Team:Tsinghua-A/Experiment
TSINGHUA-ADesign of Characters
I. Design 1.0
Figure 1. Design 1.0 of gene circuit
1. How warriors kill enemies:
AHL1 in warrior I excretes AHL1. AHL1 goes into its enemies and forms a complex with the protein AHL1R. This complex activates the promoter Pahl1, thus activating the expression of LacI. The expression of LacI inhibits the promoter Plac, thus inhibiting the expression of cmR. Because we culture our E.coli in medium containing chloramphenicol, warrior I kills its enemies. As AHL1 does not form a complex with protein AHL2R inside E.coli from warrior I’s group, thus we fulfill our desire that warrior I just kill the enemies but not bacteria from it own group. Warrior Il works in a similar way.
AHL1 in warrior I excretes AHL1. AHL1 goes into its enemies and forms a complex with the protein AHL1R. This complex activates the promoter Pahl1, thus activating the expression of LacI. The expression of LacI inhibits the promoter Plac, thus inhibiting the expression of cmR. Because we culture our E.coli in medium containing chloramphenicol, warrior I kills its enemies. As AHL1 does not form a complex with protein AHL2R inside E.coli from warrior I’s group, thus we fulfill our desire that warrior I just kill the enemies but not bacteria from it own group. Warrior Il works in a similar way.
2.How farmers provide nutrients:
RafD encodes invertase, which can hydrolyze sucrose into glucose and fructose. The signal peptide of HlyA ligated to RafD can help the invertase be secreted through the recognition of its signal sequence. This is our new part. Besides, combining with extrinsic HlyB, HlyD and intrinsic TolC from the nucleoid in E.coli together form the transporter which helps the invertase to be secreted.
This whole part is designed to produce a secretory invertase. As our E.coli can use glucose but not sucrose as its carbon source, farmers fulfill the role of providing nutrients.
However, from our orthogonality test and killing test, we find that we can just find warrior II that can be killed by warriors from the other group but not itself. Warrior I cannot satisfy this Therefore, we improved our gene circuit by choosing specific combinations of AHL, receptor and promoter.
RafD encodes invertase, which can hydrolyze sucrose into glucose and fructose. The signal peptide of HlyA ligated to RafD can help the invertase be secreted through the recognition of its signal sequence. This is our new part. Besides, combining with extrinsic HlyB, HlyD and intrinsic TolC from the nucleoid in E.coli together form the transporter which helps the invertase to be secreted.
This whole part is designed to produce a secretory invertase. As our E.coli can use glucose but not sucrose as its carbon source, farmers fulfill the role of providing nutrients.
However, from our orthogonality test and killing test, we find that we can just find warrior II that can be killed by warriors from the other group but not itself. Warrior I cannot satisfy this Therefore, we improved our gene circuit by choosing specific combinations of AHL, receptor and promoter.
II. Design 2.0
Figure 2. Improved gene circuit. The one on the top is warrior I while the one below is warrior II.
In this circuit, warrior II can only be killed by C4HSL secreted by warrior I, but not 3OC6HSL secreted by itself. (This can be shown in orthogonality test )
Without TetR, we can see that C4HSL can activate expression of LacI by binding to RhlR and LuxR from results of orthogonality test . However, we have TetR here! C4HSL forms a complex with the protein RhlR inside warrior I. This complex activate expression of TetR, TetR can then inhibit the promoter Plux/tetR, thus counteracting the self-activating effect. In this way, the expression of LacI is not activated so warrior I does not kill itself. We modify the gene circuits of beggar I and farmer I in a similar way so that warrior I no longer kill them. It feasibility can be seen in Improved gene cicuit.
Without TetR, we can see that C4HSL can activate expression of LacI by binding to RhlR and LuxR from results of orthogonality test . However, we have TetR here! C4HSL forms a complex with the protein RhlR inside warrior I. This complex activate expression of TetR, TetR can then inhibit the promoter Plux/tetR, thus counteracting the self-activating effect. In this way, the expression of LacI is not activated so warrior I does not kill itself. We modify the gene circuits of beggar I and farmer I in a similar way so that warrior I no longer kill them. It feasibility can be seen in Improved gene cicuit.
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 media to feed the bacteria without invertase
II. Experimental design
As is demonstrated in the genetic circuit, RafD is ligated to the signal peptide of HlyA. The plasmid can also express HlyB and HlyD, but the expression is controlled by PBAD. Besides, TolC originally exists in the genome of E.coli. Thus, the RafD enzyme is able to be transported to the media and hydrolyze the sucrose outside the cells.
Because the promoter of HlyB and HlyD requires arabinose to work while arabinose is a carbon source, an experiment to test whether this part or the arabinose added to activate the PBAD is what bacteria live on becomes necessary. We used two test parts, secretory invertase and Pcon_mRFP_TT. Each of them are transformed into E. coli MG1655 ΔsidA ΔlacI. E. coli MG1655ΔsidA ΔlacI with different plasmids are called Peasant and Civilian (Despite their similarity to Farmer and Beggar, we cannot call them in that way because their genetic circuits are different after all.) separately, which do not have any invertase genes in genome. Peasant and Civilian will grow in M9-sucrose culture media, a special M9 culture media in which glucose is replaced by sucrose. Then there are only two available carbon sources, sucrose, if hydrolyzed, and arabinose, if added.
Because the promoter of HlyB and HlyD requires arabinose to work while arabinose is a carbon source, an experiment to test whether this part or the arabinose added to activate the PBAD is what bacteria live on becomes necessary. We used two test parts, secretory invertase and Pcon_mRFP_TT. Each of them are transformed into E. coli MG1655 ΔsidA ΔlacI. E. coli MG1655ΔsidA ΔlacI with different plasmids are called Peasant and Civilian (Despite their similarity to Farmer and Beggar, we cannot call them in that way because their genetic circuits are different after all.) separately, which do not have any invertase genes in genome. Peasant and Civilian will grow in M9-sucrose culture media, a special M9 culture media in which glucose is replaced by sucrose. Then there are only two available carbon sources, sucrose, if hydrolyzed, and arabinose, if added.
III. Methods
1. Cultivate a pipe of Peasants and a pipe of Civilians for 12h.
2. Adjust their OD600 to be the same value.
3. Take 2ml each, and centrifuged them at 12000rpm for 1min. The supernatant was removed and each sediment was resuspended with 2ml M9-sucrose culture media.
4. Add 5ml M9-sucrose media 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. Adjust their OD600 to be the same value.
3. Take 2ml each, and centrifuged them at 12000rpm for 1min. The supernatant was removed and each sediment was resuspended with 2ml M9-sucrose culture media.
4. Add 5ml M9-sucrose media 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.
5. 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. Figure 1 shows the number of living cells in each group. Figure 2 shows the number of cells expressing mRFP in each group. Figure 3 shows the ratio of the number of cells expressing mRFP to the number of living cells.
Figure 1. The number of living cells
Figure 2. The number of cells expressing mRFP
Figure 3. The number of cells expressing mRFP/the number of living cells
V.Analysis
Figure 2 tells us that when Peasants and Civilians coexist in the media, Civilians grow more than the group in which Civilians live alone in the media. 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 Figure 1 and Figure 3 are also in line with expectation. Figure 1 demonstrates that when the concentration of Arabinose is 40μM, Civilians cannot live without Peasants, which means the cells in the media mainly live on sucrose instead of arabinose. However, Civilians can grow alone if the concentration of arabinose is up to 100 μM. 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 Figure 1 and Figure 3 are also in line with expectation. Figure 1 demonstrates that when the concentration of Arabinose is 40μM, Civilians cannot live without Peasants, which means the cells in the media mainly live on sucrose instead of arabinose. However, Civilians can grow alone if the concentration of arabinose is up to 100 μM. This comparison tells that 40μM is an appropriate concentration which is able to ask cells to live on sucrose but not arabinose.
