Part:BBa_K4115003

PcstA

This part is the promoter of E.coli DH5-alpha cstA gene. It can be activated by cAMP receptor protein(CRP) under a carbon source starvation. Compared with BBa_K118011, this part contains more sequence from E.coli genome at its 5'.

Usage and Biology

About PcstA

PcstA 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. Compared with BBa_K118011, this part contains an extra predicted Fis binding sequence (Fis1 on Figure 1).

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

Experiment and results

To check if the Fis1 in Figure 1 affects PcstA, we constructed two composite parts (BBa_K4115024 for this part, BBa_K4115025 for BBa_K118011) to test the promoter activity under different glucose concentrations. sfGFP with LVA is used as the fluorescence reporter. No significant different was shown, which proves that the first predicted Fis binding sequence (Fis1 on Figure 1) is not important for the activity of PcstA.

Figure 3: FI/OD₆₀₀ of PcstA (BBa_K118011) and PcstA longe version (this part)

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]