Part:BBa_K4609070
pET30a-DMBT1-sfGFP-HIS
We used this system to express DMBT1 protein and green fluorescent protein, and detected the expression of DMBT1 protein by detecting the expression of green fluorescent protein.
E. coli BL21 (DE3) contains a T7 RNA polymerase gene (λDE3) controlled by lacUV5 promoter, which can achieve IPTG-induced T7 RNA polymerase expression. After the use of IPTG inducers, the T7RNA polymerase is expressed and the T7 promoter is activated, thereby expressing DMBT1
1.Design
The DMBT1 protein (human) sequence was inserted into the plasmid, and the T7 promoter was inserted upstream to regulate the expression. lac operator was used to precisely control gene expression. eGFP gene sequence was inserted downstream to achieve simultaneous expression of DMBT1 protein and eGFP, and His tags were inserted into the C and N ends of the plasmid to facilitate subsequent protein purification and analysis, and codon optimization. The structure of His tag at the -n end of the T7 promoter -lac operator -DMBT1-eGFP-C end of His tag - T7 terminator is formed.
2. Build:
We sought the help of qualified companies for plasmid construction, and the relevant companies provided us with bacterial solutions with plasmids.
After extracting the plasmid, we selected BL21 as the protein expression system and used heat shock to transform the recombinant plasmid. Then the positive colonies were selected from kanamycin containing plates for amplification. They are sent for sequencing to verify conversion efficiency and avoid potential gaps or mismatch risks. The sequencing results are as follows
The results show that our design matches perfectly with the extracted plasmids, indicating that we have theoretically achieved the goal of the project.
3. Test:
Escherichia coli BL21(DE3) strain containing pET30a-DMBT1-sfGFP-His was used as engineering bacteria, and Escherichia coli BL21(DE3) strain containing pET30a-sfGFP-His was used as control group.
Protein expression verification experiment
This experiment was mainly used to verify that E. coli BL21(DE3) strain containing pET30a-DMBT1-sfGFP-His could normally express DMBT1 protein.
In the experiment, the strain was induced to express DMBT1 protein by IPTG, and Western Blot showed that the strain successfully expressed DMBT1 protein, indicating that the component could work normally.
Inhibition of convulsive movement experiment
In the experiment, we used a microscope to compare the inhibitory effect of DMBT1 protein on the movement of Pseudomonas aeruginosa by taking DMBT1 protein added as the experimental group and the Pseudomonas aeruginosa colony without DMBT1 protein as the control group. The results showed that DMBT1 protein could effectively inhibit the convulsive movement of Pseudomonas aeruginosa.
Growth and reproduction inhibition experiment
In the experiment, we took DMBT1 added to the Pseudomonas aeruginosa colony as the experimental group, and no DMBT1 added to the Pseudomonas aeruginosa colony as the control group, and compared and observed the inhibitory effect of DMBT1 protein on the growth and reproduction of Pseudomonas aeruginosa. The results showed that DMBT1 protein could effectively inhibit the growth and reproduction of Pseudomonas aeruginosa. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2133
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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