Part:BBa_K3823008

Pcstr: an artificial hydrogen sulfide sensitive promoter with binding sequences of CstR

Pcstr: an artificial promoter with two kinds of binding sequences of CstR from the natural Pcstr.It is sensitive to hydrogen sulfide density.

Introduction

CstR

CstR(BBa_K3823006)is a CsoR-like sulfur transferase repressor found in Staphylococcus aureus^[1], reported to react with HS_nH and form sulfhydrated proteins, which can turn on their regulated genes^[2] . Compared with other HS_nH sensors, it shows significant response to HS_nH induction.And it shows no response to H₂O₂, a structural analogue of HSSH and a common intracellular metabolite(Figure 1.).^[3]

Figure 1. Test of reported HS_nH-sensitive TFs in E. coli. (A) Schematic representation of FisR, CstR, and BigR testing plasmids. These plasmids were transformed into E. coli BL21. (B) CstR and BigR based plasmids responded to HS_nH induction in E. coli, while the FisR based one did not; 200 μM HS_nH was incubated with 2 mL of mid log phase cells (OD_{600 nm = 0.5) for 2 h (37 °C, 225 rpm shaking). mKate fluorescence was detected by flow cytometry. (C) BigR based plasmid responded to H2O2 (1 mM) and CstR based plasmid did not. (D) CstR based plasmid only responded to S8 and HSnH.^[3]}

The structure of Pcstr

Grossoehme, N., et al. found two strong candidate tandemly repeated CstR operator sites, CstO OP1 and OP2, between the cstR and cstA genes. These sites were characterized by a run of four consecutive GC-base pairs and flanked by AT-rich regions(Figure 2.) ^[4].

Figure 2. Reduced CstR binds to cst operator (CstO) sites with high affinity in a manner dependent of the central run of four GC base pairs. A, schematic of the cstR-cstA intergenic region highlighting the positions of the two tandem candidate CstR operator sites. B, nucleotide sequence of the cstR-cstA intergenic region, highlighting the OP1 and OP2 operator sequences (green). Underlined bases correspond to the 5′-fluorescein-labeled duplex oligonucleotides used for DNA binding experiments. C, CstR binding isotherms for OP1 (solid squares, ■), OP2 (open circles, ○), and OP1_5GC (filled circles, ●) in which an additional GC base pair was inserted into the run of four GC base pairs. Inset, competition dissociation experiments with fluorescein-labeled apo-CstR-OP1 complexes with unlabeled wild-type OP1 (solid squares, ■), OP1-GC3 (open triangles, △), and OP1_1GC (open squares, □) duplexes. Conditions used were pH 7.0, 0.2 m NaCl, at 25.0 °C.^[4]

To improve its performance in sensitivity, leaky strength, maximal strength, and strength amplitude, Liu, H., et al ^[1]constructed a series of combinations of PL and PR(Pcstr), and found the expression from elements 1, 4, and 5 was low and relatively stable (Figure 3), indicating that they are stringent enough for reducing leaky expression caused by background HS_nH. This page is the sequence of the promoter P_R3 in Figure 3.

Figure 3. Construction and test of HS_nH sensor-actuator elements. (A) Seven elements were constructed by changing CstR and mKate expression promoters (PL and PR). These elements were transformed into E. coli BL21. (B) Response of these elements to HS_nH induction. The data were fitted with the Hill equation. The Hill coefficient (n) and the dose inducing half turn-on (Km) were listed. (C) Leaky expression strength (without HS_nH induction) and maximal induced strength by 300 μM HS_nH of the elements. The folds of changes were given in numbers. (D) Performance of the elements in a whole growth circle. E. coli BL21 harboring these elements were cultivated in 200 μL of LB medium in a 96-well plate (37 °C with vibration). mKate fluorescence and cell density were automatically recorded at 30 min intervals using the Synergy H1Microplate reader. Data shown are normalized fluorescence (fluorescence intensity/OD_{600 nm}).^[1]

Results

1. To figure out how CstR works by sensing H₂S, we characterized the function of CstR with Pcstr using pTrchis2A-CstR-Pcstr-mKate-CpSQR(donated by Professor Liu from Shandong University). The expression level regulated by Pcstr at different concentrations of S^2- was shown by the fluorescence intensity of mKate(/OD_{600 nm}).

At the beginning, bacteria cultured in 5 mL were treated with 0, 6.5, 13.0, 26.0 and 39.0 mg/L Na₂S (0, 20, 40, 80 and 120 mg/L Na₂S·9H₂O) respectively, and then the fluorescence intensity of mKate was measured with a microplate reader(Figure 4.).

Figure 4. (A)The fluorescence intensity variation relative to the concentration of Na₂S; (B)The photo of the bacteria solution treated by gradient concentrations of Na₂S;(C)The curve of fluorescence intensity relative to time; (D)The curve of OD_{600 nm} relative to time

It can be seen that the presence of S^2- at different concentrations has no significant effect on the growth of bacteria. However, with the increase of S^2-, the fluorescence intensity increases at first and then decreases, indicating that when S^2- is too high, the lifting effect on CstR inhibition is weakened. We hope to find out an appropriate concentration range in which the strength of Pcstr is positively correlated with the concentration of S^2-, which tells us that we need to reduce the concentration gradient and concentration range for further characterization.

We further used 0, 3.25, 6.50, 9.75, 13.00, 16.25 mg/L Na₂S (0, 10, 20, 30, 40 and 50 mg/L Na₂S·9H₂O) to treat 5 mL bacterial solution and then measured the fluorescence intensity of mKate using a microplate reader(Figure 5.).

Figure 5. The fluorescence intensity variation relative to the concentration of Na₂S with a smaller range

It can be seen from the figure that in a smaller concentration range, there is a positive correlation between fluorescence intensity and Na₂S concentration, visible to naked eyes, which implies that we can use data within this range to regulate Pcstr by giving different concentration H₂S.

2. Also, to get this part for our plasmid construction, we successfully amplified CstR-Pcstr(Figure 6A), which was proved correct by sequencing and we has cloned it into our backbone to construct CstR-Pcstr-MazE-Dre(Figure 6B),which was also proved correct by sequencing.

Figure 6. Agarose electrophoresis

However, according to the sequencing results, we found that Pcstr was missing(not in the PCR product), probably due to the complexity of each block. Meanwhile, we found the terminator between the two rox sites was partly moved after we cultured the final plasmid. We assumed that it could be the leakage of Pcstr that turned on the expression of Dre, which then cut off the terminator. This means we need to optimize our design.

3.To optimize the design of our kill switch, we need to compare the strength of the three key promoters, PR(Pcstr), PL(PlacI), and J23110. So we tested their strength respectively by cloning them to BBa_J61002in E.coli BL21(DE3).(Figure 7.)

Figure 7. The strength of the three key promoters

From our results, we found that the intensity of Pcstr is significantly higher than J23110, and PlacI is very lower than J23110. This may explain why we encountered the leakage of Pcstr —— the inhibition of the CstR may be too low to block Pcstr while the intensity of Pcstr is in a high level. Although this is not consistent with the literature, the facts tell us that it is. Considering the time limitation and the workload of changing different promoters to test the best combination, we tried to construct a model to achieve this.

References

• [1]Grossoehme, N., et al., Control of Copper Resistance and Inorganic Sulfur Metabolism by Paralogous Regulators in Staphylococcus aureus. Journal of Biological Chemistry, 2011. 286(15): p. 13522-13531.
• [2]Giedroc, D.P., A new player in bacterial sulfide-inducible transcriptional regulation. Mol Microbiol, 2017. 105(3): p. 347-352.
• [3]Liu, H., et al., Synthetic Gene Circuits Enable Escherichia coli To Use Endogenous H(2)S as a Signaling Molecule for Quorum Sensing. ACS Synth Biol, 2019. 8(9): p. 2113-2120.
• [4]Grossoehme, N., et al., Control of Copper Resistance and Inorganic Sulfur Metabolism by Paralogous Regulators in Staphylococcus aureus. Journal of Biological Chemistry, 2011. 286(15): p. 13522-13531.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 31
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 31
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 31
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 31
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 31
  
• 1000
  COMPATIBLE WITH RFC[1000]