Team:SZ-SHD/Parts
Parts
Suicide Switch
As illustrated in
design
, self-lysis is a vital step to release the insect-toxic proteins. That must be inducible and carefully regulated thus avoid mass cell death in the early stage of protein synthesis. Out of ecological and commercial concerns, only a minor proportion of pre-investigated promoters are capable to be used in macroscale pesticide production. Among those, we recombined the UV inducible switch submitted by UT-Tokyo (2012) with the holin and lysozyme gene originated from T4 bacteriophage, to employ the bacterial lysis under UV radiation.
UV Switch
SulAp promoter (BBa_K518010) submitted by UT-Tokyo’s SMART bacteria project, applies the RecA mediated DNA repair mechanism to regulate gene expression in vivo. Consisting of both characteristics as a promoter and a silencer, SulAp allows RNA polymerase (RNAP) to attach. But usually, the access of RNAP will be blocked by a transcription regulating factor, the LexA protein, which suppressed on the SulAp to silence the downstream gene expression.[1] When exposed to UV (typically UVC, 254nm), RecA activity in the cytoplasm increases which cleaves the LexA to derepress the inhibition to SulAp and allows transcription occurs (Weaver, 669).[2]
In our design, activity of SulAp with and without UV induction has been investigated with the aid of the gene for green fluorescence protein (eGFP). We ligated the SulAp-eGFP fragment onto vector pSB1C3, which then transformed into the BL21 (E. coli).The increase in Fluorescence per OD600Commented [WE1]: Hyperlinked to Design page Commented [WE2]: Hyperlink Commented [WE3]: Walker, G. C. (1985).Inducible DNA Repair Systems. Annual Review of Biochemistry, 54(1), 425–457.doi:10.1146/annurev.bi.54.070185.002233Commented [WE4]: Weaver, Robert. Molecular Biology Fifth Edition.Janice Roerig-Blong, 2015.
fluorescencecan be observed over 8 hours after induction.Where there were3-4 times incrementsin fluorescence than normal, as displayed in the diagram above.
T4lysisDevice
The T4 lysis device behind the UV switch is a system originated from the T4 phage. Which have two major components: T4 holins and lysozymes. Which have been found to show the ability to penetrate the cell wall and lyse the bacteria [3]. Specifically, lysozymes refer to the peptidoglycan hydrolase, catalyst the breakdown of glycosidic bonds in peptidoglycan that destroys the bacterial cell wall. However, due to the presence of a plasma membrane, the cytoplasm is initially separated from the cell wall. Therefore, lysozymes shall only be able to reach the peptidoglycan layer through holins, which degrades the plasma membrane to introduce cellular contents into the periplasmic space [4].
Unexpectedly, rapid cell death was observed when plasmid transformed into competent cells. Possibly due to the high basal expression of SulAp. As a result, an arabinose pBAD operator was introduced to suppress the expression [5]. Therefore, we recombined the pBAD promoter and T4 lysis gene with a pSB1C3 vector, and the bacterial optical density (OD600) declined when induced by different arabinose concentration has been examined in the figure below.
Toxins
The control of pests using insecticidal proteins isolated from Bacillus thuringiensis (Bt) has been broadly studied in various agricultural activities. Compare to traditional chemical pesticides, Bt toxins have awesome advantages from the aspect of high biodegradability and specificity against pests [6]. Besides, novel researches have shown enhanced toxicity of Bt toxins when mixed and use with chitinase [7]. Inspired by this research, we intend to play a combo-strike of Bt toxin Cry7Ca1 that works on locust and the chitinase to improve the insecticidal activity
Cry7Ca1
The Cry7Ca1 is a differentiate of Bt toxins with a molecular mass of 129kDa, recently isolated from Bt strain BHT-13. According to the crystalline analysis, it could be inferred that this toxin displays a similar mode of action, the pre-pore forming model, like its relatives [8][9]. Which the lethality of this toxin against locusts have been confirmed at an LC50 (50% lethal concentration) value of 8.98 μg/ ml [8].
Jing, X., Yuan, Y., Wu, Y., Wu, D., Gong, P., & Gao, M. (2019). Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis. Protein Science, 28(3), 609-619.
The Bt toxin ingested by insects undergoes a series of complex digestion by protease in gut juice. That cleaves the protective shell and hence the core toxin will be released. Furthermore, the core toxin binds to the membrane-bonded protease on epithelial cells. The proteolytic degradation removes the N-terminal alpha chain, unreal the core into smaller oligomers that embed onto the cell membrane. Creating pores that increase the permeability thus mediated the cell death. [10]
Chitinolytic enzyme
Chitin, as one of the most abundant naturally occurring polymer, commonly found in fungal cell walls, the exoskeletons of invertebrates, and insect gut linings. In insects, the chitin layer provides extra support and shield the soft tissue against mechanical damages and toxins. Throughout evolution, many organisms have acquired ability to produce chitinolytic enzymes to digest chitin for nutrition, which have potency in the development of novel insecticide [11][12][13].
(the mode of action of chitinase, illustrated in Dahiya, N., Tewari, R., & Hoondal, G. S. (2006). Biotechnological aspects of chitinolytic enzymes: a review. Applied microbiology and biotechnology, 71(6), 773-782.) fig1
The gene ifcht1 has been isolated from an insect pathogenic fungi Isaria fumosorosea, which coded for an endochitinase (ifcht, 46kDa). It could be hypothesized that with the aid of ifcht, the protective chitin layer will be lysed hence increase the access of Cry7Ca1 to epithelial cells.
In addition, we have introduced another chitinase gene ~1.9kbp, originated form another insect pathogen Xenorhabdus nematophila, coded for a chitinase with molecular mass of 76kDa. Which the chitinase activity assay kit (SolarBio) was used to investigate its chitinolytic activity.
Reference:
[1]: Walker, G. C. (1985). Inducible DNA Repair Systems. Annual Review of Biochemistry, 54(1), 425–457. doi:10.1146/annurev.bi.54.070185.002233
[2]: Weaver, Robert. Molecular Biology Fifth Edition. Janice Roerig-Blong, 2015.
[3]: Josslin, R. (1970). The lysis mechanism of phage T4: Mutants affecting lysis. Virology, 40(3), 719–726. doi:10.1016/0042-6822(70)90216-3
[4]: Morita, M., Asami, K., Tanji, Y., & Unno, H. (2001). Programmed Escherichia coli Cell Lysis by Expression of Cloned T4 Phage Lysis Genes. Biotechnology Progress, 17(3), 573–576. doi:10.1021/bp010018t
[5]: Guzman, L. M., Belin, D., Carson, M. J., & Beckwith, J. O. N. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of bacteriology, 177(14), 4121-4130.
[6]: Roh, J. Y., Choi, J. Y., Li, M. S., Jin, B. R., & Je, Y. H. (2007). Bacillus thuringiensis as a specific, safe, and effective tool for insect pest control. Journal of microbiology and biotechnology, 17(4), 547.
[7]: Kramer, K. J., & Muthukrishnan, S. (1997). Insect Chitinases: Molecular Biology and Potential Use as Biopesticides. Insect Biochemistry and Molecular Biology, 27(11), 887–900. doi:10.1016/s0965-1748(97)00078-7
[8]: Wu, Y., Lei, C. F., Yi, D., Liu, P. M., & Gao, M. Y. (2011). Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis. Applied and environmental microbiology, 77(10), 3227-3233.
[9]: Jimenez-Juarez, N., Munoz-Garay, C., Gómez, I., Gill, S. S., Soberón, M., & Bravo, A. (2008). The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta. Peptides, 29(2), 318-323.
[10]: Melo, A. L. D. A., Soccol, V. T., & Soccol, C. R. (2016). Bacillus thuringiensis: mechanism of action, resistance, and new applications: a review. Critical reviews in biotechnology, 36(2), 317-326
[11]: Merzendorfer, H., & Zimoch, L. (2003). Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. Journal of Experimental Biology, 206(24), 4393-4412.
[12]: Patil, R. S., Ghormade, V., & Deshpande, M. V. (2000). Chitinolytic enzymes: an exploration. Enzyme and microbial technology, 26(7), 473-483.
[13]: Dahiya, N., Tewari, R., & Hoondal, G. S. (2006). Biotechnological aspects of chitinolytic enzymes: a review. Applied microbiology and biotechnology, 71(6), 773-782.
Basic Parts:
Isolated from insect pathogen Xenorhabdus nematophila (strain 19061), the gene ~1.9kbp in length coded for an endochitinase with molecular mass 76-kDa was discovered by Kumar, M., Kant, S., Banerjee, N. and Sarin, N. B. and submitted to GenBank® under accession no: JN222361[1]. Which its chitinolytic activity plays an important role in the degradation of chitin, one of the most abundant natural polymers, that shields the insect intestine to protect the epithelium from toxins[2][3][4].
(the mode of action of chitinase, illustrated inDahiya, N., Tewari, R., & Hoondal, G. S. (2006). Biotechnological aspects of chitinolytic enzymes: a review. Applied microbiology and biotechnology, 71(6), 773-782.)fig1
Besides, novel researches have shown enhanced toxicity of insecticides, such as
Bt toxins
, when mixed and use with chitinase [5]. Hence, we gain the inspiration to co-express the xncht and Bt toxin to increase the effectiveness of our biopesticide.
Construction:
The gene coded for this chitinase (xncht) has been codon-optimized with a 6*His tag attached to the C’ terminal for the ease of identification and purification. In addition, we want the xncht gene could be regulated by pTac system, allow protein compositions before and after expression to be compared. Therefore, we constructed the vector pET28a containing gene xncht then transformed into E. coli (strain DH10B) for expression.
fig 2, plasmid circuit pET28a-pTac-xncht (pSS8)
Expression:
Analysis:
Fig 4, result of chitinase activity analysis of 50ul bacteria lysate, after incubated with 50ul colloidal chitin solution at 37℃ for 4 hours. Which the product was heated with 3,5-dinitrosalicylic acid for 10 minutes and the absorbance to 540nm light was measured. Which suggested a chitinolytic activity of 0.434μMh-1.
Reference:
[1]: Mahmood, S., Kumar, M., Kumari, P., Mahapatro, G. K., Banerjee, N., & Sarin, N. B. (2020). Novel insecticidal chitinase from the insect pathogen Xenorhabdus nematophila. International Journal of Biological Macromolecules.
[2]: Merzendorfer, H., & Zimoch, L. (2003). Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. Journal of Experimental Biology, 206(24), 4393-4412.
[3]: Patil, R. S., Ghormade, V., & Deshpande, M. V. (2000). Chitinolytic enzymes: an exploration. Enzyme and microbial technology, 26(7), 473-483.
[4]: Dahiya, N., Tewari, R., & Hoondal, G. S. (2006). Biotechnological aspects of chitinolytic enzymes: a review. Applied microbiology and biotechnology, 71(6), 773-782.
[5]: Kramer, K. J., & Muthukrishnan, S. (1997). Insect Chitinases: Molecular Biology and Potential Use as Biopesticides. Insect Biochemistry and Molecular Biology, 27(11), 887–900. doi:10.1016/s0965-1748(97)00078-7
[6]: http://www.solarbio.com/data/pdf/0_20180809efujri.pdf
[7]: McCreath, K. J., & Gooday, G. W. (1992). A rapid and sensitive microassay for determination of chitinolytic activity. Journal of Microbiological Methods, 14(4), 229-237.
Composite Part:
As toxic proteins would be synthesized inside cytosol of recombinant E. coli, the concentration of these toxins was expected to increase but little could be released unless cell was damaged. However, cell disruption in traditional methodologies highly relies on expensive enzymes and apparatus, not suitable for large scale of production in industries. Therefore, we intend to construct a suicide circuit that results in cell lysis under certain environmental conditions.
Out of ecological and commercial concerns, only a minor proportion of pre-investigated promoters are capable to be used in macroscale pesticide production. Among those, we recombined the ultraviolet (UV) inducible switch submitted by iGEM11_UT-Tokyo 2011 (BBa_K518010), with the holin and lysozyme gene originated from T4 bacteriophage (submitted by UC Berkeley iGEM team in 2008, Parts: BBa_K112000; BBa_K112012). Together, this circuit would result the bacterial lysis under UV radiation.
The design of SulAp promoter aim to apply the RecA mediated DNA repairing mechanism in E. coli to control the downstream gene expression. Consisting of both characteristics as a promoter and a silencer, SulAp allows RNA polymerase (RNAP) to attach but could be repressed by the transcription factor LexA as well, and hence can block the access of RNAP and no mRNA would be synthesized. However, when irradiated by UV, the level of RecA co-protease increases in vivo, which its proteolytic activity cleaves the LexA dwelled on SulAp and derepresses the transcription [1][2][3].
UV inductive switch:
In our design, the SulAp promoter was evaluated with the aid of eGFP gene, coded for a protein with a fluorescence spectrum of 520nm. Thus, we constructed the circuit below and transformed into the RecA+ E. coli, strain BL21 (DH3).
When cell growth (OD600) approaches 0.6 in LB medium in dark, the medium was transferred into a sterilized petri-dish, and irradiated under ultraviolet C (UVC, 254nm) with an intensity of 15mWcm-2, over different period of time. Then, each sample was coated with aluminum foil and cultured in incubating-shaker for 8 hours which the fluorescence of the culture was recorded
Fig 4, the result of eGFP expression after irradiated by UVC, unit: fluorescence per OD. eGFP-: non-recombinant BL21 irradiated under UVC (without pSS1); eGFP- UV-: non-recombinant not exposed under UV. 0min: recombinant BL21, no UV exposure; 0.5min: recombinant BL21 irradiated under UV for 30 seconds; 1min: recombinant BL21 irradiated under UV for 1 min; 2min: the recombinant BL21 irradiated under UV for 2 min.
As demonstrated in fig 4, the expression level of eGFP increased after irradiation comparing to non-recombinant strain UV- groups, which proved the viability of our UV inductive switch.
T4 lysis device:
The T4 lysis device behind the UV switch is a system originated from the T4 phage. Which have two major components: T4 holins and lysozymes. Which have been found to show the ability to penetrate the cell wall and lyse the bacteria [4]. Specifically, lysozymes refer to the peptidoglycan hydrolase, catalyst the breakdown of glycosidic bonds in peptidoglycan that destroys the bacterial cell wall. However, due to the presence of a plasma membrane, the cytoplasm is initially separated from the cell wall. Therefore, lysozymes shall only be able to reach the peptidoglycan layer through holins, which degrades the plasma membrane to release cellular contents [5]. Hence, we hypothesized this T4 lysis model will be valid in terms of toxin release.
Unexpectedly, rapid cell death was resulted when attempting transform pSS2 into competent cells. Possibly due to the high basal expression of SulAp. As a result, an arabinose pBAD operator (BBa_I0500) was introduced to suppress the expression [6]. Therefore, we recombined the pBAD promoter and T4 lysis gene with a pSB1C3 vector, and the OD600 declined after induced by 1mM arabinose has been examined in the figure below.
Fig 6, the cell death resulted after 1mM arabinose added when OD600 ~0.6. Control: E. coli DH10B strain contain plasmid pSB1C3; DH10B contain pSB1C3-pBAD-T4 (pSS5). OD600 dropped over time indicated the bacterial death resulted by this induction.
Apart from cell death, our hypothesis has been supported by the results from protein concentration test as well. In this test, pSS5 and pSS8 were both delivered into one host competent cell (E. coli, DH10B) by co-transformation and cultured in LB till OD600 ~ 0.6. When 1mM IPTG was added to induce the protein expression of xncht at 37℃ for 4 hours, a centrifugal spin was performed to harvest the cell body, then resuspend in M9 medium (detailed in our protocols and methods). This process aims to remove the proteins and remaining IPTG in culture. Then, 1 mM arabinose was added and incubated at 37℃, where samples was isolated and centrifuged to measure the abundance of proteins in supernatant (Absorbance at 280nm).
Fig 7, the release of xncht after induction by arabinose (1mM) measured over 42 hours, a positively direct proportional relationship between induction time and protein concentration in supernatant was observed, with PMCC = 0.48
Reference:
[1]: Walker, G. C. (1985). Inducible DNA Repair Systems. Annual Review of Biochemistry, 54(1), 425–457. doi:10.1146/annurev.bi.54.070185.002233.
[2]: Weaver, Robert. Molecular Biology Fifth Edition. Janice Roerig-Blong, 2015.
[3]: Little, John W., et al. "Cleavage of the Escherichia coli lexA protein by the recA protease." Proceedings of the National Academy of Sciences 77.6 (1980): 3225-3229.
[4]: Josslin, R. (1970). The lysis mechanism of phage T4: Mutants affecting lysis. Virology, 40(3), 719–726. doi:10.1016/0042-6822(70)90216-3.
[5]: Morita, M., Asami, K., Tanji, Y., & Unno, H. (2001). Programmed Escherichia coli Cell Lysis by Expression of Cloned T4 Phage Lysis Genes. Biotechnology Progress, 17(3), 573–576. doi:10.1021/bp010018t.
[6]: Guzman, L. M., Belin, D., Carson, M. J., & Beckwith, J. O. N. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of bacteriology, 177(14), 4121-4130.
