Team:Tsinghua-A/killing test
TSINGHUA-A
Killing 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 effect. 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 fluorescent proteins to different warriors in order to differentiate them. We compare the killing effect 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 fluorescent proteins. We monitor the change of the amount of beggars with time to see how the killing effect 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 fluorescent proteins. We monitor the change of the amount of beggars with time to see how the killing effect 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 effect 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 effect 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
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.
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.
2. Test of the equality of killing: the killing effect 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 effect 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.
We calculate the ratio of Warrior I/Warrior II in group 3 and group 6. If the ratios are alike, then the killing effect 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 effect test
1. Test of killing effect
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 effect 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 effect of Warrior I is very obvious.
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 effect 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 effect of Warrior I is very obvious.
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 effect is surely too weak.
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 effect 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 effect)
Secondly, we get to the conclusion that warrior II is actually quite ideal as it does not kill itself and that its killing effect towards Beggar I is obvious. Warrior I needs some improvement because it kills itself and the killing effect 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 effect towards Beggar I is obvious. Warrior I needs some improvement because it kills itself and the killing effect 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)
VI Reference
[1]Tokyo_Tech 2014: https://2014.igem.org/Team:Tokyo_Tech/Experiment/Symbiosis_confirmation_by_co-culture
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