Team:RDFZ-China/Results
Results
Demonstration:
1. The genetic circuit construction of 3 sensing system and 1 TPH1 effector system
2. the sensitivity task of 3 sensing systems by adding PCA
3. The enzymtic curve of TPH1 with serial dilution
1. Genetic circuit construction
The functional sequence we get from the company is constructed on pUC57 backbone. But pUC57 is not attached to all of our designs. We need to rebuild the plasmid, and change the arrangement of sequence. For example, our effector system is construct on Pet28b plasmid and bind with upstream LacI sequence.
1.1 Pcau-pPCA-pUC57:
First, we build pcau and eGFP report sequence on puc57 plasmid, a high-copy number plasmid. According to the design of UANL team in 2019, the length of composite should be 2700bp.
This figure shows the consequence of our bacterial colony PCR. Subsequent sequencing analysis also shows we successfully build PcaU-eGFP on puc57.
1.2 PcaUAM-p3B5B-YFP-pet28b:
The PcaUAm activator and p3B5B promoter are construct on p15A plasmid, and use eYFP as reporter.
We use seq-PcaUAM-F1 and Seq-pcauam-R as two primers of bacterial colony. So the length should be about 2400bp, and the first, fourth,sixth sample on the figure can be approximately 2000-3000 and the sixth one should be the most ideal one. The following sequencing analysis also proved this result.
1.3 PcauAM-p3b5c-YFP-pet28b:
PcaUAM-p3B5C-YFP-pet28b is just one base pair different between PcaUAM-p3B5B-YFP-pet28b so the gel elctrophoresis can not prove anything. But sequence analysis show our point mutation Gel electrophoresis results:
Sequence analysis results:
1.4 TPH1-LacI-Pet28b:
TPH1-LacI-pet28b is the effector we design to produce tryptophan hydroxylase. Gel electrophoresis results:
Sequence analysis:
1.5 MusTPH1-LacI-Pet28b:
We also collected a mouse-sourced musTPH1 and plan for the same characterization and testing. It is known to catalyze the same reaction human TPH1 did. Although the full experimentation data haven’t been complete on this biobrick, musTPH1 is proven to have high expression in the bacteria strain E.coli MC1061 with proper activity to produce concentration greater than 1mM under certain conditions by the article Park D H, Stone D M, Kim K S, et al. Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli[J]. Molecular and Cellular Neuroscience, 1994, 5(1): 87-93.
2. Activity test
Overview:
1. Sensor activity of PcaU-pPCA, PcaUAM-p3B5B, PcaUAM-p3B5C system under different emission light.
2. The enzymatic activity of hTPH1 induced by IPTG.
2.1 Pcau-pPCA-GFP: excitation: 485nm, emission:515nm:
The data on the left of figure14 are tested data of PcaU expressor, construct with pPCA downstream promoter. Two groups of our repeat data show PcaU sensing system have good reaction with the join of PCA and activate downstream promoter. Because the intensity of fluorescence signal increases when PCA is joined.
But we can also see the leakage of GFP is also very serious, because compare to our negative empty pUC57 plasmid, Pcau-pPCA plasmid have over three to four times fluorescence intensity even PCA is not joined, denoting unwanted expression of the downstream sequence. This may be the result of having our reporter genes on a high copy plasmid, which increases the overall leakage, which is not what we want.
2.2 PcaU-pPCA-GFP: excitation: 485nm , emission:535nm:
The data in the right of figure 15 are the tested data of PcaU-pPCA-GFP under emission light 535nm. We make a serial dilution of PCA to do further tasking on PcaU-pPCA control system. We found similar property that when PCA molecular is not added, the fluorescence intensity/bacterial concentration of pcauU-pPCA is about 36000. And the GFP protein expression of the sensing systems has increased since the concentration of PCA increase, and the value of F/A is increased when reflected in the image. The data shows pPCA promoter can be controlled by the pcau activator and the concentration of PCA can be regulated and controlled by the concentration of PCA.
2.3 PcauAM-YFP: excitation:485nm, emission:535nm:
The data in the right of figure 15 are the tested data of PcaUAM-YFP under emission light 525nm. When PCA molecular is not added, the fluorescence intensity divided by bacterial concentration of pcauAM-p3b5b and pcauAM-p3b5c are both about 3000. And when PCA is added, the YFP protein expression of the two sensing systems has increased to different degrees, and the value of F/A is increased when reflected in the image. The data shows both p3b5b and p3b5c promoter can be controlled by the pcauAM activator. And under the horizontal comparison between p3b5b and p3b5c promoter, p3b5b promoter have much higher sensitivity than p3b5c promoter. However, consider our downstream system will be TPH1 sequence, we don't want the control system that is too sensitive, because too much TPH1 express might cause the 5-HTP symptom.
2.4 PcaUAM-YFP: excition:485nm, emission:525nm:
The data in the left of figure in figure 14 are the tested data of PcaUAM-YFP under emission light 535nm varied by different concentration of PCA.
Best sensor:
Comparing the leakage and sensitivity, we found that PcauAM expressor-p3b5b downstream promoter are the best sensor system considering the standard of our project. It has the relatively lower leakage comparing with PcaU-pCPA system, also with relatively low sensitivity comparing to PcaUAM-p3B5C promoter.
Effector:
Standard curve of 5-HTP:
Figures 18 above show the absolute standard curve we make in two experiments. From the figure, it is clearly that under the light absorption of 330, as the concentration of 5-HTP increases, the light absorption also increases.
The absolution data is seen varied with time, but ideally the standard curve at all times would be overlap. Because theoretically We think about it and the possible reason the temperature change during the test.
To avoid this data happen again, we plan to heat all prepare solution 20 minutes before we mix all solution together.
Cons enzymatic reaction:
TPH1:
In this experiment, we make the concentration gradient of enzyme and label TPH1 column 1/2/3/4/5/6. The line chart show the absorption in 330 nm (relate with the concentration of 5-HTP)is vary by two factor, "Time", and the concentration of enzyme.
Because the we use solution with bacteria but not pure protein solution in this experiment, the volume spontaneous fluorescence interfered with the fluorescence value. Which make the difference between adding IPTG induce and no adding very small.
It is given that the substrate concentration of tryptophan in the final reaction system is 60M, all data above this concentration are unreliable(the concentration of the product will never exceed this concentration, which would violate the conservation of atomic numbers, so the data for column 1,2 are discarded)
However, we can still prove our enzyme is working with this data. Because the concentration of 5-HTP first increases very fast at the beginning and then the reaction slow down after about 20min, which conforms to the reaction curve of a normal enzyme.
As we dilute the solution containing TPH1 from 6 to 3, we have TPH1 initial concentration increasing from 3 to 6. As the concentration of initial TPH1 increases, the initial gradient of 5-HTP also increases. This indicated that our TPH1 is functional. We also build a model to explain this occurrence. It can be found on our model page.