Difference between revisions of "Part:BBa K4817008"
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<p>Expression of luxR was regulated using the constitutive promoter J23100 ligated to RBS B0034 followed by terminator B0010. Expression of downstream sfGFP was regulated using the quorum sensing promoter PluxR ligated to RBS B0034 followed by terminator B0010.</p>
 
<p>Expression of luxR was regulated using the constitutive promoter J23100 ligated to RBS B0034 followed by terminator B0010. Expression of downstream sfGFP was regulated using the quorum sensing promoter PluxR ligated to RBS B0034 followed by terminator B0010.</p>
  
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                                    <img src="https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/4.png" style="width: 200;">
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                                    <p class="zhushi">Fig. 2 Preferability on Different AHLs of our system</p>
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                                <p style="text-align: center;width: 100%;">
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                                    <img src="https://static.igem.wiki/teams/4817/wiki/wiki/results/part4/5.png" style="width: 200;">
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                                    <p class="zhushi">Fig. 3 Effects on Different AHLs Concentration of our system</p>
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<p>The bacterial solution was incubated until OD600 reached 0.6, and 30× oxo-C6-HSL solution was pre-configured by adding 290 μl of bacterial solution and 10 μl of AHL solution to a 96-well plate. A total of 8 concentration gradients were set up as 0, 10e-10, 10e-9, 10e-8, 10e-7, 10e-6, 10e-5, and 10e-4 M. The results are shown in the figure above, and it was found that the best induction effect was achieved at the concentration of 10e-4M.</p>
 
<p>The bacterial solution was incubated until OD600 reached 0.6, and 30× oxo-C6-HSL solution was pre-configured by adding 290 μl of bacterial solution and 10 μl of AHL solution to a 96-well plate. A total of 8 concentration gradients were set up as 0, 10e-10, 10e-9, 10e-8, 10e-7, 10e-6, 10e-5, and 10e-4 M. The results are shown in the figure above, and it was found that the best induction effect was achieved at the concentration of 10e-4M.</p>

Revision as of 13:42, 12 October 2023


Quorum sensing induced suicide system(low AHLs)

Part 0

Our Part consists of two parts, the suicide system (Part1, Part2) and the quorum-sensing System (Part3), and is designed to enable chassis with the Part to survive in environments with certain AHLs concentrations and to commit suicide in environments with no AHLs or very low AHLs concentrations. The suicide system utilizes the ccdA/ccdB antitoxin-toxin system, and for the quorum sensing system, LuxR and AHLs and the promoter pLuxR activated by them were selected.

Biology and Usage

Quorum sensing induced suicide system
Function Quorum sensing induced suicide system
Use in Prokaryotes
Backbone pET-28a
Derived from Escherichia. coli DH5α

Design and Properties


Figure 1 pET-28a-luxR-ccdA/ccdB characterization verification line

We introduced the successfully characterized and validated Lux operon (BBa_K407012) from Part3 (We'll cover that in the next section) into the ccdA/ccdB antitoxin-toxin system constructed in Part2 (BBa_K4137006, BBa_P1010 ) upstream, constituting Part0.


Figure 2 Final Logic Circuit Verification

As the stress caused by CcdB to the engineering bacteria will lead to self-cutting of the ccdB gene fragment, we design short-time liquid culture induction experiments， according to the characteristics that AHL can not tolerate high temperature for a long time and is not applicable to be induced in the solid medium. We spread the plates every 1h and plotted the growth curves by the number of colonies in the plates at different times for the verification of the logical line.

Our suicide system inhibited bacterial growth in the absence of AHLs during the first two hours, and the number of colonies in the control group induced by adding AHLs was higher than that in the experimental group without AHLs throughout the incubation process. However, we found the growth of the experimental group and the control group converged to the same after 2-3h of AHL-induced expression, which indicated that the stress caused by CcdB on the engineered bacteria, as well as the excision of the ccdB fragments from the engineered bacteria, occurred in advance. We suspect that the leakage expression of CcdB intensified after the introduction of the quorum-sensing promoter. Therefore, we will continue to explore options to reduce the stress of CcdB on the host bacterium during the subsequent improvement of the project.

Experimental approach

oxo-C6-HSL induction validates complete suicide system functionality

The functional characterization of pLuxR (BBa_R0062) has been verified by Part 3 (We'll talk more about this in the next section.). We know that the optimal concentration of the oxo-C6-HSL to induce quorum sensing was 10-4 M. During the verification process of Part1, it is known that the chassis transformed with ccdB will cut ccdB ( BBa_P1010 ) when incubated in the liquid medium for more than 6h, and since AHL is not applicable to be induced in the solid medium [1], therefore we designed liquid culture induction experiments with a total duration of 4h

1. Prepare two 12 ml tubes, add 6 ml of K+ LB liquid medium, add oxo-C6-HSL dry powder to one of the tubes, then the liquid medium oxo-C6-HSL concentration will be 10-4 M, the blank control tube labeled 0, the experimental validation tube is labeled as 10-4 ;
2. Thirty plates of K+ LB solid medium plates were prepared. pET-28a-luxR-ccdA-ccdB plasmid transformed BL21 Star, 50ul of the bacterial solution was added to tubes 0 and 10-4, respectively, and mixed, and 50ul of the bacterial solution was aspirated from tubes 0 and 10-4 to spread the plates (repeated for three plates, respectively)
3. Tubes 0 and 10-4 continued to incubate at 37°C and 220 rmp, and after 1h, 100 ul of the bacterial solution was again aspirated from tubes 0 and 10-4 to spread the plates (repeated for three plates, respectively)
4. The incubation of tube 0 and tube 10-4 was continued at 37°C, 220rmp, and step 3 was repeated every 1h after that to obtain 2h, 3h, and 4h oxo-C6-HSL-induced CcdB expression characterization, respectively
5. All plates were well labeled and incubated overnight in a 37°C incubator, and the number of colony growth in different groups of plates at different moments was observed in the morning of the next day.

Reference

[1] Liang Y, Pan Y, Li Q, Wu B, Hu M. RNA-seq-based transcriptomic analysis of AHL-induced biofilm and pyocyanin inhibition in Pseudomonas aeruginosa by Lactobacillus brevis. Int Microbiol. 2022 Aug;25(3):447-456.

Part1 IPTG-induced kill switch

Part1 composed of LacO/LacI (BBa_K1624002, BBa_K3257045), pT7 (BBa_K4609008) and ccdB ( BBa_P1010 ) compositions, we constructed part1 aiming to verify the expression of CcdB toxic protein in the chassis and its characterization of bactericidal function through IPTG induction experiments. CcdB is a toxic protein in the CcdB/CcdA toxin-antitoxin system, with a half-life of more than 2h after expression, which, as an intracellular toxin, induces the formation of the unresolvable covalent by disrupting DNA deconjugating enzyme activity mainly through GyrA-DNA complex. CcdB promotes the breakage of chassis plasmid and chromosomal DNA, ultimately leading to defective chassis division and killing the chassis [1]. LacO/LacI is commonly found in the pET series of plasmids, and IPTG (Isopropyl β-D-1-Thio-galactopyranoside), a molecular analog of iso-lactose, which enters into the intracellular system as a low-toxicity exogenous substance, is not able to be metabolized by common laboratory chassis, such as Escherichia coli. IPTG is a molecular analog of iso-lactose. IPTG functions in the same way as isolactose in that it acts as an inducer in the chassis to bind to the deterrent factor in the Lac operon, which in turn prevents LacI from binding to LacO, ultimately initiating the expression of the target genes downstream of pT7; when there is no IPTG, LacI binds to LacO to deter the expression of CcdB [2].

Design and Properties

Fig. 1 CcdB characterization of validated gene circuits

The IPTG-induced suicide switch is characterized by the fact that the ccdB ( BBa_P1010), which encodes the toxin, is placed under LacO/LacI control.

Fig. 2 Results of IPTG gradient plate induction experiments

We verify the function of CcdB by IPTG gradient plates test. The initial minimum induced concentrations of IPTG on plates with different gradients were 0.08mg/ml, 0.10mg/ml (0.42mM) and 0.12mg/ml, respectively. The experimental results showed that, with the increase of IPTG concentration on plates with different gradients, The number of single colonies showed a decreasing trend, indicating that IPTG-induced CcdB could play a bactericidal role.

Fig. 3 Results of induction experiments with different concentrations of IPTG plates

After we carried out liquid culture induction experiments and gradient plate experiments, we finally experimented with different concentrations of IPTG plate induction. As shown in Figure 2, the results showed that with the increase of IPTG concentration, the number of single colonies showed an obvious decreasing trend. When the IPTG concentration was 0.02 mg/ml, the low amount of expressed CcdB began to play a more obvious bactericidal effect.

Experimental approach

(i)IPTG gradient liquid culture for growth curve measurement

1. pET-28a-ccdB plasmid was transformed into E. coli BL21 star competent cells and cultured in K+ LB liquid medium at 37℃, 220rpm overnight;
2. On the second day, the bacterial liquid was taken for sequencing, and the remaining bacterial liquid was evenly divided into 96 EP tubes with 1ml of bacterial liquid in each tube, centrifuged at 12000rpm for 2min, and the upper liquid was discarded;
3. Each tube was resuspended by adding 1 ml of fresh K+ LB liquid medium. 96 tubes were equally divided into three groups: 32 tubes were added with 0.5mg/ml IPTG, 32 tubes were added with 1mg/ml IPTG, 32 tubes were added with no IPTG, and the first two groups were taken as the experimental group, the last group was taken as a control group, after labeling, 96 tubes of EP tubes were incubated at 37℃ and 220rpm. Cultivation;
4. Collect 8 tubes of EP tubes in 3 groups every 6 hours, 4 tubes for measuring absorbance, 4 tubes for dilution spread plates (the bacterial solution will be aspirated 10ul added to 100ul mixed, and then aspirated 50ul for spreading), plates labeled with numbers, 37 ℃ incubation. 24h later, observe the growth of bacteria and count the number of colonies of different groups of plates. The remaining EP tubes were subjected to medium renewal and continued to be cultured at 37℃ and 220rpm;
5. Step 4 was carried out every 6 hours, a total of four-time nodes, 0h, 6h, 12h, 18h, OD600 was measured at different nodes, and the solid medium was spread according to 1:1000 dilution of the bacterial solution, and the growth curves were plotted in order to analyze whether CcdB played a role in inhibiting the growth of the colonies after IPTG induction.

Figure 3 Design of EP tube allocation for IPTG liquid induction experiments

*Note: After each round of spreading, the EP tubes used for coating are stored in a 4°C refrigerator.

(ii) IPTG gradient plate assay

1. Configure 400ml LB solid medium, sterilize at 121℃ for 20min, and then cool to about 50℃. Add 400ul kanamycin, pour into a petri dish, and then cushion up one end by 5mm; 2. After solidification of the solid medium, K+ LB medium with IPTG concentrations of 0.08mg/ml, 0.10mg/ml, 0.12mg/ml was prepared according to the demand, poured into different slant solid medium and spread out, and then left horizontally to solidify to obtain IPTG gradient plate. 3. Transform E. coli BL21 star with pET-28a-ccdB plasmid, take 50ul of the bacterial solution and spread it evenly onto different IPTG gradient plates respectively, and incubate it overnight at 37℃ in a constant temperature incubator; 4. Count the density of single colonies at different concentrations.

(iii) Induction experiments with different concentrations of IPTG plates

1. Configure LB solid medium, sterilize at 121℃ for 20min, and cool to about 50℃. Add kanamycin solution and different amounts of liquid IPTG to the medium. IPTG concentration of 0mg/ml, 0.02mg/ml, 0.04mg/ml, 0.06mg/ml, and 0.08mg/ml, 0.10mg/ml of K+ LB medium was subsequently labeled as 0, 0.02, 0.04, 0.06, 0.08, 0.10 according to the concentration, respectively;
2. As the concentration of IPTG masterbatch used was 1M, a small amount of IPTG masterbatch was first taken and diluted using ddH2O to reduce the consumption of masterbatch. Finally, the diluted IPTG solution was added to the culture medium for further preparation with different IPTG concentrations. To exclude the effect of adding a certain amount of ddH2O to the medium on the growth of the chassis, we also set up another control group of K+ LB medium with only a certain amount of ddH2O added proportionally, which was subsequently labeled as 0*;
3. Transform E. coli BL21 star with pET-28a-ccdB plasmid, take 50ul of bacterial solution and spread it evenly onto seven sets of plates with different IPTG concentrations respectively, and incubate them overnight at 37℃(there are three replicate plates for each marker);
4. Seven sets of IPTG plates with different concentrations were observed throughout 12-24h, and the density of single colonies in plates with varying concentrations of IPTG was counted.

Reference

[1] Bahassi EM, O'Dea MH, Allali N, Messens J, Gellert M, Couturier M. Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA. J Biol Chem. 1999 Apr 16;274(16):10936-44.
[2] Swint-Kruse L, Matthews KS. Allostery in the LacI/GalR family: variations on a theme. Curr Opin Microbiol. 2009 Apr;12(2):129-37.

Part 2 IPTG-induced ccdA/ccdB system

During the characterization and validation of part1, we found an expression leakage of ccdB in the gene circuits. To better solve this problem, we improved the suicide system and constructed part 2 by adding the ccdA antitoxin gene (BBa_K4137006) to part 1. CcdA is the antitoxin in the ccdA/ccdB system, which acts as an unstable antitoxin with a half-life of about 1h in the presence of CcdB. CcdA binds to CcdB and blocks its activity, and DNA deconjugated activity is restored by the formation of a CcdA-CcdB complex that removes bound CcdB protein from the CcdB-GyrA complex [1].

Design and Properties

Fig. 1 ccdA/ccdB characterization verification line

We added the ccdA gene (BBa_K4137006) to Part 1.

Fig. 2 Comparison of CcdB-induced results with CcdA&CcdB-induced results

We compared the results of CcdB induction with those of CcdA&CcdB. The results are shown in Fig. 2. We found that the effects of CcdB induction differed significantly from those of CcdA&CcdB induction between the highest IPTG-inducing concentration and the lowest IPTG-inducing concentration. Still, the impact of CcdB induction was almost the same in the highest IPTG-inducing concentration and the lowest IPTG-inducing concentration, which indicated that when CcdB-induced expression reached a certain value, the presence of CcdA would not affect CcdB to maximize its inhibitory effect.

Experimental approach

Induction experiments with different concentrations of IPTG plates

1. Configure LB solid medium, sterilized at 121°C for 20min and cooled to about 50°C, add kanamycin solution at 1:1000, add different amounts of liquid IPTG to the medium according to the demand, and prepare IPTG concentration of 0mg/ml, 0.02mg/ml, 0.04mg/ml, 0.06mg/ml, 0.08mg/ml, 0.10mg/ml of K+ LB medium and subsequently labeled as 0, 0.02, 0.04, 0.06, 0.08, 0.10 according to the concentration, respectively;
2. As the concentration of IPTG masterbatch used was 1M, in order to reduce the consumption of masterbatch, a small amount of IPTG masterbatch was first taken and diluted using ddH2O. Finally, the diluted IPTG solution was added to the culture medium for further preparation of the culture medium with different IPTG concentrations. In order to exclude the effect of adding a certain amount of ddH2 O to the medium on the growth of the chassis, in addition to the setup of a 0mg/ml control group, we also set up another control group of K+ LB medium with only a certain amount of ddH2O added proportionally, which was subsequently labeled as 0*;
3. Transform E. coli BL21 star receptor with pET-28a-ccdA-ccdB plasmid, take 50ul of the bacterial solution and spread it evenly onto seven sets of plates with different IPTG concentrations respectively, and incubate them overnight at 37℃ in a constant temperature incubator (there are three replicate plates for each marker);
4. Seven sets of IPTG plates with different concentrations were observed for 12-24h, and the density of single colonies in plates with different concentrations of IPTG was counted.

Reference

[1] Aghera NK, Prabha J, Tandon H, Chattopadhyay G, Vishwanath S, Srinivasan N, Varadarajan R. Mechanism of CcdA-Mediated Rejuvenation of DNA Gyrase. Structure. 2020 May 5;28(5):562-572.e4.

Part3 AHL induced expression system

We needed to validate the effect of our quorum sensing regulatory system [1] on the growth and metabolism of chassis organisms as well as the sensitivity of the system, so we constructed Part 3 for characterization and validation.

Design and Properties

Fig. 1 Genetic circuits of the group sensing verification system

Expression of luxR was regulated using the constitutive promoter J23100 ligated to RBS B0034 followed by terminator B0010. Expression of downstream sfGFP was regulated using the quorum sensing promoter PluxR ligated to RBS B0034 followed by terminator B0010.

Fig. 2 Preferability on Different AHLs of our system

Fig. 3 Effects on Different AHLs Concentration of our system

The bacterial solution was incubated until OD600 reached 0.6, and 30× oxo-C6-HSL solution was pre-configured by adding 290 μl of bacterial solution and 10 μl of AHL solution to a 96-well plate. A total of 8 concentration gradients were set up as 0, 10e-10, 10e-9, 10e-8, 10e-7, 10e-6, 10e-5, and 10e-4 M. The results are shown in the figure above, and it was found that the best induction effect was achieved at the concentration of 10e-4M.

Experimental approach

Measurement of AHL-induced concentrations

1. Configure 30× oxo-C6-HSL solution.
2. The bacterial solution was incubated until OD600 reached 0.6, and 290 μl of bacterial solution and 10 μl of AHL solution were added to a 96-well plate. A total of 8 concentration gradients were set up for the ahl solution, namely, 0, 10e-10, 10e-9, 10e-8, 10e-7, 10e-6, 10e-5, and 10e-4M, respectively.
3. Two clear wells and two black wells were set up for each concentration in the experimental group; in the control group, the same concentration gradient of ahl was added to 290 μl of the medium, and one clear well and one black well were set up for each concentration.
4. Absorbance and fluorescence values were measured every 10 min for 3h using an enzyme labeling apparatus.

Reference

[1] Liu L, Zeng X, Zheng J, Zou Y, Qiu S, Dai Y. AHL-mediated quorum sensing to regulate bacterial substance and energy metabolism: a review. Microbiol Res. 2022 Sep;262:127102. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4029
    Illegal NheI site found at 4052
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1813
    Illegal BamHI site found at 3645
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1263
    Illegal BsaI.rc site found at 3095