Identification of the Optimal Fluorescent Protein Reporter Gene in Methylobacterium extorquens AM1

doi:10.13560/j.cnki.biotech.bull.1985.2018-0892

Abstract

Abstract: Fluorescent protein has been a commonly used molecular marker in genetic engineering over the recent years,detailed interpretation of the expressions of different fluorescent protein genes in Methylobacterium extorquens is for identifying the most suitable fluorescent reporter gene. Genes of 5 fluorescent proteins,the frequently-used green fluorescent protein eGFP,red fluorescent protein mCherry,and yellow fluorescent protein YFP,as well as green fluorescent protein wGFP and red fluorescent protein RFP from other sources were cloned,the corresponding expression vectors were constructed,and their expressions in M. extorquens AM1 were achieved. Fluorescence protein expression in the transformants was identified via fluorescence imaging observation and SDS-PAGE protein gel electrophoresis. Furthermore,bacteria-growth curve,total fluorescence amount and relative fluorescence intensity were detected in all transformants to illustrate the fluorescence stability and expression intensity. As results,all target fluorescent proteins in the transformants were detected,while RFP expressed in a trace and no imaging was observed,and YFP presented the most stable and the highest relative fluorescence intensity as the 1.6 times of common eGFP,and 3.4 times of mCherry. In sum,YFP is the optimal fluorescent protein for M. extorquens AM1.

Key words: Methylobacterium extorquens, fluorescence reporter gene, relative fluorescence intensity

ZHU Li-ping, LIU Cong-cong, SONG Shu-zhen, DIAO Bin. Identification of the Optimal Fluorescent Protein Reporter Gene in Methylobacterium extorquens AM1[J]. Biotechnology Bulletin, 2019, 35(4): 43-50.

References

[1] Chistoserdova L, Vorholt JA, Thauer RK, et al.C1 transfer enzymes and coenzymes linking methylotrophic bacteria and methanogenic Archaea[J]. Science, 1998, 281:99-102.
[2] Lee MC, Chou HH, Marx CJ.Asymmetric, bimodal trade-offs during adaptation of Methylobacterium to distinct growth substrates[J]. Evolution, 2009, 63:2816-2830.
[3] Chou HH, Chiu HC, Delaney NF, et al.Diminishing returns epistasis among beneficial mutations decelerates adaptation[J]. Science, 2011, 332:1190-1192.
[4] Vu HN, Subuyuj GA, Vijayakumar S, et al.Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth[J]. J Bacteriol, 2016, 198:1250-1259.
[5] Marx CJ, Lidstrom ME.Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria[J]. Microbiology, 2001, 147:2065-2075.
[6] Chubiz LM, Purswani J, Carroll SM, et al.A novel pair of inducible expression vectors for use in Methylobacterium extorquens[J]. BMC Res Notes, 2013, 6:183.
[7] Matz MV, et al.Fluorescent proteins from nonbiolumi-nescent Anthozoa species[J]. Nat Biotechnol, 1999, 17:969-973.
[8] Shaner NC, Campbell RE, et al.Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein[J]. Nat Biotechnol, 2004, 22:1567-1572.
[9] Shu X, Royant A, Lin MZ, et al.Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome[J]. Science, 2009, 324:804-807.
[10] Day RN, Davidson MW.The fluorescent protein palette:tools for cellular imaging[J]. Chem Soc Rev, 2009, 38:2887-2921.
[11] Michener JK, et al.Phylogeny poorly predicts the utility of a challenging horizontally transferred gene in Methylobacterium strains[J]. J Bacteriol, 2014, 196:2101-2107.
[12] Figueira MM, Laramee L, et al.Production of green fluorescent protein by the methylotrophic bacterium methylobacterium extorquens[J]. FEMS Microbiol Lett, 2000, 193:195-200.
[13] Marx CJ, Lidstrom ME.Development of an insertional expression vector system for Methylobacterium extorquens AM1 and generation of null mutants lacking mtdA and/or fch[J]. Microbiology, 2004, 150:9-19.
[14] Sy A, Timmers AC, Knief C, et al.Methylotrophic metabolism is advantageous for Methylobacterium extorquens during colonization of Medicago truncatula under competitive conditions[J]. Appl Environ Microbiol, 2005, 71:7245-7252.
[15] Liang WF, Sun MY, Cui LY, et al.Cre/loxP-Mediated Multicopy Integration of the Mevalonate Operon into the Genome of Methylobacterium extorquens AM1[J]. Appl Biochem Biotechnol, 2018, 185:565-577.
[16] Choi YJ, Morel L, Bourque D, et al.Bestowing inducibility on the cloned methanol dehydrogenase promoter(PmxaF)of Methylobacterium extorquens by applying regulatory elements of Pseudomonas putida F1[J]. Appl Environ Microbiol, 2006, 72:7723-7729.
[17] Guiziou S, Sauveplane V, Chang HJ, et al.A part toolbox to tune genetic expression in Bacillus subtilis[J]. Nucleic Acids Res, 2016, 44:7495-7508.
[18] Singh A, Azad M, Shymko MD, et al.The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation[J]. PLoS One, 2018, 13:e0204792.
[19] Yang YM, Chen WJ, Yang J, et al.Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route[J]. Microb Cell Fact, 2017, 16:179.
[20] Heim R, Cubitt AB, Tsien RY.Improved green fluorescence[J]. Nat, 1995, 373:663-664.