Part:BBa_K4115032

PyciG-sfGFP-B0015

A carbon starvation reporter based on PyciG. sfGFP without LVA is used in this part. This composite part indicates the endogenous signal of RpoS through fluorescence signal.

Usage and Biology

We constructed two report genes based on two different fluorescence reporters shown in Figure 1: sfGFP (BBa_K4115000) and sfGFP with LVA (BBa_K4115001). LVA degradation tag can mediate a faster degradation and reduce the half-life of sfGFP.(Figure 1)

Figure 1. 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/OD₆₀₀ of PyciG abnormally (Figure 2). 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 3.

Figure 2. Use sfGFP or sfGFP with LVA to indicate the promoter activity

Figure 3. Summarized data of PyciG, High [Glucose]=4 g/L, Low [Glucose]=0.25 g/L

In conclusion, there might be some crosstalk between PyciG and LVA degradation tag. So you should avoid expressing proteins with ssrA degradation tags with PyciG. Compared with BBa_K4115033 (based on sfGFP with LVA), this composite part reflects the nutrient situation more realistically.
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]