Plasmid system for comprehensively representing strength of escherichia coli terminator and method

A technology of Escherichia coli and terminators, applied in the field of synthetic biology, can solve the problems affecting the use of terminators to regulate gene expression, and the lack of comprehensive terminator characterization

Active Publication Date: 2020-06-23
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existing studies are not comprehensive enough to characterize t

Method used

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  • Plasmid system for comprehensively representing strength of escherichia coli terminator and method
  • Plasmid system for comprehensively representing strength of escherichia coli terminator and method
  • Plasmid system for comprehensively representing strength of escherichia coli terminator and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Establishment of a rational strategy for terminator characterization

[0053] Terminator probe plasmid construction strategies such as figure 1 .

[0054] 1. Construction method of terminator probe plasmid pTK.

[0055] Directly synthesized fragments tac, gfp1 and rfp1, the nucleotide sequence of tac is shown in SEQ ID NO.18, the nucleotide sequence of gfp1 is shown in SEQ ID NO.19, and the nucleotide sequence of rfp1 is shown in SEQ ID NO .20 shown. The primer tac-F / gfp-R1 was used for overlapping PCR to obtain the fragment tac-gfp1, and then the primer tac-F / rfp-R was used for overlapping PCR to obtain the fragment tac-gfp-TTS1-rfp.

[0056] tac-F: 5'-CAGTGGTGGTGGTGGTGGTGGACACCATCGAATGGTGCAA (SEQ ID NO. 6)

[0057] gfp-R1: 5'-TTTCGCTAGCTTACTTGTACAGCTCGTCCA (SEQ ID NO. 7)

[0058] rfp-R: 5'-ACTCTTTTGTTTATTTTTCTTAGGCGCCGGTGGAGTGG (SEQ ID NO. 8)

[0059] Use the following primers to amplify plasmid pK (SEQ ID NO.4), then insert the fragment tac-gfp1-TTS1...

Embodiment 2

[0081] Example 2: Characterization and analysis of terminator characteristic parameters.

[0082] 1. Use three characteristic parameters, apparent termination efficiency η, transcription termination degree α, and upstream gene mRNA protection ability β, to fully characterize the terminator strength, and calculate the terminator by constructing corresponding probe plasmids pTK, pTK-dR and pTK-sR The characteristic parameters of , the formula is as follows:

[0083]

[0084]

[0085]

[0086] Among them, RFP and GFP in the formula represent the expression intensity of red fluorescent protein and green fluorescent protein respectively, p1, p2 and p3 represent the probe plasmids pTK, pTK-dR and pTK-sR containing the terminator to be tested respectively, c1 and c2 and c3 represent the probe plasmids pTK, pTK-dR and pTK-sR of the control group (without the terminator to be tested), respectively. The result is as image 3 As shown, the upstream gene mRNA protection ability...

Embodiment 3

[0088] Example 3: Effect of GC base ratio in the terminator sequence on its characteristic parameters.

[0089] The base composition of the terminator sequence will affect the terminator strength, so the difference analysis is performed on the AU bases and GC bases in the terminator sequence. From Figure 5 In A, it can be seen that the ratio of GC bases is higher than that of AU bases, and its P value is between 0.0001 and 0.0002, which has a significant difference. It shows that the strong interaction force between GC bases is more conducive to the formation and stability of terminator structure. In addition, the GC bases in the terminator sequence are mainly concentrated in the stem structure, so the relationship between the proportion of GC bases in the stem structure and the terminator characteristic parameters was further analyzed. From Figure 5 As can be seen in B and 5C, the proportion of GC bases in the stem has a certain positive effect on the degree of transcrip...

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Abstract

The invention discloses a plasmid system for comprehensively representing the strength of an escherichia coli terminator and a method. According to the functions of terminator termination transcription and upstream gene mRNA protection, the characteristic parameter transcription termination degree (alpha), the upstream gene mRNA protection capability (beta) and the apparent termination efficiency(eta) are set. The invention also discloses a building method of probe plasmids used for representing the characteristic parameters of the terminator and application of the probe plasmids. The built terminator probe plasmids are pTK, pTK-dR and pTK-sR, and respectively correspond to characteristic parameters of alpha, beta and eta; an effective tool is provided for comprehensively representing thestrength of the escherichia coli terminator; and an accurate element module is provided for synthetic biology.

Description

technical field [0001] The invention relates to a plasmid system and method for comprehensively characterizing the terminator strength of Escherichia coli, belonging to the technical field of synthetic biology. Background technique [0002] Gene transcription refers to a process in which RNA polymerase (RNAP) is recruited to the promoter to start mRNA synthesis, dissociates when encountering a terminator and repeats the cycle. Terminator is an important regulatory element in optimizing metabolic pathways and genetic engineering. It can not only regulate gene transcription rate, but also regulate mRNA degradation rate, thereby regulating the initial expression of protein. [0003] Terminators can be divided into endogenous terminators and rho factor-dependent terminators. About 80% of transcription termination in Escherichia coli is due to the action of endogenous terminators, and compared with rho factor-dependent terminators, the structure and mechanism of endogenous termi...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/65C12N15/11C12N15/66G01N33/68
CPCC12N15/70C12N15/65G01N33/68C12N2830/36Y02A50/30
Inventor 许正宏何志云张晓娟段艳婷张晓梅史劲松
Owner JIANGNAN UNIV
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