Construction method and application of glycine ribose switch gene regulation circuit
A technology of riboswitch and construction method, applied in the field of synthetic biology, can solve the problem of narrow sensing range and so on
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0052] A method for constructing a gene regulatory circuit of a glycine riboswitch and screening for mutants, which is characterized by comprising the following steps:
[0053] 1): Using BSGlyRS-F shown in SEQ ID No.1 and BSGlyRS-R shown in SEQ ID No.2 as primers, and using pUC-BSGlyRS shown in SEQ ID No.3 as a template, by PCR method, get SEQ ID NO. BSGlyRS fragment shown in ID No.4;
[0054] 2): Using tetA-F shown in SEQ ID No.5 and tetA-R shown in SEQ ID No.6 as primers, pUC-TETA shown in SEQ ID No.7 as a template, by PCR method, get SEQ ID No. The tetA fragment shown in ID No.8;
[0055] 3): Using GFP-F shown in SEQ ID No.9 and GFP-R shown in SEQ ID No.10 as primers, using pUC-GFP shown in SEQ ID No.11 as template, by PCR method, get SEQ ID No. GFP fragment shown in ID No.12;
[0056]4): using pUC18-F shown in SEQ ID No.13 and pUC18-R shown in SEQ ID No.14 as primers, and pUC18 shown in SEQ ID No.15 as a template, amplified by PCR to obtain the pUC18 plasmid backbone; ...
Embodiment 2
[0078] The fermentation characterization method of the glycine riboswitch gene circuit, as follows:
[0079] (1) Activated strains: the strain MG1655-BSTG with wild-type glycine element, the strain MG1655-BS-ON14 with BSglyON14, and the strain MG1655-BS-OFF6 with BSglyOFF6 were cultured on LB solid containing ampicillin resistance Streak in the base, culture at 37°C for 10-20h to rejuvenate the strain;
[0080] (2) Cultivation of the seed liquid: the activated bacteria in step (1) of Tiaoyihuan were inoculated into the LB liquid medium, cultivated at 37°C and 220rpm for 10-20h, and then the resulting bacterial liquid was prepared according to the initial OD 600 =0.02 inoculum amount was inoculated into M9 medium, cultured at 37°C and 220rpm for 10-20h to obtain seed solution;
[0081] (3) Fermentation characterization: the seed liquid obtained in step (2) was 600 =0.02 was inoculated into a 50mL Erlenmeyer flask, and ampicillin was added to make the concentration 80-100μg / L....
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More