Part:BBa_K4115029

PcsiE-B0034-sfGFP(with LVA)-B0015

A carbon starvation reporter based on PcsiE (BBa_K4115016). This composite part indicates the endogenous signals of RpoS and cAMP receptor protein through fluorescence signal.

Usage and Biology

To see the detailed description of PcsiE, you can go to BBa_K4115016. 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

In conclusion, this composite part can indicate the nutrient conditions more realistically compared with BBa_K4115031 (based on sfGFP).
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]