Part:BBa_K4115016

PcsiE, a starvation induced promoter

This part is the promoter of E.coli DH5-alpha csiE gene. PcsiE has a higher activity under carbon source starvation conditions and stationary phase.

Usage and Biology

Although the function of E. coli csiE is unknown until now, its promoter has been well-characterized. RpoS and cAMP receptor protein (CRP) are the main regulatory factors for PcsiE. These two regulators are involved in two pathways of starvation response, respectively. RpoS is an alternative sigma factor. As with other sigma factors, RpoS binds to the core RNA polymerase and controls the expression of a specific but large set of genes related to stress resistance. Under the conditions of nutrient deprivation or other stresses, the intracellular abundance of RpoS will increase, which will accelerate the transcription initiation of the RpoS-dependent genes (Figure 1A). The other starvation response pathway uses CRP as a regulator and the housekeeping sigma factor RpoD for transcription initiation. Under 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 this CRP-dependent promoter (Figure 1B).

Figure 1. Two pathways for PcsiE regulatory (A) RpoS-dependent (B) CRP-dependent

Experiments and results

To quantify the promoter activity of PcsiE under high or low carbon source concentration, we constructed reporter genes based on sfGFP (BBa_K4115028) or sfGFP with LVA (BBa_K4115029).(Figure 2)

Figure 2. Reporter gene based on sfGFP and sfGFP with LVA

sfGFP has a very long half-life in the cytoplasm, which makes it not suitable for indicating some immediate change in gene expression. LVA tag can reduce the half-life. So in principle, using sfGFP with LVA tag as the reporter can more realistically reflect changes in promoter activity under different carbon source concentrations. PcsiE is a starvation promoter, so we hope to see a higher fluorescence intensity under a lower glucose concentration.J23101 (a constitutive promoter) is used as the positive control. The background signal of no GFP expression culture has been removed from FI/OD₆₀₀ of all groups shown in this figure. (LVA) indicates that in this group, the reporter is sfGFP with LVA. Data of PcsiE is normalized with that of J23101 under 4 g/L glucose. Data of PcsiE (LVA) is normalized with that of J23101 (LVA) under 4 g/L glucose. All the constructs were cloned into pUC high-copy number backbone.As we expected, the promoter activity of PcsiE under low glucose concentration increase compared with that of high concentration. Also, the activity fold-changs at different glucose concentrations increased with the addition of the LVA tag (Figure 3). The detailed values are summarized in Figure 4.

Figure 3. Use sfGFP or sfGFP with LVA to indicate the promoter activity

Figure 4. Summarized data of PcsiE, High [Glucose]=4 g/L, Low [Glucose]=0.25 g/L

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]