Team:Tsinghua-A/Experiment
TSINGHUA-ADesign of Characters
I. Design 1.0
Fig. 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
Fig. 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 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 media 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 (Peasant), and another with a constitutive expression of RFP (Civilian). We changed the ratio between Peasant and 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 (Peasant), and another with a constitutive expression of RFP (Civilian). We changed the ratio between Peasant and 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 BBa_K2250003 (Peasant) and constitutively expressed mRFP (Civilian) separately into E. coli MG1655 ΔsidA ΔlacI. Let them grow in M9-sucrose culture media, a special M9 culture media 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 media.
5. 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. 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 media.
5. 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.
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 media, 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 media 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 media 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.
Test of the Orthogonality of the Parts
I. Background
In our project, we hope that the warriors only kill the bacteria from the other side, but not his own side. In other words, the orthogonality of the gene circuit must be good. Necessarily, it means that the AHL molecules secreted by warrior I can just activate the promoter inside warrior II, but cannot activate the promoter inside itself and vice versa. (Note: In this text, we mean the same when we refer to response intensity of AHL promoter and killing effect of warriors, that is to say, if the response intensity of promoter to AHL secreted by warrior A inside warrior B is high---A may equals to B, it means that the warrior A can be effectively killed by warrior B and vice versa.)
To help us choose appropriate circuit of two warriors, we should know how specific receptor-promoter combination respond to specific AHL.
This is characterized by ETH_Zurich 2014. However, the way they characterize the crosstalk may cause inconvenience for us to use. Detailed explanation is shown below:
The response concentration of some receptor-promoter combinations to AHL molecule may be too high but the concentration range that ETH_Zurich considered is limited, however, in real situation, bacteria we used may be able to secrete higher concentration of AHL molecule. What is more, the reverse situation may also happen. In other words, bacteria we used may be unable to secrete so much AHL molecule to activate gene expression. For example, in our project, we want to design two kinds of E. coli which can synthesize two kinds of AHL and respond to AHL secreted by another but not by itself. Therefore, according to results from ETH_Zurich 2014, shown below (Fig. 1), we may design gene circuit of our E. coli as follows. (Fig. 2)
To help us choose appropriate circuit of two warriors, we should know how specific receptor-promoter combination respond to specific AHL.
This is characterized by ETH_Zurich 2014. However, the way they characterize the crosstalk may cause inconvenience for us to use. Detailed explanation is shown below:
The response concentration of some receptor-promoter combinations to AHL molecule may be too high but the concentration range that ETH_Zurich considered is limited, however, in real situation, bacteria we used may be able to secrete higher concentration of AHL molecule. What is more, the reverse situation may also happen. In other words, bacteria we used may be unable to secrete so much AHL molecule to activate gene expression. For example, in our project, we want to design two kinds of E. coli which can synthesize two kinds of AHL and respond to AHL secreted by another but not by itself. Therefore, according to results from ETH_Zurich 2014, shown below (Fig. 1), we may design gene circuit of our E. coli as follows. (Fig. 2)
Fig. 1 Results from ETH_Zurich 2014. From this results, we may think that gene circuit designed in Fig. 2 can satisfy our needs. However, actually, from our results, we can see since E. coli can actually synthesize much more C4HSL than maximal concentration considered above by ETH_Zurich 2014, gene circuit in Fig. 2 cannot satisfy our needs.
Fig. 2 Designed gene circuit by results from ETH_Zurich 2014
In conclusion, it is inconvenient for us to choose our gene circuit just according to results from ETH_Zurich 2014 since it does not consider the ability of E. coli synthesizing AHL. Therefore, we designed our experiment as follows, which consider E. coli’s ability to synthesize AHL.
II. Experimental design
To test which kind of AHL, receptor and promoter we can choose to keep the gene circuit orthogonal during the time period of our experiment (For example, in our final experiment, we will co-culture six characters in 3ml medium for 48h), we should detect the killing effect of two warriors in real time.
However, here for simplicity, we used RFP to indicate amount of LacI inside bacteria, so its intensity inside each bacteria in real time can be used as a relative measurement of the response intensity of specific receptor-promoter to specific AHL and the total killing effect of two warriors in real time.
What is more, we let E. coli secrete and receive AHL itself to mimic real situation of our system approximately.
Below are detailed plasmid construction design.
First, we cloned three AHL synthases---luxI, lasI and rhlI to the low copy backbone---pSB3K3. At the same time, we cloned nine receptor-promoter combinations to pSB6A1. The three receptors are luxR, lasR and rhlR while the three promoters are Plux, Plas and Prhl. RFP is attached to the promoters, which is used to detect the response of the promoters to AHL molecules via the receptors (Fig. 3). After that, we cotransform the plasmids containing AHL synthases and plasmids containing receptor-promoter combinations. As a result, we get 27 different combinations.
However, here for simplicity, we used RFP to indicate amount of LacI inside bacteria, so its intensity inside each bacteria in real time can be used as a relative measurement of the response intensity of specific receptor-promoter to specific AHL and the total killing effect of two warriors in real time.
What is more, we let E. coli secrete and receive AHL itself to mimic real situation of our system approximately.
Below are detailed plasmid construction design.
First, we cloned three AHL synthases---luxI, lasI and rhlI to the low copy backbone---pSB3K3. At the same time, we cloned nine receptor-promoter combinations to pSB6A1. The three receptors are luxR, lasR and rhlR while the three promoters are Plux, Plas and Prhl. RFP is attached to the promoters, which is used to detect the response of the promoters to AHL molecules via the receptors (Fig. 3). After that, we cotransform the plasmids containing AHL synthases and plasmids containing receptor-promoter combinations. As a result, we get 27 different combinations.
Fig. 3 Illustration of plasmid construction
III. Methods
1. Transform MG1655ΔlacI ΔsdiA with 27 kinds of combinations of plasmids mentioned above, which will be used in the orthogonality test.
2. Pick bacterial clones from the petri plate,then shake it overnight in the LB medium (3ml) with 50μg/ml Ampicilin and 30μg/ml Kanamycin at 37℃. For each combination, 4 clones are picked.
3. Dilute the overnight culture to 1/50 in fresh LB medium (5ml) containing 50μg/ml Ampicilin and 30μg/ml Kanamycin.
4. Incubate the fresh cultures at 37℃ until OD600 reaches 0.2.
5. Add 1ml culture obtained from Procedure 4 to 2ml fresh LB medium containing 50μg/ml Ampicilin and 30μg/ml Kanamycin. Incubate the fresh cultures at 37℃.
6. Take out 200μl cultures obtained from Procedure 5 and measure the fluorescent intensity and OD600 after 17, 20, 24, 34, 38, 44h. (The cultures will not be put back again.) The excitation wavelength is 584nm, emission wavelength is 607nm.
2. Pick bacterial clones from the petri plate,then shake it overnight in the LB medium (3ml) with 50μg/ml Ampicilin and 30μg/ml Kanamycin at 37℃. For each combination, 4 clones are picked.
3. Dilute the overnight culture to 1/50 in fresh LB medium (5ml) containing 50μg/ml Ampicilin and 30μg/ml Kanamycin.
4. Incubate the fresh cultures at 37℃ until OD600 reaches 0.2.
5. Add 1ml culture obtained from Procedure 4 to 2ml fresh LB medium containing 50μg/ml Ampicilin and 30μg/ml Kanamycin. Incubate the fresh cultures at 37℃.
6. Take out 200μl cultures obtained from Procedure 5 and measure the fluorescent intensity and OD600 after 17, 20, 24, 34, 38, 44h. (The cultures will not be put back again.) The excitation wavelength is 584nm, emission wavelength is 607nm.
IV. Data
Fig. 4 Response of each receptor-promoter combinations to AHL
V. Analysis
From results above, how could we design our two warriors? Let’s show for you!
First, we need choose two kinds of AHL secreted by each warrior, right? Let’s try to make warrior I secrete C4HSL and warrior II secrete 3OC6HSL. Then which receptor-promoter should we put into two warriors to make them just killed by another warrior but not itself. Let’s first try to determine warrior I, so can we find a receptor-promoter combinations that just respond to 3OC6HSL but not C4HSL? Unfortunately, we can’t! So we cannot choose C4HSL and 3OC6HSL to construct two warriors that we want.
Similar results of other 2 AHL pairs can be obtained by similar analysis.
However, we do not need to be so frustrated, since we can determine part of two warriors that satisfy our needs of warrior as follows (Fig. 5) and then our model can tell us how to improve this gene circuit based on this circuit. (Improved gene circuit)
First, we need choose two kinds of AHL secreted by each warrior, right? Let’s try to make warrior I secrete C4HSL and warrior II secrete 3OC6HSL. Then which receptor-promoter should we put into two warriors to make them just killed by another warrior but not itself. Let’s first try to determine warrior I, so can we find a receptor-promoter combinations that just respond to 3OC6HSL but not C4HSL? Unfortunately, we can’t! So we cannot choose C4HSL and 3OC6HSL to construct two warriors that we want.
Similar results of other 2 AHL pairs can be obtained by similar analysis.
However, we do not need to be so frustrated, since we can determine part of two warriors that satisfy our needs of warrior as follows (Fig. 5) and then our model can tell us how to improve this gene circuit based on this circuit. (Improved gene circuit)
Fig. 5 Gene circuit designed from orthogonality test results. From experiment results, we know that warrior II just response to C4HSL secreted by warrior I, but not 3OC6HSL secreted by itself, and we cannot design a warrior I that can be killed by warrior II but not killed by itself regardless of which receptor-promoter we put inside warrior I
What is more, because we use RFP to indicate level of killing effect of warriors inside cell during orthogonality test, we are not sure about if the results will be the same (namely, if this indication is reasonable) when we use a complete circuit, so we have Killing test to verify it.
Note: The needs of us about warrior can be decomposed to four parts:
(1)Warrior I can be killed by warrior II
(2)Warrior I cannot be killed by itself
(3)Warrior II can be killed by warrior I
(4)Warrior II cannot be killed by itself
We can just satisfy 3 of these needs now, like circuit mentioned in Fig. 5. In other words, the best design we can achieve now by no more other genes is to construct one warrior that can be only killed by the other but not two warriors. Of course, circuit in Fig. 5 is not uniqueness for us to achieve 3 of these needs. However, since it can already satisfy our needs to verify the credibility of orthogonality test in killing test and give a better gene circuit based on it. Therefore, during our project, we use it when we mentioned these two needs, not other circuit.
(1)Warrior I can be killed by warrior II
(2)Warrior I cannot be killed by itself
(3)Warrior II can be killed by warrior I
(4)Warrior II cannot be killed by itself
We can just satisfy 3 of these needs now, like circuit mentioned in Fig. 5. In other words, the best design we can achieve now by no more other genes is to construct one warrior that can be only killed by the other but not two warriors. Of course, circuit in Fig. 5 is not uniqueness for us to achieve 3 of these needs. However, since it can already satisfy our needs to verify the credibility of orthogonality test in killing test and give a better gene circuit based on it. Therefore, during our project, we use it when we mentioned these two needs, not other circuit.
Killing Ability Test
I. Background
From our orthogonality test, we find that no matter how we construct, warrior I can’t fulfill our desire that it is only killed by warrior II but not itself. However, our orthogonality test uses RFP to represent the level of LacI, thus representing the killing ability. We want to find out if it is really the case in our warriors, so we construct our warriors and carry out the killing test.
II. Experimental design
To test how our warriors kill the enemies and if they kill themselves, we let equal amounts of the two kinds of warriors to grow together, with the warriors growing separately as negative controls. We attach different fluorescins to different warriors in order to differentiate them. We compare the killing ability of the warriors by monitoring the ratio of the two kinds of the warriors.
To test how efficient our warriors can kill the enemies, we do a gradient experiment. We let warriors and beggars from different sides to grow together with different starting ratios,with beggars growing alone as negative controls. The warriors and beggars from different sides are attached to different fluorescins. We monitor the change of the amount of beggars with time to see how the killing ability is.
To test how efficient our warriors can kill the enemies, we do a gradient experiment. We let warriors and beggars from different sides to grow together with different starting ratios,with beggars growing alone as negative controls. The warriors and beggars from different sides are attached to different fluorescins. We monitor the change of the amount of beggars with time to see how the killing ability is.
III. Methods
1. Construct our warriors and beggars by co-transformation. (the gene circuits are as follows)
*if you want to know the details of how we assemble the parts together, see our protocol “DNA construction”(download）
*if you want to know the details of how we assemble the parts together, see our protocol “DNA construction”(download）
Fig. 1 gene circuits of warriors and beggars from both sides
2. Pick bacterial clones from the petri plate, then shake it overnight in the LB medium (3ml) with 50μg/ml Ampicilin and 30μg/ml Kanamycin at 37℃. For each combination, 3 clones are picked. The clones are numbered from 1 to 3.
3. Dilute the overnight culture to 1/50 of the original density in fresh LB medium (5ml) containing 50μg/ml Ampicilin and 30μg/ml Kanamycin.
4. Incubate the fresh cultures at 37℃ until OD600 reach 0.2.
A. Self-killing test
5. Construct the following 6 groups and then incubate the cultures at 37℃,shaking at 220rpm.
For each group, 3 replicates are constructed, numbered from 1 to 3. Each replicate contains the bacteria with the same number.
3. Dilute the overnight culture to 1/50 of the original density in fresh LB medium (5ml) containing 50μg/ml Ampicilin and 30μg/ml Kanamycin.
4. Incubate the fresh cultures at 37℃ until OD600 reach 0.2.
A. Self-killing test
5. Construct the following 6 groups and then incubate the cultures at 37℃,shaking at 220rpm.
For each group, 3 replicates are constructed, numbered from 1 to 3. Each replicate contains the bacteria with the same number.
6. Take out 200ul cultures obtained from Procedure 5 and measure the fluorescent intensity and the amount of bacteria using flow cytometry after 15h.
B. Killing ability test
5. Construct the following 8 groups and then incubate the cultures at 37℃, shaking at 220rpm.
For each group, 3 replicates are constructed, numbered from 1 to 3. Each replicate contains the bacteria with the same number. All the medium contain 100ug/ml chloramphenicol.
B. Killing ability test
5. Construct the following 8 groups and then incubate the cultures at 37℃, shaking at 220rpm.
For each group, 3 replicates are constructed, numbered from 1 to 3. Each replicate contains the bacteria with the same number. All the medium contain 100ug/ml chloramphenicol.
6. Take out 200ul cultures obtained from Procedure 5 and measure the fluorescent intensity and the amount of bacteria using flowcytometry after 15h.
IV. Results
A. Self-killing test
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1. Test of self-killing: Warrior I kills itself but warrior II doesn’t We compare the amount of warriors in medium containing A+K with the amount of warriors in medium containing C. If the amount is apparently lower in the medium containing C, the warrior must have killed itself. We find that the amount of warrior I is apparently lower in C group, which means that warrior I kills itself. The figure shows the result. The ratio represents the amount of warriors in C medium divided by in A+K medium.(group 4/group 1, group 5/group 2) *A+K stands for ampicillin + kanamycin, C stands for chloramphenicol. |
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2. Test of the equality of killing: the killing ability of warrior II is far stronger than warrior I We calculate the ratio of Warrior I/Warrior II in group 3 and group 6. If the ratios are alike, then the killing ability of the warriors are similar. The figure shows the result. We can see that in the medium containing A+K, the amount of the warriors from two sides are roughly equal. At the same time, in the medium containing C, the amount of Warrior II is much larger. This is easy to explain as not only does Warrior II kills warrior I, but Warrior I kills itself as well. *A+K stands for ampicillin + kanamycin, C stands for chloramphenicol. |
B. Killing ability test
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1. Test of killing ability We calculate the ratio of Beggar/Warrior in each group and the results can be seen from the figures. We can see that when we mix Warrior I with Beggar II, the amount of Beggar II after 15h is much more than the amount of Warrior I. This may partly because Warrior I kills itself and partly because its killing ability towards Beggar II is weak. In contrast with the result above, we find that when we mix Beggar I and Warrior II together, the amount of Warrior II surely surpasses that of Beggar I. This means that the killing ability of Warrior I is very obvious. |
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2. Killing significance of warriors Furthermore, we compared the amount of beggars when they are cultured alone with the amount of beggars when they are cultured with warriors. We calculated the amount of beggars in group 7/group 8 to see the change in beggar I and in group11 /group 12 to see the change in Beggar II. From this figure, we can see that the amount of beggar I drops drastically when it is cultured with Warrior II, which means that Warrior II really work. As the amount of Beggar II does not have an apparent change, we may say that warrior I’s killing ability is surely too weak. |
V. Analysis
Firstly, we ensure that our warriors can really kill. The amount of beggars do decrease when the warriors from the other side is added and the more warriors, the less beggars. (tset of killing ability)
Secondly, we get to the conclusion that warrior II is actually quite ideal as it does not kill itself and that its killing ability towards Beggar I is obvious. Warrior I needs some improvement because it kills itself and the killing ability is rather weak.
These results are consistent with our orthogonality test, as Warrior II is truly ideal but warrior I is not. Therefore, it is reasonable that we use RFP to indicate killing effect of warriors in orthogonality test.
In order to improve Warrior I, we have to modify our gene circuit so that warrior I does not kill itself and at the same time, effectively kills Beggar II. (see improved gene circuit)
Secondly, we get to the conclusion that warrior II is actually quite ideal as it does not kill itself and that its killing ability towards Beggar I is obvious. Warrior I needs some improvement because it kills itself and the killing ability is rather weak.
These results are consistent with our orthogonality test, as Warrior II is truly ideal but warrior I is not. Therefore, it is reasonable that we use RFP to indicate killing effect of warriors in orthogonality test.
In order to improve Warrior I, we have to modify our gene circuit so that warrior I does not kill itself and at the same time, effectively kills Beggar II. (see improved gene circuit)
