Difference between revisions of "Part:BBa K4291005"

Revision as of 09:23, 26 September 2022

Trc pro- Lac oper- tnaC- Rho -RBS_tnaC_amilGFP

Characterization by Canton_HS

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.

Error creating thumbnail: Invalid thumbnail parameters

Figure 2. Map of pTrc99k-trp-amilGFP plasmid

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

@@ Line 38: / Line 38: @@
 ====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 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.
+[[File:T--Canton HS--BBa K4291005-figure 2.png|500px|thumb|center|Figure 2. Map of pTrc99k-trp-amilGFP plasmid]]