Part:BBa_K4291005

Trc pro- Lac oper- tnaC- Rho -RBS_tnaC_amilGFP

Trc pro- Lac oper- tnaC- Rho -RBS_tnaC_amilGFP

BBa_K4291005

Name: Trc pro- Lac oper- tnaC- Rho -RBS_tnaC_amilGFP

Base Pairs: 1109 bp

Origin: E. coli, genome

Properties: a tool for monitoring the tryptophan concentration

Usage and biology

The existing tryptophan-sensor is composited by one regulation sequence upstream of the tryptophanase (tnaA) operon in wild-type E. coli which encodes a 24-residue nascent peptide, and a transcription termination factor (Rho) recognition site. In our project, we developed another tryptophan-sensor with tnaC, and we fused it with the amilGFP gene to characterize our biosensor.

Construct design

The expression of tnaC was regulated by tnaCAB operon in the pTrc99K vector. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp).

Figure 1. The principle of our tryptophan biosensor

The components of biosensor are described as follows:

BBa_K4291000

Name: Trc promoter

Base Pairs: 17 bp

Origin: E.coli

Properties: strong promoter

Usage and biology

BBa_K4291000 is an encoding sequence of combination tac with lac protomers. The promoter is stronger in comparison of lac promoter. Thus, the hybrid promotor significantly improves the expression of exogenous gene.

BBa_K4291002

Name: tnaC Base Pairs: 349 bp Origin: E. coli, genome Properties: tnaC regulatory gene controls the tna operon transcription

Usage and biology

BBa_K4291002 is an encoding sequence of tnaC regulatory gene from the tna operon of E. coli which controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination.

BBa_K4291003

Name: Rho binding site Base Pairs: 207 bp Origin: E. coli, genome Properties: termination of RNA synthesis

Usage and biology

BBa_K4291003 is an encoding sequence of Rho binding site. The Rho transcription termination factor is responsible for the termination of RNA synthesis.

Experimental approach

1.1 Construction of biosensor expression plasmids DNA fragment of tnaC was amplified from the MG1655 genomic DNA and the amilGFP DNA fragment was amplified from a plasmid containing this fragment (Figure 2A). Then, two fragments linked by overlap extension PCR (Figure 2B) and inserted the fused gene fragment into the NcoI and HindIII sites of the pTrc99k vector which formed recombinant plasmid pTrc99k-trp-amilGFP.

Figure 2. Map of pTrc99k-trp-amilGFP plasmid

1.2 Sanger sequencing of recombinant plasmid We extracted the plasmid and send it to the company for Sanger sequencing. The results of recombinant plasmid sequencing are shown as Figure3.

Figure 3. Sanger sequencing of pTrc99k-trp-amilGFP plasmid

1.3 Tryptophan detection Single colonies of the engineered strain containing the recombinant plasmid pTrc99K-tnaC-amilGFP were picked, inoculated in a triangular flask containing 10mL of fresh LB medium with 100mg/L ampicillin, and incubated at 37°C, 220 rpm overnight. 1 mL of bacterial cultured medium was added to a triangular flask containing 100mL of fresh LB medium with 100mg/L ampicillin and incubated at 37°C, 220 rpm until the OD600 is around 0.6. The solution was divided into conical flasks, adding 50ml of cultured medium and L-tryptophan to a final concentration of 0.75mmol/L, 1mmol/L, 1.5mmol/L, 2mmol/L, 2.5mmol/L, and 3mmol / L and cultured at 22°C, 220 rpm for 7 hours, and samples were tested at 1h, 2h, 4h, 5h, 6h, 7h. Fluorescence intensity was detected immediately by using bacterial solution samples and tryptophan to a 96-well black microplate plate, and the final volume is 200 μL (Figure 4).

Figure 4. Fluorescence intensity result of L-tryptophan

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 400