Regulatory

Part:BBa_K4115027

Designed by: Kaijun Wang   Group: iGEM22_ShanghaiTech_China   (2022-09-30)


PcstA_Mutant2

This part is the promoter of E. coli DH5-alpha cstA gene with two designed base replacements. One is located on the CRP-binding sequence of PcstA, the other is on a predicted Fis-binding sequence. It can be activated under a carbon source starvation.


Usage and Biology

PcstA (BBa_K118011the original version) is the promoter of cstA gene. cstA encodes a pyruvate transporter. PcstA is characterized as a carbon starvation promoter. It has a higher strength during a carbon source starvation or stationary phase. Unlike some other starvation promoters using sigma-54, PcstA is a sigma-70-dependent promoter. PcstA has been reported to be regulated by a repressor Fis (factor for inversion stimulation) and an activator CRP.

Figure 1: Fis competes with CRP for the CRP binding site.


Sigma-54 is up-regulated by a series of stresses, like heat, hyperosmolarity, and stationary phase(starvation). But the abundance of sigma-70 keeps stable in E.coli. The activation of PcstA is based on related repressors and activators, but not the abundance of sigma factor. So, in principle, PcstA can respond to carbon source conditions more specifically and orthogonally compared with those sigma-54-dependent promoters.

Mechanism of starvation response

PcstA uses RpoD as its sigma factor. cAMP receptor protein (CRP) is an activator of PcstA. When undergoes carbon source starvation conditions, the intracellular cAMP concentration will increase and further activate CRP. Then CRP binds to a CRP-binding sequence located upstream of the transcription start site and activates of transcription initiation of PcstA (Figure 2A). Furthermore, PcstA is down-regulated by the factor for inversion stimulation (Fis) under nutrient-rich conditions (Figure 2B). Under starvation conditions, the Fis abundance in cells will decrease, and the inhibition will be removed.

CRP contains a cAMP binding N-terminal domain and a DNA binding C-terminal domain, connected via a short hinge region. After binding with cAMP, CRP is activated and then binds to a consensus sequence(5'-AAATGTGA-N6-TCACATTT-3'), which is called the CRP-binding sequence (CBS). Positions 4–8 and 15–19 in CBS (underlined) are the most important for CRP/cAMP-DNA interaction. The original CRP binding sequence is 5'-CGGAGTGA-TCGAGT-TAACATTG-3'.

Figure 2: Regulators used by PcstA


PcstA_Mutant2

PcstA_Mutant2 contains two base replacements. One is located on the CRP-binding sequence of PcstA, the other is on the predicted Fis-binding sequence (Figure 3).

Figure 3: Regulation-related sequence of PcstA variants

The mutation on the CRP-binding sequence is designed to improve its affinity to CRP. This mutation will increase the promoter activity of PcstA, which has been proved on PcstA_Mutant1 (BBa_K4115004). Two Fis-binding sequences are predicted and shown in Figure 1. However, according to our data of PcstA long version (BBa_K4115003) and PcstA (BBa_K118011). Fis1 seems to have no effect on PcstA. So we choose Fis2 to be the target for mutation. The A to C replacement is expected to increase the affinity to Fis so that Fis can decrease the leakage expression under rich-nutrient conditions.

Experiments and results

We used sfGFP with LVA as the reporter of PcstA variants. The reporter gene of PcstA_Mutant2 is BBa_K4115059. The fluorescence intensity (FI)/OD600 indicates the promoter activity under a specific glucose concentration.These constructs were cloned into pET low copy number plasmid.

Figure 4: sfGFP with LVA is used to indicate the activities of PcstA variants.

The protocol that we used for this starvation response experiment is shown below:
1. Preparation of M9 minimal medium: In 1L M9 minimal medium 33.9g Na2HPO4, 15g KH2PO4, 2.5g NaCl, 5g NH4Cl, 0.36g MgSO4, glucose with a concentration gradient is supplied as the sole carbon source.
2. Dilute the E.coli cultures that are stored at 4 degrees 1:100 into 3ml LB medium overnight.
3. Then the cultures were diluted 1:100 into 3mL LB medium and cultured at 37℃ with shaking at 220rpm for about 3 hours. Use these cultures(OD600</subb> 0.6~0.8) as seeds.
4. Dilute the seeds 1:100 to 600ul M9 minimal medium and culture for 6h. Add 200ul into each well of 96-well plate. Measure OD600 and fluorescence intensity of each sample with a multimode plate reader.

Results

Only the activities of PcstA_Mutant1 and PcstA_Mutant2 were significantly different at 0.5 g/L glucose. These data show that, our A to C replacement on Fis-binding sequence may actually decrease its affinity to Fis according to the higher activity under 0.5 g/L glucose. Anyway, the mutation failed to reach our expectations. The data of relative promoter activities and fold changes of PcstA variants are summarized in Figure 6.

Figure 5:FI/OD600 of PcstA variants. All the constructs are cloned into pET low-copy number backbone.
Figure 6: Summarized data of PcstA variants. High [Glucose]=2 g/L, Low [Glucose]=0.25 g/L. Relative promoter activities are normalized with FI/OD600 of J23101 at 2 g/L glucose.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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