Killling Ability Test
I. Background
The ideal killing ability of the warriors includes the two following characters. First, the killing ability of the warriors from the two sides must be roughly equal. Second, the killing ability of the warriors must be adequate——neither too strong nor too weak.
II.Experimental design
To test if the killing ability of the warriors from two sides are the same, we let equal amount of the two kinds of warriors to grow together. 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 if the killing ability is adequate or not, we do a gradient experiment. We let warriors and beggars from different sides to grow together with different starting ratios. 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 if the killing ability is ideal or not.
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”
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.Test the equality of the killing ability
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 flowcytometry after 15h.
B.Test if the killing ability is adequate
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.
A.Test the equality of the killing ability:
1.warrior 1 kills itself but warrior 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 1 is apparently lower in C group, which means that warrior 1 kills itself. Figure1 shows the result. The ratio represents the amount of warriors in C medium divided by in A+K medium.(group4/group1,group 5/group 2) *A+K stands for ampicilin+kanamycin,C stands for chloramphenicol.
2.The killing ability of the two warriors are not equal We calculate the ratio of warrior 1/warrior 2 in group 3 and group 6. If the ratios are alike, then the killing ability of the warriors are similar. Figure 2 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 2 is much larger.This is easy to explain as not only does warrior 2 kills warrior 1, but warrior 1 kills itself as well. *A+K stands for ampicilin+kanamycin,C stands for chloramphenicol.
IV.Results:
B.Test if the killing ability is adequate
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 warrior1 with beggar 2, the amount of beggar 2 after 15h is much more than the amount of warrior 1.This may partly because warrior1 kills itself and partly because its killing ability towards beggar 2 is weak.
In contrast with the result above, we find that when we mix beggar1 and warrior 2 together, the amount of warrior2 surely surpasses that of beggar1. Besides, the killing ability of warrior2 is adequate. The killing ability is neither too strong when the ratio is 1:1, nor is it too weak when the ratio is 1:10.
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 beggar1 and in group11/group12 to see the change in beggar2.From figure 4, we can see that the amount of beggar1 drops drastically when it is cultured with warrior2, which means that warrior2 really work. As the amount of beggar2 does not have an apparent change, we may say that warrior1’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.(figure 3) Secondly,we get to the conclusion that the killing ability of the two warriors are apparently not equal. The killing ability of warrior2 is actually quite ideal as it does not kill itself and that its killing ability towards beggar1 is obvious but not excessively strong. The killing ability of warrior1 is rather weak. The weakness is partly because that warrior1 kills itself, and partly because it does not effectively kill beggar2. This result is consistent when our orthogonality test, as the orthogonality of warrior2 is quite well, but the orthogonality of warrior1 is not very good. In order to solve this problem, we have to modify our gene circuit so that warrior 1 does not kill itself and at the same time, effectively kill beggar2.
TSINGHUA-A