Construction and Evaluation of Self-Directing Expression System Using Regulatory Elements of Cry Gene of Bacillus Thuringiensis

An expression system based on the cry gene regulatory elements was constructed. The Terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1 was cloned in pSG1151 plasmid downstream to gfp mut1 . The promoter region of the cry gene was amplied to give three different reading frames. The Promoter region of cry gene was cloned in pSG1151T plasmid upstream to gfp mut1 . The expression of GFP under the promoter/terminator expression system was evaluated by checking the expression of gfp mut1 under the same promoter. The GFP content of pSG1151 and three constructs; pDSA1, pDSA2 and pDSA3 were compared by uorescence spectroscopy. The uorescent intensity of pSG1151 and pDSA1 were compared at time interval of 6 hours upto 72 hours. Both the samples showed detectable uorescence that increased with time up to 12 hours, but the increase in the uorescence of pDSA1 was 3 times higher as compared to pSG1151. A cold peptidase gene was cloned under the control of the cry promoter. The transformed E.coli DH5α colonies were patched on skim milk agar plates and the clones of pSG1151CP and pDSA1CP were compared on the basis of zone of clearance. The zone of clearance of pDSA1CP was much higher as compared to that of pSG1151CP. The cell-free supernatant of Bacillus sp. S1DI 10 and recombinant pDSA1CP collected at different time points was assayed for the specic activity of the extracellular protease. At 72 hours the protease activity in pDSA1CP was 2.7 fold higher compared to that of wild Bacillus sp. S1DI 10.


Introduction
The accumulation of Cry toxin protein inside the bacillary body leads to the formation of crystal inclusions in B. thuringiensis. The possible mechanism behind the accumulation of crystal protein by B. thuringiensis is the expression of crystal protein gene via a strong promoter. The promoter is active during both in logarithmic and in stationary phase resulting in the overproduction of the crystal protein. The strains of Bacillus sp. respond to nutritional de cient conditions by either becoming; dormant or by sporulation. On this basis, the cry genes that are expressed in the stationary phase can be divided into two categories; cry genes contingent on spore formation and cry genes that are not related to spore formation .
The endospore formation in Bacillus sp. takes place in a sporangium which consists of two cellular compartments called as the forespore and the mother cell. The process of endospore development is regulated by a primary sigma factor of vegetative cell and ve other sigma factors that appear successively during sporulation. The recognition of gene promoters selectively depends upon the binding of these sigma factors to RNA Polymerase (Helmann et al., 1988, Moran, 1993. The Cry toxin secreted by cryIA gene is an example of cry gene expressed in mother cell of B. thuringiensis and is associated with sporulation. In addition to sigma factors, two overlapping promoters (BtI and BtII) for cryIA were mapped by Wong et al., (1983) which is active during different time points in sporulation phase. The initiation of transcription by the RNA Polymerase from these two promoters carries separate sigma factors (Calogero et al., 1989, Brown andWhiteley, 1990). The different sigma factor mutants of cryIA-lacZ fusion in B. thuringiensis gave either diminished or no β-galactosidase activity. However, a very high β-galactosidase activity was observed in wild cryIA-lacZ fusion of B. thuringiensis, which suggests that strong promoters are involved in the expression of cryIA gene . Various studies have shown that some cry genes in different strains of B. thuringiensis carry only BtI or both the promoters and others have regions similar to those in BtI and BtII promoters (Brown and Whiteley, 1988, Brown, 1993, Dervyn et al., 1995, Hajime et al., 1993. Thus, these cry genes can be categorized as sporulation dependent cry genes. The cryIIIA gene is an example of sporulation independent cry genes as its expression was independent of the sigma factors present during sporulation in both B. thuringiensis and B. subtilis cry gene Lereclus, 1994a, Salamitou et al., 1996). The expression of cryIIIA was increased in mutant strains of B. thuringiensis which were not able to commence sporulation. (Lereculus et al., 1995). Smith, (1993) identi ed two different regulators in stationary phase which are required for gene expression in B. subtilis.
Further, the location of a gene on plasmid affects its expression level as the copy number of plasmid has been exploited in recent times for overexpression of a protein. The cry genes are also carried by plasmids which naturally lead to a huge quantity of toxins in various B. thuringiensis strains. The presence of different cry genes varies in different strains of B. thuringiensis which differs in size and shape of the crystal (Lereculus et al., 1993). However, the cloning of cryIAc gene in a strain having other cryI genes lead to lower production of Cry protein as compared to its cloning in cry − strain while there was no decline in its expression when cloned into strains with cryIIIA gene (Baum et al., 1990, Lecadet et al., 1992, Lereculus et al., 1992. Therefore, the expression of cry gene may not be related to the high copy number of plasmid. mRNA has a speci c half life and its degradation affects the expression of a gene. The increase in expression of a protein requires production of a stable mRNA as evident from ompA mRNA that encodes a membrane protein in Escherichia coli having longer half life of 20 minutes as compared to 2-3 minutes for the other mRNAs in E. coli (Nilsson et al., 1984). The mRNAs that encode crystal protein in B. thuringiensis have a half life of ~10 minutes (Glatron et al., 1972). In addition to that, the 3' terminal region of the cryIAa gene acts as a positive retro-regulator in B. thuringiensis as its heterologous expression with a gene increased the mRNAs half life as well as the expression (Wong andChang, 1986, Wong et al., 1983). The terminal region consists of inverted repeats forming stem loop structure that prevents degradation of mRNA from exonucleases like exoribonuclease PNPase in E. coli (Causton et al., 1994). An ~600bp upstream promoter containing two distinct regions is involved in expression of the cryIIIA gene in B. thuringiensis (De Souza et al., 1993, Agaisse andLereclus, 1994b). The upstream region was reported to be involved in transcription while the downstream region acts as a 5' mRNA stabilizer which increased the stability of mRNA as well as the expression of protein. (Bechhoffer et al., 1993, Hue et al., 1995, Agaisse and Lereculus, 1996. Therefore, the present study was undertaken to construct promoter/terminator based expression system harboring promoter and terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1.

Isolation of genomic DNA and plasmid DNA
The reference strain B. thuringiensis subsp. kurstaki HD-1 was grown overnight for the isolation of genomic DNA according to the method described in a previous study with slight modi cations (Valicente et al., 2008, Singh et al., 2019. The strains carrying plasmid (pSG1151) were grown overnight for isolation of plasmid DNA using High Speed Plasmid Mini Kit (IBI, Scienti c, USA) following the manufacturer's instructions.

PCR ampli cation of terminator region of cry gene
The DNA isolated from B. thuringiensis subsp. kurstaki HD-1 was used as template for the ampli cation of promoter and terminator region of the cry gene. The primers for the terminator region of cry gene (TFP1 5'-GCTCTAGACGTGGACA GCGTGGAATTA-3' and TRP2 5'-TCCCCGCGGTAAGTTGCTCTATACATA-3') were designed on the basis of the sequence determined in a previous study (Wong and Chang, 1986). The terminator region was ampli ed in a 20 µL reaction mixture containing Phusion High Fidelity buffer (5X), 10 µM each of forward and reverse primer, 10 mM dNTPs, 20 ng/µL DNA, and 2U Phusion DNA Polymerase (NEB, USA). The PCR reaction was performed in a Thermocycler (Agilent Technologies, USA) with an initial denaturation step of 98˚C for 30 sec, followed by 35 cycles of denaturation at 98˚C for 10 seconds, annealing at 62˚C for 30 seconds and extension at 72˚C for 20 seconds. A nal step of extension was performed at 72˚C for 10 min. The PCR-ampli ed product was puri ed using a PCR/Gel Puri cation Kit (IBI, Scienti c, USA) following the manufacturer's instructions.

Cloning of terminator region of cry gene
The PCR ampli ed product was puri ed and digested with restriction enzymes; XbaI and SacII (NEB, USA) at 37˚C for 4 hours. pSG1151 plasmid was digested overnight with the aforementioned restriction enzymes at 37˚C. The digested PCR product and vector were ligated using T4 DNA Ligase (NEB, USA) at 16˚C for 16 hours and transformed into DH5α strain of E. coli. The transformed cells were plated on ampicillin (100 µg/ml) supplemented LB Agar plates. After incubation at 37°C for 18 hours, clones with successful recombination showing ampicillin resistance were selected and multiplied.

PCR ampli cation of promoter region of cry gene:
The primers for the promoter region of cry gene (Table 1) were designed from a previous study that determined nucleotide sequence of promoter region of cry gene (Wong et al., 1983). The promoter region was ampli ed in a 20 µL reaction mixture containing Phusion High Fidelity buffer (5X), 10 µM each of forward and reverse primer, 10 mM dNTPs, 20 ng/µL DNA, and 2U Phusion DNA Polymerase (NEB, USA).
The PCR reaction was performed in a Thermocycler (Agilent Technologies, USA) with an initial denaturation step of 98˚C for 30 sec, followed by 35 cycles of denaturation at 98˚C for 10 seconds, annealing at 60˚C for 30 seconds and extension at 72˚C for 30 seconds. A nal step of extension was performed at 72˚C for 10 min. The PCR-ampli ed product was puri ed using a PCR/Gel Puri cation Kit (IBI, Scienti c, USA) following the manufacturer's instructions. 2.8 GFP expression analysis by uorescence microscopy: The bacterial cells were monitored at different time intervals for the uorescence at 507nm (with excitation at 488nm for strain carrying gfp mut1 plasmid) by Fluorescent microscopy using Fluorescent microscope (Nikon, Japan).

Cloning and expression of peptidase under cry regulatory elements
The primers with restriction enzyme site added to the 5' end (PDFP Sal1

Cloning of terminator region of cry gene:
The ampli cation of terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1 resulted in ã 209bp PCR product. Figure 1 depicts the PCR product of terminator region of cry gene. The plasmid map of pSG1151 with restriction enzymes selected for cloning of terminator and promoter region are shown in Figure 2. S1 depicts the digested products from the transformed pSG1151T plasmid.

Cloning of promoter region of cry gene:
The promoter region of the cry gene was ampli ed in such a way to give three different reading frames. The ampli cation of promoter region of cry gene from B. thuringiensis subsp. kurstaki HD-1 resulted in ã 350bp PCR product for all three frames. Figure 3 depicts the PCR product of promoter region of cry gene for all three frames. S2 depicts the digested products from the transformed plasmids. The constructs carrying three frames were named as pDSA1, pDSA2 and pDSA3 for gfp mut1 . Figure 4 represents the structural organizations of these constructs.

GFP expression analysis by uorescence spectroscopy and microscopy:
The expression of GFP under the promoter/terminator expression system was evaluated by checking the expression of gfp mut1 under the same promoter/terminator. The results obtained from the uorescence spectroscopic and microscopic studies of pSG1151 were compared with the modi ed constructs of gfp mut1 . The GFP content of pSG1151 and three constructs; pDSA1, pDSA2 and pDSA3 were compared, out of which pDSA1 had higher GFP content and gave maximum uorescence ( Figure 5 and 6). The uorescent intensity of pSG1151 and pDSA1 was also compared up to 72 hours at a time interval of 6 hours. Both the sample showed detectable uorescence which increased with time up to 12 hours, but there was a much higher increase in the uorescence of pDSA1 as compared to pSG1151 that enhanced slightly with time (Figure 7 and 8).

Cloning and expression of peptidase:
In order to study the expression of an enzyme under cry promoter and terminator, cold peptidase gene previously isolated from Bacillus sp. S1DI 10 (Singh et al., 2019) was cloned in the constructed vectors. The ampli cation of cold peptidase gene from S1DI 10 resulted in a ~2400bp PCR product. Figure 9 depicts the PCR product of peptidase gene and Figure 10 represents the plasmid map of pDSA1 showing restriction enzymes selected for cloning of cold peptidase. S3 depicts the digested products from the transformed pSG1151CP and pDSA1CP plasmids. The transformed E.coli DH5α colonies were patched on skim milk agar plates and the clones of pSG1151CP and pDSA1CP were compared on the basis of zone of clearance. The zone of clearance of pDSA1CP was much higher as compared to that of pSG1151CP and wild Bacillus sp. S1DI 10 ( Figure 11). The expression of peptidase in pDSA1CP was also compared to the expression of peptidase in wild Bacillus sp. S1DI 10. The cell free supernatant of Bacillus sp. S1DI 10 and recombinant pDSA1CP collected at different time points was assayed for the speci c activity of the extracellular peptidase. There was 2.7 fold higher protease activity of in recombinant pDSA1CP than that of wild Bacillus sp. S1DI 10 at 72 hours ( Figure 12). The expression of extracellular peptidase from pDSA1CP at different time intervals was also higher than that of wild Bacillus sp. S1DI 10 as observed from the SDS-Page ( Figure 13). In order to check the expression of a heterologous bacterial gene encoding for an enzyme in the promoter/terminator expression system, cold peptidase enzyme isolated from a Bacillus sp S1D1 10 was ued in our expression system. (Singh et al., 2019). Enzyme puri cation and characterization are essential basic requirements for effective industrial application of enzymes. The strength of the promoter/terminator expression system was further analysed by checking the expression of an extracellular cold peptidase under the in uence of the promoter and terminator. The cell free supernatant of Bacillus sp. S1DI 10 and recombinant pDSA1CP collected at different time points was assayed for the speci c activity of the extracellular peptidase. The results revealed that there was 2.7 fold higher activity (at 72 hours) of pDSA1CP than that of wild Bacillus sp. S1DI 10. The expression and activity of an extracellular lipase increased signi cantly under the regulation of P 43 and P AE promoters in B. subtilis

Conclusion
The promoter/terminator based expression system constructed in the present work can be used to express the industrially important proteins which will further cut the cost of inducer, as the novel approach developed for this expression system does not require induction by an inducer.  Peptidase production by pDSA1CP and Bacillus sp. S1DI 10 at different time intervals. Figure 13