Team:Tsinghua-A/orthogonality test
TSINGHUA-AOrthogonality test
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 these 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.
The detailed plasmid construction design is shown below.
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.
The detailed plasmid construction design is shown below.
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 the results above, how could we design our two warriors? Let's see!
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.
VI Reference
[1]Tokyo_Tech 2014: https://2014.igem.org/Team:Tokyo_Tech/Experiment/Plux_and_Prhl_reporter_assay
