Part:BBa_K4609071
pET21a（+）-HSP 27
This part is mainly used to induce the expression of DMBT1 protein. In the process of synthesizing Hsp27, Escherichia coli consumes glucose, the glucose metabolic pathway is regulated, and the lactose pathway is activated, increasing the lactose concentration in Escherichia coli, thus leading to protein expression. Or Hsp27 interacts with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in E. coli. GAPDH is encoded by Escherichia coli gapA gene, and the affinity between Hsp27 and GAPDH is strong. GAPDH is the sixth enzyme in the glycolytic pathway. Disturbance of this enzyme can directly alter glycolysis, thus altering the entire bacterial metabolism, transforming the glycolytic pathway into the lactose pathway, increasing lactose concentration in E. coli, and ultimately leading to protein expression.
In addition, Hsp27 has a protective effect on cornea in the eye. Phosphorylated Hsp27 is highly expressed in corneal surface epithelial cells during corneal epithelial wound healing, and phosphorylation of Hsp27 plays a role in cell component reconstruction and cell migration. This phosphorylated protein has anti-apoptotic properties, and its surface localization confers innate corneal defense and corneal barrier functions. Yoo et al. also evaluated and affirmed the role of Hsp27 in epithelial cell migration and apoptosis during corneal epithelial wound healing.
1. Design
The SILEX system is the first recombinant protein overexpression system to date that uses lac operons to induce plasmodies through self-induction without any culture-medium adaptation for easy expression screening, and SILEX relies on metabolic modifications driven by interactions with the metabolizing E. coli GAPDH enzyme. Our self-induction system is designed to express the target proteins of Hsp27 and DMBT1 by designing two plasmids. In the HSP27-SILex system, Hsp27 has a high affinity and can interact with endogenous GAPDH, indirectly leading to a large amount of its self-induction. hsp27 protein is involved in the glucose metabolism pathway in Escherichia coli. This part involves the self-induced expression plasmid pET21a-Hsp27, which can continuously produce hsp27 protein, increase the lactose concentration in Escherichia coli, achieve the effect of inducing the expression of DMBT1 protein, and make the engineered bacteria produce more DMBT1 protein. Due to the chaperone action of Hsp27, this self-induction system can promote the correct folding of proteins and protect the cornea, while greatly improving the toxic side effects of previously used IPTG inducers.
2. construct
In this part, we sought the help of qualified companies for plasmid construction, and the relevant companies provided us with bacterial solution with plasmids.
3. Sequencing
In order to ensure the correct sequence of constructed plasmids, we sequenced the plasmids, and the sequencing results were 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.
4. Verify
Growth curve measurement
The overnight Escherichia coli liquid 100μl was inoculated into a centrifuge tube containing 10mlLB and cultured under shock on a shaking table of 180rpm at 37℃. The test tubes were removed at an interval of 2 hours successively and 200μl bacterial liquid was taken into 96-well plates. The concentration of bacterial solution (OD value) was determined with biotek epoch at the wavelength of 600 nm. OD values of each group measured were plotted with their corresponding culture time, and the following figure was obtained for comparison by reference to culture time and induction
Identification of inducible expression proteins
Fluorescence imaging
10ul hsp27-GFP co-transferred overnight Escherichia coli bacteria solution was mixed with 100UL 10% glycerol solution and added into 96-well plate, which was dried and fixed in a 37℃ incubator overnight. Fluorescence imaging was performed using cytation3 fluorescence enzyme spectrometer. The fluorescence and bright field photos were overlated, and the fluorophore and Escherichia coli bacteria were highly overlated. It is proved that Hsp27-SILEX system can make Escherichia coli successfully express the target gene
western-blot
1.5ml bacterial solution with OD600 of about 0.5 was taken for overnight culture and centrifuged to precipitate the bacterial solution. Active protein extraction reagent was added. After full reaction, the solution was centrifuged and supernant was taken to obtain soluble protein solution. loading Buffer was added to the loading buffer and heated at 100℃ for 10 minutes to denaturate the protein. Hsp27 primary antibody and sfGFP primary antibody were incubated overnight, and horseradish peroxidase secondary antibody was incubated on the second day. PVDF membrane was covered with ECL Plus hypersensitive luminescent solution, and the images were exposed on the ECL luminescence instrument.
Self-induction effect verification
Three control groups and one experimental group with the same concentration were set up, namely PET-30a-sfGFP, PET-30a-sfGFP +0.5mmol IPTG, PET-30a-sfGFP +1.0mmol IPTG, and the experimental group was PET-21a-HSP27. At the same time, the bacteria solution was cultured in a shaking table at 37℃ at 200rmp, and OD values were detected every 1 hour. The results were as shown in the figure below. The group protein expression level of 0.5mmol IPTG group was <PET-21a-HSPB1(HSP27) group, and the group protein expression level of hsp27 group was similar to that of 1mmol IPTG group. The characteristics of Hsp27-SILEX system on self-inducible expression of protein were fully demonstrated
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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