Team:RDFZ-CHINA/Engineering
Brief Introduction
On this page, we will introduce the experiments that tested the validity of all components. However, because we have limited time in the laboratory, most experiments remained in the design state.
1.Xanthomonas Cultivation
Since Xanthomonas. Oryzea and Xanthomonas. Campestris are significantly different from E.coli, we firstly tested that whether Xoo and Xcc can grow in normal NA media.
These two images show the results of xanthomonas media after 12h in 28˚C shaker. The xanthomonas orzyae, xanthomonas campestris, and control group were tested separately. We repeated this experiment twice.
Because in both experiments the mean OD600 of Xoo and Xcc were smaller than 0.5 and the value had no difference between control groups, we concluded that the bacteria didn't grow. Therefore, we decided to change the nutrients in the liquid medium in order to culture Xoo and Xcc.
List of improved nutrients components is provided.
In this image the OD600 value of both the experimental group and control group were still under 0.5. We speculated this because the low bacteria activity caused by the long time preservation in the 4-degrees-Celsius refrigerator. We had no chance to preserve Xoo and Xcc bacteria samples in -80 degrees Celsius immediately after they were sent to our laboratory because the laboratory was forced to shut down due to the pandemic.
Future plan: We will verify our hypothesis about bacteria activity by asking the company. Finding and configuring medium with new nutrient components. The results of this experiment can help us to find the most effective and cheapest media which can be used in massive industrial production in the future.
In this image the OD600 value of both the experimental group and control group were still under 0.5. We speculated this because the low bacteria activity caused by the long time preservation in the 4-degrees-Celsius refrigerator. We had no chance to preserve Xoo and Xcc bacteria samples in -80 degrees Celsius immediately after they were sent to our laboratory because the laboratory was forced to shut down due to the pandemic.
Future plan: We will verify our hypothesis about bacteria activity by asking the company. Finding and configuring medium with new nutrient components. The results of this experiment can help us to find the most effective and cheapest media which can be used in massive industrial production in the future.
2. DSF testing experiment(in Xanthomonas)
DSF part test experiment
The sensitivity of Vc2riboswitch can be tested by the reporting EYFP circuit we constructed downstream. As the Vc2riboswitch is activated, the EYFP will be expressed, emitting a fluorescent signal. The fluorescent signal can then be tested by the machine for the further evaluation of its sensitivity.
Assumption of result are shown on the right.
1. engineered DSF-pBBR plasmid
a.DSF+
b.DSF-
In this graph, we assume there is change in the intensity of Xanthomonas's fluorescence signal after the introduction of DSF-sensor circuit. DSF-pBBR represents the pBBR plasmid with vc2riboswitch pathway transformed into Xoo. Xoo-pBBR represents the plasmid pBBR without any construct transformed into Xoo. Xoo represents the Xanthomonas without with any plasmid. The actual result that we do not have time to achieve should be in accordance with this prediction. If the Vc2riboswith has a strong leaking expression, all the groups should present high protein expression in 0μM. If the 1.a group presented low protein concentration through the separate concentration of DSF, the Vc2riboswitch may not work because it cannot adapt to the difference in RpfC and RpfG protein between two different bacteria. If the control groups(3.a and 3.b) presented high protein expression, which gives its own fluorescence signals, can interfer the EYFP Flurescene signal. By comparing 2.a and 2.b with 1.a, we can conclude that the pBBR plasmid we designed will not interfer with the function and EYFP expression. 2. Xoo-pBBR plasmid
a. DSF+
b. DSF- 3. Xoo
a. DSF+
b. DSF-
1. engineered DSF-pBBR plasmid
a.DSF+
b.DSF-
In this graph, we assume there is change in the intensity of Xanthomonas's fluorescence signal after the introduction of DSF-sensor circuit. DSF-pBBR represents the pBBR plasmid with vc2riboswitch pathway transformed into Xoo. Xoo-pBBR represents the plasmid pBBR without any construct transformed into Xoo. Xoo represents the Xanthomonas without with any plasmid. The actual result that we do not have time to achieve should be in accordance with this prediction. If the Vc2riboswith has a strong leaking expression, all the groups should present high protein expression in 0μM. If the 1.a group presented low protein concentration through the separate concentration of DSF, the Vc2riboswitch may not work because it cannot adapt to the difference in RpfC and RpfG protein between two different bacteria. If the control groups(3.a and 3.b) presented high protein expression, which gives its own fluorescence signals, can interfer the EYFP Flurescene signal. By comparing 2.a and 2.b with 1.a, we can conclude that the pBBR plasmid we designed will not interfer with the function and EYFP expression. 2. Xoo-pBBR plasmid
a. DSF+
b. DSF- 3. Xoo
a. DSF+
b. DSF-
3. Overexpression of GdpX1 protein in Xanthomonas influences in plant's leaf (in Xanthomonas)
In this experiment, we plan to test the effects of overexpression of gdpx1 protein on Xoo and Xcc.Ideal consequence and experiment we plan to repeat are shown on the lower left.
These images are cited from the article (Yang et al., 2016). Number 1: Xanthomonas Oryzae PXO099 with no execution. Number 2: gdpX1 protein with Xanthomonas. Number 3: gdpx1 system on pHM plasmid（a high expression plasmid in Xanthomonas）. Number 4: the empty pBBR plasmid with Xanthomonas. Number 5: the overexpression of plasmid pBBR-gdpx1 in Xanthomonas Oryzae cell. Image b displays the lengths of the lesions of each group. It is obvious that the length of lesions of number 5 leaf is the shortest, which indicates that number 5 leaf is least affected by Xanthomonas. Taking into consideration that only in leaf 5 are genes encoding gdpx1 present, we could come to the conclusion that only with gdpx1 present could the virulence of Xanthomonas oryzae be eliminated.
Experiment we design:
1. Xoo with GdpX1+LacI plasmid
a. IPTG+
b. IPTG-
We use IPTG inducing system to express the gdpx1 protein. the virluence task of 1.a and 1.b can let us conclude the function of gdpx1. Comparing the results of 1.a and 1.b lead to the conclusion that the expression of gdpx1 protein could reduce the toxicity of Xanthomonas. Comparing the results of 1.a with the results of 2.a and 2.b proves that it is not the pBBR plasmid who decrease the virulence. 2. Xoo with no plasmid
a. IPTG+
b. IPTG-
These images are cited from the article (Yang et al., 2016). Number 1: Xanthomonas Oryzae PXO099 with no execution. Number 2: gdpX1 protein with Xanthomonas. Number 3: gdpx1 system on pHM plasmid（a high expression plasmid in Xanthomonas）. Number 4: the empty pBBR plasmid with Xanthomonas. Number 5: the overexpression of plasmid pBBR-gdpx1 in Xanthomonas Oryzae cell. Image b displays the lengths of the lesions of each group. It is obvious that the length of lesions of number 5 leaf is the shortest, which indicates that number 5 leaf is least affected by Xanthomonas. Taking into consideration that only in leaf 5 are genes encoding gdpx1 present, we could come to the conclusion that only with gdpx1 present could the virulence of Xanthomonas oryzae be eliminated.
Experiment we design:1. Xoo with GdpX1+LacI plasmid
a. IPTG+
b. IPTG-
We use IPTG inducing system to express the gdpx1 protein. the virluence task of 1.a and 1.b can let us conclude the function of gdpx1. Comparing the results of 1.a and 1.b lead to the conclusion that the expression of gdpx1 protein could reduce the toxicity of Xanthomonas. Comparing the results of 1.a with the results of 2.a and 2.b proves that it is not the pBBR plasmid who decrease the virulence. 2. Xoo with no plasmid
a. IPTG+
b. IPTG-
4. dcas12a repression (in bacillus coil)
We use IPTG and cumate inducing system to repress a particular protein on the phage.
CymR expression pathway, LacI expression pathway, major tail protein expression pathway, and endolysin expression pathway are transferred into all the bacteria involved in the experiment.
Ideal consequences of protein concentration are shown in the picture on the upper right.
The results indicate that IPTG is responsible for dcas12a protein expression, and cumate is responsible for dcas12a protein expression.
1. Xoo with dcas12a and sgrna pathway
a. IPTG+，cumate+
b. IPTG+，cumate-
c. IPTG-，cumate+
d. IPTG-，cumate-
By comparing the data from 1.a and 1.b, we can exclude the interference that may caused by dcpf1. When the difference between results of 1.d and 1.a increases, the leakage of our phage releasing system decreases. The protein expression is hence inhibited to maximum level.
Further plan: So far, we constructed the dcas system in E.coli but we had no time to test the vaildity. In the future, we plan to edit RBS by PCR and reconstruct the whole system in pBBR plasmid, which will allow us to test our parts in Xanthomonas. If the endolysin and major tail protein expression remain low, we can conclude that our system is effective. 2. Xoo with no plasmid
a. IPTG+,cumate+
b. IPTG-,cumate-
1. Xoo with dcas12a and sgrna pathway
a. IPTG+，cumate+
b. IPTG+，cumate-
c. IPTG-，cumate+
d. IPTG-，cumate-
By comparing the data from 1.a and 1.b, we can exclude the interference that may caused by dcpf1. When the difference between results of 1.d and 1.a increases, the leakage of our phage releasing system decreases. The protein expression is hence inhibited to maximum level.
Further plan: So far, we constructed the dcas system in E.coli but we had no time to test the vaildity. In the future, we plan to edit RBS by PCR and reconstruct the whole system in pBBR plasmid, which will allow us to test our parts in Xanthomonas. If the endolysin and major tail protein expression remain low, we can conclude that our system is effective. 2. Xoo with no plasmid
a. IPTG+,cumate+
b. IPTG-,cumate-
5. AcrVA1 protein reactivation (in bacillus coil)
WAs long as experiment 5 is completed, the AcrVA1 gene expression system will be transformed into Xoo to confirm the validity of AcrVA1.
AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. After the strands of DNA are reconjugated, RNA polymerase in Xanthomonas can bind to the genetic material of the phage and express it. The first generation of phage will rapidly come in contact with and infect other harmful Xanthomonas. The generations of phages produced from the first generation of phages can reduce the concentration of Xanthomonas in 5~10 hours.
Reference
1. Yang, F., Qian, S., Tian, F., Chen, H., Hutchins, W., Yang, C.-H. ., & He, C. (2016). The GGDEF-domain protein GdpX1 attenuates motility, exopolysaccharide production and virulence in Xanthomonas oryzae pv. oryzae. Journal of Applied Microbiology, 120(6), 1646–1657. https://doi.org/10.1111/jam.13115
