Part:BBa_K4115028

PcstA_Mutant1-B0034-sfGFP(with LVA)-B0015

A carbon starvation reporter based on PcstA_Mutant1 (BBa_K4115004). This composite part indicates the endogenous signal of cAMP receptor protein through fluorescence signal.

Usage and Biology

To see more detailed descriptions of PcstA_Mutant1, you can go to BBa_K4115004. The original PcstA is BBa_K4115003.

Results

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

We used the following protocol for the characterization of fluorescence reporter genes:
1. Preparation of M9 minimal medium: In 1L M9 minimal medium 33.9g Na2HPO4, 15g KH2PO4, 2.5g NaCl, 5g NH4Cl, 0.36g MgSO4, glucose with a concentration gradient is supplied as the sole carbon source.
2. Dilute the E.coli cultures that are stored at 4 degrees 1:100 into 3ml LB medium overnight.
3. Then the cultures were diluted 1:100 into 3mL LB medium and cultured at 37℃ with shaking at 220rpm for about 3 hours. Use these cultures(OD600 0.6~0.8) as seeds.
4. Dilute the seeds 1:100 to 600ul M9 minimal medium and culture for 6h. Add 200ul into each well of 96-well plate. Measure OD₆₀₀ and fluorescence intensity of each sample with a multimode plate reader.

Based on the data of PcstA (wild-type promoter without mutations), we suppose that sfGFP is the better reporter for PcstA. Because the reporter gene based on sfGFP with LVA shows a higher fluorescence fold-change. We speculate that sfGFP with LVA will also be the better reporter for PcstA_Mutant1.

Figure 2: Using sfGFP or sfGFP with LVA as the fluorescence reporter of PcstA

Then we constructed the reporter gene of PcstA_Mutant1. The reporter gene (this composite part) has different performances on different copy number backbones. On high-copy number backbone, it has a high expression level but a small fold-change (Figure 3). This is caused by the high leakage expression under high glucose concentrations. Additionally, it has some side effects on the growth of transformed cells (Figure 4).

Figure 3: FI/OD₆₀₀ of PcstA and PcstA_Mutant1. All the constructs are cloned into pUC high-copy number backbone.

Figure 4: Growth curve of Empty and PcstA_Mutant1 strain in M9 minimal medium containing 4.0 g/L glucose as the sole carbon source.

On low copy number plasmid, PcstA_Mutant1 has a high expression level (compared with other promoters) as well as a large fold-change. However, the expression level all decreased to about 1/10 of that high-copy number constructs.

Figure 5: FI/OD₆₀₀ of PcstA and PcstA_Mutant1. All the constructs are cloned into pET low-copy number backbone.

Figure 6: Summarized data of PcstA variants. High [Glucose]=2 g/L, Low [Glucose]=0.25 g/L. Relative promoter activities are normalized with FI/OD₆₀₀ of J23101 at 2 g/L glucose

We recommend using this reporter on low copy number plasmid to reflect the nutrient condition sensitively.

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]