Study On The Expression Of The IeGFP And ImCherry Fusion Fluorescent Proteins In Prokaryotic Bacteria

In order to study the Cry1 Ia protein secretion signal peptide(Iasp)of Bacillus thuringiensis(Bt),it was fused to the N-terminus of e GFP and m Cherry,respectively,to form two fusion fluorescences of Ie GFP and Im Cherry.protein.The prokaryotic expression of the two fusion proteins was examined to study the effect of Iasp on the expression of two fluorescent proteins.In the early stage,the laboratory has studied the expression of the fusion protein Ie GFP in Gram-positive Bt cells,and the results show that Iasp can significantly enhance the fluorescence intensity of e GFP in Bt cells.However,whether the Ie GFP fusion fluorescent protein has the same or similar expression effect in Gram-negative bacteria,such as E.coli,is still unclear.Therefore,the plasmids p Ac Ie GFP expressing Ie GFP and p Ace GFP expressing e GFP were transformed into E.coli TG1 cells to obtain TAc Ie GFP and Tace GFP.Strains,and conduct research.The TAc Ie GFP and Tace GFP strains were separately cultured,and cell color observation and fluorescence signal intensity monitoring were performed.The test results showed that the cell color of TAc Ie GFP(expressing Ie GFP)was more significant than that of TAce GFP cells(expressing e GFP).At five monitoring time points(4,8,10,12 and 24 h),the fluorescence signal of TAc Ie GFP cells was higher than that of TAce GFP.Especially after 10 h of culture,the fluorescence signal intensity of the two was always more than 2 times.The results of the Western blot analysis are consistent with the trend of monitoring fluorescence signal intensity.To study the effect of Iasp on e GFP protein itself,Ie GFP and e GFP proteins were purified and quantified separately.The difference in fluorescence signals of the equal amounts of Ie GFP and e GFP was determined,and the p H stability of the two proteins was analyzed.The results showed that the fluorescence intensity of Ie GFP protein was significantly higher than that of equimolar e GFP.The fluorescence signal changes of Ie GFP were consistent with e GFP protein under different p H conditions.To further verify the effect of Iasp on other fluorescent protein signals,it was fused to the N-terminus of m Cherry to obtain Im Cherry.The expression of Im Cherry protein in Escherichia coli and Bacillus thuringiensis showed that the performance of Im Cherry was better than that of m Cherry.The cell color was easier to observe and the fluorescence signal intensity was higher,which was consistent with the expression of Ie GFP.In summary,Ie GFP and Im Cherry fluorescent proteins can replace their respective parental proteins,e GFP and m Cherry,as a novel fluorescent indicator molecule for use in prokaryotic cells.This study also showed that the Cry1 Ia signal peptide Iasp can promote the functional expression of fluorescent protein,which can be used to improve other fluorescent proteins.