Part:BBa_K4115004

PcstA Mutant1

This part is the promoter of E. coli DH5-alpha cstA gene with a designed mutation at its cAMP receptor protein (CRP) binding site. It can be activated by CRP under a carbon source starvation.

Usage and Biology

About PcstA

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 sequence.

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_Mutant1

To ensure a good signal-to-noise ratio for glucose concentration sensing, we made efforts on improving the activity and fold-change. To achieve these goals, we try to mutate the PcstA. In principle, if the CRP-binding sequence of PcstA has higher similarity to the consensus sequence, PcstA will have a higher affinity to CRP. The consensus sequence of the CRP-binding site is AAATGTGA-N6-TCTCATTT. The nucleotides with underline are the core of the CRP-binding sequence. G and C with underlines provide most of the binding energy. We hypothesize that if the promoter has a higher affinity to CRP, it will have a larger fold change. Based on these rules, we replaced one base pair on PcstA and named it PcstA_Mutant1 (this part).

Figure 3: The CRP-binding sequences of PcstA and PcstA_Mutant1

Emperiments and results

Methods

To test the promoter activities of PcstA and PcstA_Mutant1 under different glucose concentrations, we used sfGFP with LVA as the reporter and constructed two composite parts (BBa_K4115025for PcstA, BBa_K4115028for PcstA_Mutant1) for testing. The fluorescence intensities normalized by OD ₆₀₀ indicate the relative promoter activities.

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 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

The reporter gene constructs are first cloned into pUC high-copy number backbone. A significant promoter activity increase was observed on PcstA_Mutant1 and the starvation response function remained (Figure 5). However, some unwanted phenomena also appeared. First, the leakage expression of PcstA_Mutant1 under high glucose concentration increases, which results in the decrease of fold-change. Second, the high copy of PcstA_Mutant1 in cells has some side effects on growth (Figure 6). We hypothesize that the high copy of PcstA_Mutant1 causes these side effects. CRP regulates the gene expression related to central metabolisms like glycolysis and pentose phosphate pathway. The mutant on the CRP-binding sequence of PcstA_Mutant1 increases its affinity to CRP. If PcstA_Mutant1 keeps a high copy in cells, it may compete the CRP with the endogenous CRP-dependent promoter. Under the high glucose concentration, even though the abundance of CRP in cells is low, the high copy and high affinity of PcstA_Mutant1 cause the small amount of CRP to be still used to activate PcstA_Mutant1. This might be a possible explanation of the higher leakage activity of PcstA_Mutant1. Less CRP proteins can be used to regulate endogenous gene expression and cause some side effects on global metabolism.

Figure 5: FI/OD₆₀₀ of PcstA and PcstA_Mutant1. All the constructs are cloned into pUC high-copy number backbone.

Figure 6: Growth curve of Empty and PcstA_Mutant1 strain in M9 minimal medium containing 4.0 g/L glucose as the sole carbon source.

To test our hypothesis and remove the side effects, we shifted all constructions to the low-copy number pET backbone and repeat the experiment. The bacteria growth is back to normal in the PcstA_Mutant1 group. PcstA_Mutant1 shows significantly higher activity as well as larger fold-change with low-copy number backbone (Figure 7). The data of relative promoter activities and fold changes of PcstA variants in this cycle are summarized in Figure 8. All the relative promoter activity is normalized with J23101, a widely used and well-characterized constitutive promoter. The parameters in Figure 8 might be useful for modeling as well,

Figure 7: FI/OD₆₀₀ of PcstA and PcstA_Mutant1. All the constructs are cloned into pET low-copy number backbone.

Figure 8: 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

In conclusion, carbon starvation response is best achieved using PcstA_Mutant1 on low-copy plasmids.It can be used to indicate the nutrient situation in culture medium if you add some downstream reporters. If you want your strain to express some genes under starvation conditions specifically, you can use this promoter as the signal input.
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]