Difference between revisions of "Team:OUC-China/Demonstrate"

Revision as of 11:19, 11 October 2018

Team OUC-China: Main

Demonstrate

The result of first system

Plasmid construction

First, we use an inducible promoter Ptac to regulate the expression of Csy4. Without the IPTG, the circuit of Csy4 closes at the same time. On the contrary, Csy4 enzyme produce and help regulate the expression of downstream genes of miniToe structure in another plasmid as a part of our first system. Also, we use the promoter J23199 from Anderson family which is a constitutive promoter to regulate the reporter circuit. So if our system works well, we will get some curves for fluorescence intensity as our expectation.

Fig.1-1 The two plasmids of miniToe test system.

Selective Medium Assay

After circuit construction to get the two plasmids we mentioned before, we transformed both of them into E.coli DH5 Alpha and got the miniToe testing strain successfully. Because the promoter Tac has high leakage in LB medium so we culture our recombinant strain in M9 medium. We measured the growth rate of both our engineered strain and the negative control as preliminary experiment. As a result, the curve well demonstrates that the strain with our system has almost some OD600 with the negative control strain during the entire cultivation period. It means that our system has no big negative influence on the growth of strain. The metabolic stress by two plasmids is not harm to our recombinant strain.

Fig.1-2 Growth curve of strains of our recombinant strain (with the whole miniToe system including two plasmids) and negative control groups. Error bars represent standard deviation of four biological replicates. (Measured by microplate reader)

Proof of function

We use microplate reader to test the fluorescence intensity of sfGFP which is changed over time. getting data to support our idea directly that our system can control the downstream gene expression during the whole cultivation period.
The following chart shows the dynamic curve measured by microplate reader. We test our system every two hours. The yellow line is the symbol of test group which is a recombinant strain (with the whole miniToe system including two plasmids) with IPTG (0.125mM). The blue line shows the change of fluorescence intensity by a recombinant strain(with the whole miniToe system including two plasmids) without IPTG (0mM). The green line is also a control group in our system, it shows the fluorescence intensity of sfGFP by a strain with only one kind of plasmids (pReporter) which only has miniToe structure without the Csy4. The result by this curve help us to prove two functions in miniToe system.

Fig.1-3 The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups which means three different strains we tested in the chart. The yellow line is a test group with IPTG (0.125mM). The blue line is a control group without IPTG (0mM). The green line is a control group with only one plasmid (pReporter).

The first problem is whether our miniToe structure fold exactly. So first we predict our secondary structure by using mfold(http://unafold.rna.albany.edu/?q=mfold) and RNAfold(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). We predict the whole structure of our circuit and structure of miniToe to see if our structure can fold directly on the level of RNA. By the result of prediction, we just find our structure can fold directly after transcription.

Fig.1-4 The structure prediction of the whole circuit and miniToe. The structure of miniToe is on the right of picture and the structure of whole circuit is on the left of picture. The red frame indicates the miniToe structure in the whole circuit.

But in fact, we also need to prove that our miniToe can fold directly in reality by experiments. As the result showed in Fig1-3, one of the control group (the green line) is relatively stable during the whole process comparing with two other strains. This means the miniToe without Csy4 folds well in secondary structure on the level of RNA and also keep the OFF state so we can’t detect the changes of fluorescence intensity by sfGFP because the translation of sfGFP is closed.

Fig.1-3 The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups which means three different strains we tested in the chart. The yellow line is a test group with IPTG (0.125mM). The blue line is a control group without IPTG (0mM). The green line is a control group with only one plasmid (pReporter).

The second problem we need to prove is that whether our miniToe system can work successfully as a switch to regulate the downstream genes. Obviously, in the Fig1-3 we can find that there is a rise in expression of sfGFP between two lines in the whole process. The yellow line is the test group with the IPTG and the blue line is a control group without IPTG. It is not difficult to find that the fluorescence intensity of control group (the blue line) is always lower than test group (the yellow line). This means our system can work successfully.
At the same time, we find the control group without IPTG (the blue line) has leakage compared with other two group. Because the control group with only one plasmid (the green line) has the stable and low expression of sfGFP, the leakage may result from the Inductive promoter Ptac. Even though the control group has leakage of sfGFP, we can prove the function of our system successfully. But in the future, we may have more time to find a better promoter which is also suitable for our system.
We also test our system by Flow cytometric, the blue group showed in the Fig.1-5 is the test group when the white group is a control group. It’s easy to distinguish the two groups and the test group has the obvious increase compare to the control group. The result shows the same conclusions we mentioned before.

Fig.1-5 Flow cytometric measurement of fluorescence of sfGFP. Histograms show distribution of fluorescence in samples with test group with IPTG (green) and control group without IPTG(white). Crosscolumn number shows fold increase of sfGFP fluorescence. The strain we use in test group is a recombinant strain (with the whole miniToe system including two plasmids) with IPTG (0.125mM). And the control group is a recombinant strain (with the whole miniToe system including two plasmids) without IPTG (0 mM).

另一个问题，曲线为什么会下降？

Discussion

Combining the biology and math, we discuss the dynamics of GFP in the Fig.1-3 now. In order to explain in detail, we present the dynamics of all species in the miniToe system in Fig.1-6.

Fig.1-6 The dynamics of all species in the miniToe system

In the Fig.1-3, the red line which represents the dynamics of GFP which increases in the beginning and then drop down to a stable level. The reason is that the capability of Csy4’s cleavage is stronger. And the capability of mRNA’s production() is relate weaker which results in the decline of after 10 hours. Before we add IPTG to induce the Ptac, the is accumulated because it is under controlled by a constitutive promoter. After we add IPTG, the initial concentration of plays an important role in the production of GFP during the first 10-hour. Even though the rate of cleavage is faster than the production of , the concentration of mRNA keeps increasing. But once the original is consumed, the stop increasing and drop down to a stable level. So the balance of the product rate and decay rate can kept. This is the reason why the level of sfGFP keep stable finally in Fig1-3.
See more details in model! Click here!
(https://2018.igem.org/Team:OUC-China/miniToe)

collaboration：结果对比正确/SDU/SK

Fig.1-7 The result from other four teams which have proved our conclusions.

We also have collaborations with other 4 teams, and they help us in proving our results by experiments in their labs. Thank you! See more details here! (https://2018.igem.org/Team:OUC-China/Collaborations)