Difference between revisions of "Team:Jilin China/Geneguard"

key achievements

1. Verify toxicity of CbtA towards bacteria, which stagnates the growth of bacteria

2. verify CbeA of its growth-promoting effects towards bacteria

3. verify when both CbtA and CbeA exist, bacteria can grow normally

4. verify after poisoned by CbtA for a while, growth of bacteria can be covered with the expression of CbeA

Introduction

Geneguard system is the most important system in our project (What is Genegraud system?). For the verification of the system, we designed the characterization construct shown in figure 1, and we named it as pET23a-TA.

Fig.1. This is the characterization construct. We constructed it to verify the function of Geneguard system.

We used the lactose operon in pET28a so that the expression of CbeA can be regulated by IPTG. What is more, after referring to the structure of BBa_I0500 （superlink to BBa_I0500), we added AraC and pBAD, so that we could also control the expression of CbtA by adding L-arabinose. So the construct can be simplified into figure 2. We can tell from the structure that the addition of Laracinose and IPTG can promote the expression of CbtA and CbeA,respectively.

Fig.2. Simplied Characterization construct. We can tell from the structure that the addition of L-arabinose and IPTG can promote the expression of CbtA and CbeA, respectively.

Experimental Design

The plasmid pET28a-TA and the vector, pET28a were transformed into BL21(DE3)pLysS. After single and double enzymes digestion, the plasmids were sent for sequencing. The groups of bacteria with the two plasmids transformed were named TA group and Vector group, respectively. We designed three experiments to verify the function.

ONE. plate streaking and monoclone growth observation According to others' work (Heller DM et al.,2017), we selected the appropriate concentrations for inducers and did the pre-experiments to confirm them. Medium with different types of inducers were made and we observed the growth of the bacteria in them.

	Plate A	Plate B	Plate C	Plate D
0.1%Ara	—	—	+	+
0.1mMIPTG	—	+	—	+

TWO. drawing Abs600-time curve under different inducers (read more in protocol)

The population and growth condition can be told by Abs600, so through the curve of Abs600-time, the effect of CbeA and CbtA towards bacteria can be displayed.

1. CbtA function validation: L-Arabinose was added into TA group bacteria solution to induce the expression of CbtA and Abs600 values were measured along culturing. The Abs600-time curve was drawn according to the values. To exclude the impact of L-arabinose towards bacteria, we used the vector group as the control.

2. CbeA function validation: IPTG was added into TA group bacteria solution to induce the expression of CbeA and Abs600 values were measured along culturing. The Abs600-time curve was drawn according to the values. To exclude the impact of IPTG towards bacteria, we used the vector group as the control.

3. The recovering effect of CbeA towards CbtA validation: L-Arabinose was added into TA and vector groups bacteria solution to induce the expression of CbtA. After the bacteria displayed poisoning features, IPTG was added. Abs600 values were measured along culturing. The Abs600-time curve was drawn according to the values. To exclude the impact of inducers towards bacteria, we used the vector group as the control.

THREE. cellular morphology observation

Results and Discussion

ONE. plate streaking and colony growth observation

Result: This experiment was carried out by BIT-China (read more). The results show that addition of arabinose, leading to the expression of toxin, can greatly suppress the growth of bacteria. In the contrary, the addition of IPTG or both inducers show no significant effect, which confirm that the expression of antitoxin can relieve the suppression of toxin in bacteria.

Discussion: Since no colony froms in the medium with L-Arabinose, we can conclude that the expression of CbtA can suppress the growth of bacteria. When IPTG and L-Arabinose are added together, which means that both CbtA and CbeA exist in the bacteria, the bacteria can growth and reproduce normally. Work of Masuda H et al. thought that the function of CbeA acts as an antagonist, rather than binding to CbtA, which is different from other Type-II Toxin-Antitoxin system.

Fig.3.Using plates with different kinds of inducer, we detected the growth conditions of bacteria with TA system. (A) No inducer was added into the medium, after 16 hours of streak cultivation, the bacteria grew normally. (B) 0.1mM of IPTG was added to induce the expression of antitoxin, after 16 hours of streak cultivation, the bacteria grew normally. (C) 0.1% of arabinose was added to induce the expression of toxin, after 16 hours of streak cultivation, the bacteria couldn't grow in the medium. (4) 0.1mM of IPTG and 0.1% of arabinose was added to induce the expression of toxin and anti-toxin, respectively. After 16 hours of streak cultivation, the bacteria grew normally.

TWO. drawing Abs600-time curve with different inducers

1. CbtA function validation:

Results: Abs600 values were measured from 0 to 14 hours in two groups and the figures were drawn as figure 4. The growing rate of vector group bacteria after adding L-Arabinose becomes larger for a short time and then slows down. At last, the trend is recovered just like that without L-Arabinose. However, TA group bacteria, with the addition of L-Arabinose, showed the same OD and Abs600 values from the 4th hour for a while. After that, Abs600 values display a little increase. Besides, turbidities of bacteria in different groups can be told by eyes after 14 hours of culturing (fig.5).

Discussion: (1) The reason why the growth of vector group bacteria accelerates after adding L-Arabinose may because that E.coli can turn L-Arabinose into DXylulose 5-phosphate, which is an intermediate product in pentose phosphate pathway and can be used in bacteria as a carbon resource. What is more, the decrease after that maybe related with Carbon catabolite repression(CCR) Görke B et al. referred to in 2008. (2) According to the characterization of pBAD, the addition of L-Arabinose with the concentrations of 0.1%, 0.2% and 0.6% will not influence the expression of CbtA. Therefore, the difference among different concentrations of L-Arabinose in TA group maybe because of the metabolism of L-arabinose. with the consuming of L-Arabinose, the concentration becomes lower. The expression of CbtA decreases, subsequently, so the bacteria can recover from the toxicity of CbtA automatically. Also the concentrations of 0.1% and 0.2% are too low for TA group to be poisoned by CbtA after the 6th hour. (3) Comparing the growth trends of two groups of bacteria, we conclude that the addition of L-Arabinose leads to the expression of CbtA, so the the bacteria stop reproducing, which proves the toxicity of CbtA(We think that Western Blot can better prove it but the time is not enough and we add this experiment into our future work).

Fig.4. (A) Different concentrations of L-Arabinose were added into vector group and the growth curve was drawn to made sure the effect of L-Arabinose towards bacteria. According to the results, the addition of L-Arabinose would first slightly enhance and then suppress the growth. However, this effect would disappear after a while. (B) Different concentrations of L-Arabinose were added into TA group and the growth curve was drawn to made sure the effect of CbtA(induced by L-Arabinose) towards bacteria. According to the results, the addition of LArabinose the growth would be stagnated at first and then recover. Fig.

Fig.5. (A) and (B) The two figures were taken to compare the turbidities of TA group after 14 hours od culturing, and the difference could be told by eyes.

2. CbeA function validation

Result: We measured the Abs600 values of TA and vector group from 0 to 16 hours and the Abs600- time curve was drawn(fig.6). The Abs600 values with different concentrations of IPTG in TA-pET28a group were higher than pET28a group. Without the induction of IPTG, the growth curves were almost the same.

DIscussion: (1) The growth of vector group after adding IPTG will be stagnated compared with that without IPTG. This is because IPTG shows toxicity towards bacteria. (2) Comparing the Abs600 values between TA group and vector group, the growth condition of TA group is better than that of vector group. The reason for it is that the expression of CbeA is induced by IPTG, and CbeA promotes the assembly reactions of MreB and FtsZ (Masuda H et al.2012). Therefore, we speculate that CbeA can promote the reproduction of bacteria. (3) Comparing TA group with IPTG addition to that without addition, we can tell that the growth rate is also decreased. We think that it is because the effect of CbeA cannot offset the toxicity of IPTG.

Fig.6. Investigate the effect of CbeA towards bacteria after induced by IPTG. From the curve we can know that CbeA expression can promote bacteria growth. However, the toxicity of IPTG cannot be offset by CbeA.

3.The recovering effect of CbeA towards CbtA validation

Result: The Abs600 values were measured within TA group and pET28a group from 0 to 16 hours and the Abs600 curves were drawn (fig.7A and B). After adding IPTG, the growth rate of TA group is significantly larger than that without the addition of IPTG. However, in pER28a group, the growth rate is smaller.

Discussion: After adding L-Arabinose, bacteria in TA group is poisoned. After that, the addition of IPTG accelerate the growth of TA group. So we think that CbeA can release the toxicity of CbtA in some degree. At last, the value of Abs600 is smaller than that of normal growth group. We think it is because the measuring time is not long enough. Just like previous observation, in vector group, the addition of L-Arabinose first accelerate then slow down the growth rate. Then the growth rate recovers to normal condition. However, the existence of IPTG lower the growth rate.

Fig.7. After orderly adding L-Arabinose and IPTG, the expression of CbtA and CbeA was induced and its effect on the growth of bacteria was shown. When OD value reached 0.15, L-Arabinose was added to induce the expression of CbtA. After the OD values remained unchanged for a while, IPTG was added simultaneous to induce the expression of CbeA. (A) The growth rate of TA group with IPTG addition is larger than that without IPTG addition. (B) The growth rate of vector group with IPTG addition is smaller than that without the addition of IPTG.

THREE

Experience

Compare with the vector group, the bacteriolysis phenomenon often happened to TA group bacteria. We assume that it is because CbtA will express without the addition of IPTG. This is relative with the feature of lactose operon, which bring us some troubles.

Future work

1. Repeated experiment：Since the time limitation and the appearance of bacteriolysis, more times of repetition are needed in the future.

2. Western blot: Just as it referred in discussion part, we want to further confirm the expression of CbeA and CbtA.

3. More constructs are needed to verify the Geneguard system. So we need to design more structures to further valid Geneguard system.