Team:Tsinghua-A/invertase assay
TSINGHUA-A
Invertase assay
I Background
RafD, which is primitively from the raffinose operon of E. coli, can express a β-fructofuranosidase. It can hydrolyze raffinose into fructose and meliose, and sucrose into glucose and fructose. HlyA is a secretory protein in bacteria, which can be secreted through the recognition of its signal sequence at the C terminal by a transporter constructed by HlyB, HlyD and TolC. This part is designed to produce a secretory invertase, which can hydrolyze sucrose in the medium to feed the bacteria without invertase.
Tips: BBa_K2250003 just contains RafD and HlyA signal peptide!
II Experimental design
As demonstrated in the genetic circuit, RafD is ligated to the signal peptide of HlyA. The plasmid can also express HlyB and HlyD, which is controlled by PBAD to tune the secretion process of RafD. Besides, TolC originally exists in the genome of E. coli. Thus, the RafD enzyme is able to be transported to the medium and hydrolyze the sucrose outside the cells.
To test the function of this part, we constructed two kinds of E. coli, one with this part BBa_K2250003 and a transport system (We call it "Peasant" to distinguish it with farmer, which also has parts that can sense AHLs.), and the other with a constitutive expression of RFP (We call it "Civilian" to distinguish it with beggar, which also has parts that can sense AHLs.). We changed the ratio of Peasant to Civilian and the concertration of arabinose, which can active PBAD to control the invertase’s secretion. RFP was measured to represent the abundance of Civilian, i.e. the ability how this part (Peasant) can feed bacteria without any invertase genes.
To test the function of this part, we constructed two kinds of E. coli, one with this part BBa_K2250003 and a transport system (We call it "Peasant" to distinguish it with farmer, which also has parts that can sense AHLs.), and the other with a constitutive expression of RFP (We call it "Civilian" to distinguish it with beggar, which also has parts that can sense AHLs.). We changed the ratio of Peasant to Civilian and the concertration of arabinose, which can active PBAD to control the invertase’s secretion. RFP was measured to represent the abundance of Civilian, i.e. the ability how this part (Peasant) can feed bacteria without any invertase genes.
III Methods
1.Transform invertase and its transport system (Peasant) and constitutively expressed mRFP (Civilian) separately into E. coli MG1655 ΔsidA ΔlacI. Let them grow in M9-sucrose culture medium, a special M9 culture medium in which glucose is replaced by sucrose.
2. Cultivate a pipe of Peasants and a pipe of Civilians for 12h.
3. Adjust their OD600 to be the same value.
4. Take 2ml each, and centrifuged them at 12000rpm for 1min. The supernatant was removed and each sediment was resuspended with 2ml M9-sucrose culture medium.
5. Add 5ml M9-sucrose medium and 100μl bacterial liquid, in which Peasants and Civilians are mixed, so the total number of them is fixed, nevertheless of their ratio. The concentration of Arabinose was set by adding concentrated Arabinose solution.
The experimental groups and control groups are listed as follows. The number “1” and “3” in the boxes are the numbers of repeats.
2. Cultivate a pipe of Peasants and a pipe of Civilians for 12h.
3. Adjust their OD600 to be the same value.
4. Take 2ml each, and centrifuged them at 12000rpm for 1min. The supernatant was removed and each sediment was resuspended with 2ml M9-sucrose culture medium.
5. Add 5ml M9-sucrose medium and 100μl bacterial liquid, in which Peasants and Civilians are mixed, so the total number of them is fixed, nevertheless of their ratio. The concentration of Arabinose was set by adding concentrated Arabinose solution.
The experimental groups and control groups are listed as follows. The number “1” and “3” in the boxes are the numbers of repeats.
6. All of the groups were cultured for 20h. The growth was measured by flow cytometry. The number of cells is averaged.
IV Results
The number of cells were counted and illustrated in the figures. Fig. 1 shows the number of living cells in each group (Peasant + Civilian). Fig. 2 shows the number of cells expressing mRFP in each group(Civilian). Fig. 3 shows the ratio of the number of cells expressing mRFP to the number of living cells (Civilian / (Peasant + Civilian)).
Fig.1 The number of living cells
Fig.2 The number of cells expressing mRFP
Fig.3 The number of cells expressing mRFP/the number of living cells
V Analysis
Fig. 2 tells us that when Peasants and Civilians coexist in the medium, Civilians grow more than the group in which Civilians live alone. Also, the number of Civilians becomes larger than the negative control group if the primitive fraction of peasant is larger. Contrarily, the number of Civilians does not become larger if the primitive quantity of Peasants is too big while the original number of Civilians is too small. And this is considered acceptable. Besides, as is revealed, Civilians grows fastest when the primitive ratio of Peasants and Civilians is 1.
The results in Fig. 1 and Fig. 3 are also in line with expectation. Fig. 1 demonstrates that when the concentration of Arabinose is 40μM, Civilians cannot live without Peasants, which means the cells in the medium mainly live on sucrose instead of arabinose. However, Civilians can grow alone if the concentration of arabinose is up to 100 μM, which may provide extra carbon sources. This comparison tells that 40μM is an appropriate concentration which is able to ask cells to live on sucrose but not arabinose.
The results in Fig. 1 and Fig. 3 are also in line with expectation. Fig. 1 demonstrates that when the concentration of Arabinose is 40μM, Civilians cannot live without Peasants, which means the cells in the medium mainly live on sucrose instead of arabinose. However, Civilians can grow alone if the concentration of arabinose is up to 100 μM, which may provide extra carbon sources. This comparison tells that 40μM is an appropriate concentration which is able to ask cells to live on sucrose but not arabinose.
VI Reference
[1] Aslanidis C, Schmid K, Schmitt R. Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli.[J]. Journal of Bacteriology, 1989, 171(12):6753-63.
[2] Edinburgh 2008: https://2008.igem.org/Team:Edinburgh/Plan/Cellulolysis
[2] Edinburgh 2008: https://2008.igem.org/Team:Edinburgh/Plan/Cellulolysis
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