5071 | 12 | 0 |
Result of Micro plate Reader in the black 96-well plate. Tube 1 and 2 are experimental group (with E.coli without plasmid), and tube 3 is the control group (with E.coli with plasmid pET28b of co-expression system).
2.3. Bacteria tracking in vitro
Escherichia coli BL21 - Co-expression
Laser confocal microscopy was use to observe these bacteria directly. Excitation wavelength is 640nm.
2.4. Bacteria tracking in vivo
Escherichia coli BL21 - Co-expression
The picture shows relative fluorescent intensity after doing intragastric administration (10^8 and 10^11 CFU) for 5.5h. The left is the control one (with E.coli without plasmid), the right is the experimental one (with E.coli with plasmid pET28b, 10^11 CFU). The result successfully showed that our system was executable and excellent. And smURFP has very compatible persistence and penetrability.
Trend of RFU in vivo. M2 is the mice with 10^8 CFU bacteria, and M3 is the mice with 10^11 CFU. We can see the fluorescence is relatively high, and the RFU is still over 3000 after 300min when the mice with 10^11 CFU bacteria.
3.1. Protein purification
a) Nickel Column Purification
To test the purification efficiency and condition of Nickel Column Purification is via SDS-PAGE method. (M) The marker. (1) The 20 μl sample of E.coli crushing fluid before Nickel Column Purification with the target part of smURFP and his-sumo tag, around 26 kD. (2) The 20 μl sample of the solution with the target part is after Nickel Column Purification. (3)The 20 μl sample of the solution after is Nickel Column Purification is before the sumo protease digestion, with sumo protease around 13 kDa. (4) The 20 μl sample of the Nickel medium is after 12 h sumo protease digestion with the target protein smURFP, around 12 kDa. (5) The 20 μl sample of the eluent is with the protein smURFP. (6) The 20 μl sample of the Nickel medium after the elution to test whether the target protein is all in the solution.
b) Purification of ion exchange column & molecular sieve column.
Result of purification of ion exchange column.
Result of purification of molecular sieve column.
To test the purification efficiency and condition of AKTA Ion Exchange Chromatography and Molecular Sieve Purification. (a) The 20 μl sample of the solution before Ion Exchange Chromatography Purification is as a compare. (b)~ (h) The 20 μl sample of the solution is after Ion Exchange Chromatography Purification. (i) The marker. (j) The 20 μl sample of the solution before Molecular Sieve Purification is as a compare. (l)~ (o) The 20 μl sample of the solution is after Molecular Sieve Purification.
3.2. Protein crystallization
Result of proper condition for crystallization.
