Difference between revisions of "Part:BBa K3796000"

Revision as of 13:15, 17 October 2021

Corynebacterium glutamicum strong constitutive promoter P0864

This is an endogenous strong promoter in the genome of Corynebacterium glutamicum ATCC13032. This promoter is linked upstream of the gene and can be expressed constitutively in Corynebacterium glutamicum with high expression intensity by transformation into it.

Usage and Biology

P0864 is an endogenous constitutive expression promoter of Corynebacterium glutamate, which can start the transcription of downstream sequences with high intensity. The measured data of expression amount can be seen below. When in use, connect a target fragment to be expressed downstream of the promoter and add a corresponding terminator.

Characterization

1.Identification

From the chemically synthesized sequence, we obtained the fragment from the plasmid by PCR and recovered the gel.

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2.Strength identification

In order to detect the activity of the new promoter p0864 in Corynebacterium glutamicum and Escherichia coli, we constructed the related circuit in pxmj19 plasmid. The expression of p0864 was characterized by fluorescence size.

<img src="https://2021.igem.org/wiki/images/c/c6/T--CAU_China--Part01_pmc.svg" alt=""/>

Since pxmj19 itself contains an inducible promoter PTAC (bba_m31370), and the inducible promoter may be leaked, it is necessary to construct a gene line without p0864 as a control.

<img src="https://2021.igem.org/wiki/images/5/5a/T--CAU_China--Part02_mc.svg" alt=""/>

The transformed strains were cultured on LBG chloramphenicol resistant solid medium for 24 hours. In E. coli, two gene circuits showed different fluorescence. It has different performance under the irradiation of different light sources. The fluorescence of circuit containing p0864 is significantly brighter than that of another circuit. Under the irradiation of natural light, the colonies showed purplish red.

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Corynebacterium glutamicum and Escherichia coli were cultured in LBG chloramphenicol liquid medium at 35 ℃ for 26h. The bacteria were collected by centrifugation and observed under light. Then the bacteria were resuspended, the fluorescence relative value of the bacterial solution was measured by fluorescence spectrophotometer, and its OD600 was measured.

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T-test was performed on the three groups of data.

<img src="https://2021.igem.org/File:T--CAU_China--Parts_ttest.png" alt=""/>

The mean value of experimental group was significantly different from that of control group. The data showed that P0864 could be expressed in E. coli, and the expression amount was significantly different from that in the control group. As a constitutive promoter, it can be used as a standardized element.

Sequence and Features