Team:Shanghai United HS/Results
Results
Group 1
Experimental bacteria：E. coli Nissle 1917
Experimental plasmid:
plasmid A (puc57-kan-mini-J23101-OmpA-araB-TT).
PROCEDURE
1) Electroporation
Plasmid A (~ 100 ng) was transformed to E. coli Nissle 1917 by electroporation.
2) AraB activity assay
In the engineering cells simultaneously containing plasmid A and plasmid B, the expression of ClyR in plasmid B is to be induced by arabinose, which can be supplied by AraB encoded by plasmid A via the hydrolysis of araboxylan. So, for the sake of ClyR expression, plasmid A must express active AraB. We tried to monitor the concentration of the reducing sugar arabinose and thus access the activity of AraB.
The concentration of reducing sugar was determined by the DNS kit and the A540 were recorded accordingly.
Plasmid A (~ 100 ng) was transformed to E. coli Nissle 1917 by electroporation.
2) AraB activity assay
In the engineering cells simultaneously containing plasmid A and plasmid B, the expression of ClyR in plasmid B is to be induced by arabinose, which can be supplied by AraB encoded by plasmid A via the hydrolysis of araboxylan. So, for the sake of ClyR expression, plasmid A must express active AraB. We tried to monitor the concentration of the reducing sugar arabinose and thus access the activity of AraB.
The concentration of reducing sugar was determined by the DNS kit and the A540 were recorded accordingly.
RESULT
Table 1. The A540 data of the culture supernatant of E. coli with plasmid A and B.
Figure 1. The concentration assay of the reducing sugar in bacteria transformed with plasmid A.
Figure 1. The concentration assay of the reducing sugar in bacteria transformed with plasmid A.
CONCLUSION
All the data at “0 h” with different concentration of araboxylan was referred as
blank, and the concentration of the reducing sugar could be calculated.
As can be seen from figure 1, in the transformed bacteria with plasmid A, the concentration of the reducing sugar increased with the elongation of the incubation time. The plasmid A can work normally and possess hydrolyzing arabinoxylan activity, which supply the arabinose for the expression of ClyR.
As can be seen from figure 1, in the transformed bacteria with plasmid A, the concentration of the reducing sugar increased with the elongation of the incubation time. The plasmid A can work normally and possess hydrolyzing arabinoxylan activity, which supply the arabinose for the expression of ClyR.
Group 3
Experimental bacteria：E. coli Nissle 1917
Experimental plasmid:
plasmid A (puc57-kan-mini-J23101-OmpA-araB-TT).
plasmid C (pBAD-Myc-HisA-OmpA-amilGFP).
PROCEDURE
1) Electroporation
Plasmid A and plasmid C were co-transformed to E. coli Nissle 1917 by electroporation.
2) AmilGFP expression
Based on the data of Group 1, the cells containing plasmid A could hydrolyze araboxylan to arabinose. To test the produced arabinose could then induce the expression of genes under the control of pBAD. We choose green fluorescent protein AmilGFP as a reporter protein. In the cells simultaneously containing plasmid A and plasmid C, if the active AraB from plasmid A work effectively, the AmilGFP would be monitored.
3) AmilGFP assessment
The existence of AmilGFP was monitored by the fluoroscopic examination using SpectraMax i3x..Read the fluoroscopic data of the supernatant, and recorded the average data. After this timepoint, 100 ul of the culture was sampled and marked as “6 h”, “8 h”, “10 h”, “12 h” and “13 h”, respectively, and the fluoroscopic data were recorded accordingly.
Plasmid A and plasmid C were co-transformed to E. coli Nissle 1917 by electroporation.
2) AmilGFP expression
Based on the data of Group 1, the cells containing plasmid A could hydrolyze araboxylan to arabinose. To test the produced arabinose could then induce the expression of genes under the control of pBAD. We choose green fluorescent protein AmilGFP as a reporter protein. In the cells simultaneously containing plasmid A and plasmid C, if the active AraB from plasmid A work effectively, the AmilGFP would be monitored.
3) AmilGFP assessment
The existence of AmilGFP was monitored by the fluoroscopic examination using SpectraMax i3x..Read the fluoroscopic data of the supernatant, and recorded the average data. After this timepoint, 100 ul of the culture was sampled and marked as “6 h”, “8 h”, “10 h”, “12 h” and “13 h”, respectively, and the fluoroscopic data were recorded accordingly.
RESULT
1) AmilGFP expression in cells transformed with plasmid A
Figure 2. The fluoroscopic data of cells transformed with plasmid A.
In the cells transformed with only
plasmid A, the
fluoroscopic data of cells with the addition of different concentration of araboxylan showed no difference
with that of cells without the addition of araboxylan. This data suggested no AmilGFP was produced in cells
with or without addition of araboxylan.
2) AmilGFP expression in cells transformed with plasmids A and C
Figure 3. The fluoroscopic data of cells transformed with plasmids A and C.
In the cells transformed with plasmids A and C, the
fluoroscopic data of cells increased with the addition of araboxylan. Meanwhile, the fluoroscopic data
become larger with the prolongation of incubation time.
3) AmilGFP expression in cells transformed with plasmid C
Figure 4. The fluoroscopic data of cells transformed with plasmid C with the addition of
arabinose.
In the cells transformed with plasmid C, the fluoroscopic
data of cells increased with the addition of arabinose. Meanwhile, the fluoroscopic data become larger with
the prolongation of incubation time.
CONCLUSION
AmilGFP was successfully expressed, suggesting the
feasibility and possibility of the inducible secretory expression of ClyR.
Group 2
Experimental bacteria：E. coli Nissle 1917
Experimental plasmid:
plasmid A (puc57-kan-mini-J23101-OmpA-araB-TT)
plasmid B (pBAD-Myc-HisA-OmpA-ClyR-6His-TT)
PROCEDURE
1) Electroporation
Plasmid A and plasmid B were co-transformed to E. coli Nissle 1917 by electroporation.
2) ClyR expression
The araboxylan with final concentration 0, 0.3%, 0.6%, 1.0%, 1.5% and 2.0% (w/v) was added into the culture to induce ClyR expression.
For the comparison, E. coli Nissle 1917 with plasmid B was cultured by the same way and the expression of ClyR was induced by the addition of arabinose with final concentration of 0 μM, 10 μM,30 μM,0.1 mM, 0.2 mM,0.5 mM and 2 mM.
3) The exported protein concentration assay
The protein concentration was monitored at 595 nm using Multiscan Spectrum (BioTek). Read the data for three times, recorded the average of the A595 data..
4) SDS-PAGE analysis of ClyR
Gels were scanned with the ImageQuant™ LAS 4000 mini (GE Healthcare).
Plasmid A and plasmid B were co-transformed to E. coli Nissle 1917 by electroporation.
2) ClyR expression
The araboxylan with final concentration 0, 0.3%, 0.6%, 1.0%, 1.5% and 2.0% (w/v) was added into the culture to induce ClyR expression.
For the comparison, E. coli Nissle 1917 with plasmid B was cultured by the same way and the expression of ClyR was induced by the addition of arabinose with final concentration of 0 μM, 10 μM,30 μM,0.1 mM, 0.2 mM,0.5 mM and 2 mM.
3) The exported protein concentration assay
The protein concentration was monitored at 595 nm using Multiscan Spectrum (BioTek). Read the data for three times, recorded the average of the A595 data..
4) SDS-PAGE analysis of ClyR
Gels were scanned with the ImageQuant™ LAS 4000 mini (GE Healthcare).
RESULT
Table 2. The protein concentration of the culture supernatant of E. coli with plasmids A and B.
Figure 5. The SDS-PAGE of supernatant.
Figure 5. The SDS-PAGE of supernatant.
The A595 of the culture supernatant of E. coli with plasmid
A and B suggested the possible expression of ClyR (Table 2). The A595 data at “0 %” was referred as blank,
and the protein concentration could be calculated referred to the standard formula. As can be seen from
figure 5, in the transformed bacteria with plasmid A and B, the concentration of the reducing sugar
increased with the elongation of the incubation time.
During the expression process, bacteria lysis occurred with
the elongation of the incubation time. But the ClyR band could obviously be obtained as is shown in figure
5.
CONCLUSION
In the engineering cells containing plasmid A and plasmid B, the expression of ClyR
was successful.
In vitro activity assay of ClyR
Experimental bacteria：E. coli Nissle 1917 transferred with plasmid A
and B, Lactobacillus casei subsp. casei (ATCC334)
PROCEDURE
1) The culture of Lactobacillus casei subsp. casei (ATCC334)
2) In vitro assay
The value of OD600 was monitored using Multiscan Spectrum (BioTek).
2) In vitro assay
The value of OD600 was monitored using Multiscan Spectrum (BioTek).
RESULT
Figure 6. The in vitro assay of ClyR.
The OD600 of the Lactobacillus casei subsp.
casei cell suspension gradually reduced with the addition of supernatant (figure 6).
CONCLUSION
We have successfully obtained the ClyR protein in the
culture supernatant of cells carrying plasmids with araB and clyR genes. The concentration of ClyR was low,
so more efforts are still needed in future. It is worth to note that the expression of ClyR protein may
cause the host to suicide, which can be supported by the obvious cell lysis and the presence of flocculent
precipitate.