Part:BBa_K4115018

PyciG, a stress induced promoter

This part is the promoter of E.coli DH5-alpha yciG gene. PyciG is controlled by RpoS, so its activity will be upregulated by a series of stress. The native RBS is contained in this parts

Usage and Biology

PyciG uses RpoS as its sigma factor for transcription initiation. 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 1). Transcription of yciG has been used as a reporter for analyzing metabolic shifts in response to substrate perturbations.

Figure 1: Stress response mechanism of PyciG

Experiments and results

To quantify the promoter activity of PyciG under high or low carbon source concentration, we constructed reporter genes based on sfGFP (BBa_K4115032) or sfGFP with LVA (BBa_K4115033).(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. PyciG 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 PyciG is normalized with that of J23101 under 4 g/L glucose. Data of PyciG (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 PyciG under low glucose concentration increase compared with that of high concentration. Unexpectedly, LVA tag increases the relative FI/OD (600) of PyciG abnormally (Figure 3). This may be due to LVA tag competitively inhibiting the degradation of sigmaS, for RpoS degrades in the same pathway with LVA-tagged proteins. 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 PyciG, High [Glucose]=4 g/L, Low [Glucose]=0.25 g/L

In conclusion, we characterized the starvation response properties of PyciG. Also, there might be some crosstalk between PyciG and LVA degradation tag. So you should avoid expressing proteins with ssrA degradation tags with PyciG.
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]