1. Certification of the CbtA toxicity towards bacteria, which stagnates the growth of bacteria.
2. Demonstration of CbeA growth-promoting effects towards bacteria.
3. Certification bacterial normal growth on condition that CbtA-CbeA co-existence.
4. Verification of CbtA reversible toxicity blocked by CbeA.
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.
Figure 1. Characterized construction of TA.
We used the lactose operon in pET28a so that the expression of CbeA (antitoxin) can be induced by IPTG. What is more, after referring to the structure of BBa_I0500, we added AraC and pBAD, so that we could also induce the expression of CbtA (toxin) by L-arabinose. So the construct can be simplified into Figure 2. We can know from the structure that the addition of Laracinose and IPTG can enhance the expression of CbtA and CbeA, respectively.
Figure 2. Simplified characterized construction. Addition of L-arabinose and IPTG can enhance the expression of CbtA and CbeA, respectively.
The plasmid pET28a-TA and the vector pET28a were transformed into BL21(DE3)-pLysS. After digestion and sequencing. Two groups of bacteria with the two plasmids transformed were named TA group and Vector group, respectively. Three experiments were designed to verify the function.
plate streaking and monoclone growth observation
According to others' work , 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.This experiment was carried out by BIT-China (read more)
|Plate A||Plate B||Plate C||Plate D|
dDrawing Abs600-time curve under different inducers
The population and growth condition can be reflected by Abs600, so through the curve of Abs600-time, the effect of CbeA and CbtA towards bacteria can be displayed. (read more in protocol)
1. CbtA function validation: L-Arabinose was added into TA group bacteria 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 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 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.
Cellular morphology observation
Two significant changes will happen when Geneguard takes effect. One is the delaying in bacterial growth, the other one is transformation in bacterial morphology. The two experiments we designed above certified geneguard's function through delaying in bacterial growth. We also wanted to demonstrate it through transformation in bacterial morphology. However, we lacked appropriate equipment for observation. Thus, we seek help from our partner NKU(read more). We designed the morphology experiment according to our pervious work. Groups of control, 0.2% arabinose induction, 0.2% arabinose and IPTG co-induction were set. Bacterial morphology was observed in 1.5, 3 and 6 hrs.
plate streaking and colony growth observation
Result: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 forms in the plate with L-Arabinose, we can conclude that the expression of CbtA can suppress the growth of bacteria. When CbeA and CbtA are co-induced by IPTG and L-Arabinose, the bacteria can growth and reproduce normally. It is reported that CbeA acts as an antagonist, rather than binding to CbtA, which is different from other Type-II Toxin-Antitoxin system.
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 toxication curves were drawn as shown in Figure 4. The growth rate of vector group bacteria after adding L-Arabinose sped up for a short time and then slowed down. At last, the trend was consistent with that without L-Arabinose. However, TA group, with the addition of L-Arabinose, showed the same OD and Abs600 values from the 4th hour for a while. After that, Abs600 values displayed a little increase. Besides, after culturing for 14 hrs, difference in turbidities of bacteria in different groups was visible .(Figure 5.)
Discussion: (1) The reason why the growth of vector group bacteria accelerates after addition of L-Arabinose is probably 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). (2) According to the characterization of pBAD, the addition of L-Arabinose with the concentrations of 0.1%, 0.2% and 0.6% shows no obvious difference in induction level of CbtA. Therefore, the difference among various 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 gradually, 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 toxicity of cbtA induced by L-Arabinose can make bacteria stop reproducing. (We think that Western Blot can better prove it but the time is limited and we add this experiment into our future work).
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.(Figure 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 addition. 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. It is because that CbeA induced by IPTG promotes the assembly reactions of MerB and FtsZ. 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.
3.The recovering effect of CbeA towards CbtA validation
Result:The Abs600 values were measured within TA group and vector group from 0 to 16 hours and the Abs600 curves were drawn (Figure 7.A and B). After adding IPTG, the growth rate of TA group is significantly larger than that without the addition of IPTG. However, in vector 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. However, the value of Abs600 is smaller than that of normal growth group at last. 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.
cellular morphology observation
Result:As Figure 8. shows, bacterial shape in control group didn’t have evident change in 1.5,3 and 6 hrs, all in rhabditiform. Arabinose induction group appeared bacilliform in 1.5 hrs. In 3 hrs observation, part of bacterial turned into roundness. In 6 hrs observation, most of bacteria became rounded. The arabinose and IPTG co-induction group appeared bacilliform, in 3 hrs observation, part of bacterial turned into roundness. In 6 hrs observation, most of bacteria were bacilliform.
Discussion:E.coli in control group didn’t express inducted heterologous protein. Thus, they appeared bacilliform. While in arabinose induction group, part of bacteria became rounded in 3 hrs and most of them turned into roundness in 6 hrs. we can consider that toxin CbtA led to the transformation in bacterial morphology.Part of bacteria in Arabinose and IPTG co-induction group turned rounded in 3 hrs observation. That’s because the toxin addicted time was too short that only part of bacteria expressed cbtA. While expression of cbeA made most bacteria appeared in bacilliform in 6h observation. Because the experiment time was limited, we didn’t get the distinct result. We think that more solid result will appear if we extend the observation duration.
1. Western blot: Just as it referred in discussion part, we want to further confirm the expression of CbeA and CbtA.
2. More constructs are needed to verify the Geneguard system. So we need to design more structures to further valid Geneguard system.
3. We can also apply our Geneguard into other fields like petroleum industry and fermentation industry through well- designed circuits.