Difference between revisions of "Part:BBa K3633015"

Revision as of 10:55, 12 October 2022

T7 promoter

T7 promoter (19 bp)

Description

This is a common promoter that can be induced by IPTG. The iGEM20_Shanghai_SFLS_SPBS use the promoter to accomplish experiments including production of betacyanins and laccase.

Experiments and Results

Successful production of betacyanins in E.coli BL21(DE3)(T7 promoter)

The team members constructed the plasmid T7-4,5-DODA using Gibson Assembly. They added 0.1 mM IPTG for induction. Again, after 20 h culture at 37℃, 220 rpm, we centrifuged the cells, discarded the LB medium, and resuspended the cell pellet in sterilized water. They then cultured the cells at 20℃, 120 rpm for 102 h and acquired expected results. They did not test the production in Vibrio natriegens because the bacterial strain we acquired (ATCC 14048) does not have the T7 promoter.

Fig 2. Production of dopaxanthin and indoline-betacyanin in E. coli BL21(DE3) after induction. (A) Production of dopaxanthin, after resuspension in sterilized water at 20℃, 120 rpm in 102 h. The horizontal axis is time (hours), and the vertical axis is absorbance of the bacterial solution at 415 nm. (B) Production of indoline-betacyanin, after resuspension in sterilized water at 20℃, 120 rpm in 102 h. The horizontal axis is time (hours), and the vertical axis is absorbance of the bacterial solution at 525 nm. (C) Production of dopaxanthin from 0-102 h. (D) Production of indoline-betacyanin from 0-102 h.

Successful production of laccase in E.coli BL21(DE3)(T7 promoter)

The plasmid was transformed into E. coli BL21(DE3). 0.1 mM IPTG induction was added when the culture reached an OD600 of 1.0. Then, the shake flask was cultured at 25℃ for 20 h (cultured at 25℃ instead of 18℃ because no more shakers were available in the laboratory). Then, the bacterial solution was centrifuged, the cell pellet was resuspended in PBS (pH=7.2-7.4), and the resuspension was sonicated.

Sequence & Features

Contribution by CU-Egypt 2022

We measured the expression yield of the T7 promoter with different genes [BBa_K3396000, BBa_K4165003] to compare the expression yield and validate our Dry-Lab mathematical model with the Wet-Lab experimental results while keeping the promoter, RBS, lac operator, and terminator unchanged in the biobrick.

GST-DOC vs GST-COH

        Figure 1. This figure shows the extraction of GST-DOC vs GST-COH using a Chemical method and sonication method.

His-DOC vs His-COH

        Figure 2. This figure shows the extraction of His-DOC vs His-COH using a Chemical method and sonication method.