Difference between revisions of "Part:BBa K4291005"
 
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<partinfo>BBa_K4291005 short</partinfo>
 
<partinfo>BBa_K4291005 short</partinfo>
  
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Trc pro- Lac oper- tnaC- Rho -RBS_tnaC_amilGFP
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== Characterization by Canton_HS ==
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== Profile ==
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== BBa_K4291005 ==
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Name: Trc pro- Lac oper- tnaC- Rho -RBS_tnaC_amilGFP
 
Name: Trc pro- Lac oper- tnaC- Rho -RBS_tnaC_amilGFP
  
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===BBa_K4291000===
 
===BBa_K4291000===
 
Name: Trc promoter
 
Name: Trc promoter
 +
 
Base Pairs: 17 bp
 
Base Pairs: 17 bp
 +
 
Origin: E.coli
 
Origin: E.coli
 +
 
Properties: strong promoter
 
Properties: strong promoter
 
====Usage and biology ====
 
====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_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===
 
===BBa_K4291002===
 
Name: tnaC
 
Name: tnaC
 +
 
Base Pairs: 349 bp  
 
Base Pairs: 349 bp  
 +
 
Origin: E. coli, genome
 
Origin: E. coli, genome
 +
 
Properties: tnaC regulatory gene controls the tna operon transcription
 
Properties: tnaC regulatory gene controls the tna operon transcription
 
====Usage and biology ====
 
====Usage and biology ====
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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_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===
 
===BBa_K4291003===
 
Name: Rho binding site
 
Name: Rho binding site
 +
 
Base Pairs: 207 bp  
 
Base Pairs: 207 bp  
 +
 
Origin: E. coli, genome
 
Origin: E. coli, genome
 +
 
Properties: termination of RNA synthesis  
 
Properties: termination of RNA synthesis  
 
====Usage and biology ====
 
====Usage and biology ====
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BBa_K4291003 is an encoding sequence of Rho binding site. The Rho transcription termination factor is responsible for the termination of RNA synthesis.  
+
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 ==
 
== Experimental approach ==
 
1.1 Construction of biosensor expression plasmids
 
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.
 
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.
 
[[File:T--Canton HS--BBa K4291005-figure 2(1).jpg|500px|thumb|center|Figure 2. Map of pTrc99k-trp-amilGFP plasmid]]
 
[[File:T--Canton HS--BBa K4291005-figure 2(1).jpg|500px|thumb|center|Figure 2. Map of pTrc99k-trp-amilGFP plasmid]]
  
 
1.2 Sanger sequencing of recombinant 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.
 
We extracted the plasmid and send it to the company for Sanger sequencing. The results of recombinant plasmid sequencing are shown as Figure3.
 
[[File:T--Canton HS--BBa K4291005-figure 3.jpg|500px|thumb|center|Figure 3. Sanger sequencing of pTrc99k-trp-amilGFP plasmid]]
 
[[File:T--Canton HS--BBa K4291005-figure 3.jpg|500px|thumb|center|Figure 3. Sanger sequencing of pTrc99k-trp-amilGFP plasmid]]
  
 
1.3 Tryptophan detection
 
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).
 
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).
 
[[File:T--Canton HS--BBa K4291005-figure 4.png|500px|thumb|center|Figure 4. Fluorescence intensity result of L-tryptophan]]
 
[[File:T--Canton HS--BBa K4291005-figure 4.png|500px|thumb|center|Figure 4. Fluorescence intensity result of L-tryptophan]]
  
  
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===Usage and Biology===
 
  
 
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Latest revision as of 07:38, 30 September 2022


Trc pro- Lac oper- tnaC- Rho -RBS_tnaC_amilGFP


Profile

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