Coding

Part:BBa_K5103003

Designed by: Nikoo Mansourian   Group: iGEM24_Guelph   (2024-09-18)


Cry8Da with HindIII and XhoI cut sites introduced using primers

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 987
    Illegal EcoRI site found at 1066
    Illegal EcoRI site found at 2478
    Illegal EcoRI site found at 3141
    Illegal PstI site found at 118
    Illegal PstI site found at 1559
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 987
    Illegal EcoRI site found at 1066
    Illegal EcoRI site found at 2478
    Illegal EcoRI site found at 3141
    Illegal PstI site found at 118
    Illegal PstI site found at 1559
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 987
    Illegal EcoRI site found at 1066
    Illegal EcoRI site found at 2478
    Illegal EcoRI site found at 3141
    Illegal XhoI site found at 3446
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 987
    Illegal EcoRI site found at 1066
    Illegal EcoRI site found at 2478
    Illegal EcoRI site found at 3141
    Illegal PstI site found at 118
    Illegal PstI site found at 1559
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 987
    Illegal EcoRI site found at 1066
    Illegal EcoRI site found at 2478
    Illegal EcoRI site found at 3141
    Illegal PstI site found at 118
    Illegal PstI site found at 1559
    Illegal AgeI site found at 1333
    Illegal AgeI site found at 2215
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Name: Cry8Da with primer cut sites (HindIII and XhoI)
Base Pairs: 3451 bp
Origin: Cry8Da modified to be compatible with a yeast derived expression vector created by Invitrogen[1,2]
Properties: Cry8Da with specific cut sites (HindIII and XhoI)

Usage and Biology

This part represents the fragment of the Cry8Da gene (Part:BBa_K5103000) cut with HindIII (Part:BBa_K5103006) and XhoI (Part:BBa_K5103007) restriction enzymes that has undergone PCR. Primers were designed to modify the location of HindIII and XhoI restriction sites on Cry8Da so it can be cloned into pYES2 plasmid, an expression vectors for yeast expression.[1,2] See Part:BBa_K5103002.

It is a new part that was created to facilitate cloning Cry8Da into S. cerevisiae, which needed the location of cut sites to be added/adjusted (depending on the cut site) to be able to be cloned into both pYES2 and PRS-316-PGK. Cloning was successful for pYES2 and not pRS-316-PGK.

To create this part, we PCR'd the fragments. See Part:BBa_K5103006 and Part:BBa_K5103007 for more PCR cycle details. We then digested the fragment to create the staggered ends and then ran a gel to filter everything out, cut it out of the gel, purified it and then ligated it with cut pYES2. To confirm this ligation, we then did another digestion with the same enzymes (HindIII and XhoI) and ran another gel. The part is not a Type IIS enzyme which means that it can be implemented for any expression with HindIII and XhoL cut sites.

We are the first team to attempt to clone Cry8Da into pYES2 (see Part:BBa_K5103004) and the first team to use pYES2 (see Part:BBa_K5103002) within iGEM, and we had successful results! This success is monumental for iGEM and synthetic biology. The Cry8Da gene, which encodes a protein known for its insecticidal properties, specifically targeting members of the Coleoptera order, including the Emerald Ash Borer (EAB).[3,4] The Cry8Da protein binds to receptors[3,4] and this proteolytic cleavage[5,6] in the insect's midgut, leads to pore formation and cell death.[3,4] See Part:BBa_K5103000 for more details about the cytotoxic activity of the Cry8Da protein.

Furthermore, the destination organism for this part is a shuttle plasmid that is capable of insertion in S. cerevisiae.[1,2] S. cerevisiae has been shown to produce volatile compounds that attract insects, which it uses to disperse itself into new environments.[7] Additionally, S. cerevisiae naturally inhabits the bark of trees, including ash trees, making it an ideal candidate for this project[8] and will subsequently revolutionize the biopesticide industry by harnessing the strengths of nature to target invasive pests.

Furthermore, there are very few studies exploring the expression of Cry proteins in yeast, and none look at the looked at the expression of Cry8Da. One study looks at the expression of Cyt2Aa1 in Pichia pastoris, using a synthetic version of the cytotoxic gene[9], but this study does not also use the pYES2 plasmid, thus proving our work including chassis selection is unique and as a result, its successful insertion of this part into S. cerevisiae will prove its potential for revolutionizing biopesticides and theorizes an alternative solution to the widespread Emerald Ash Borer infestation. See (Part:BBa_K5103004) for more details.

Proof of Concept

We successfully PCRd the Cry8DA fragment (Part:BBa_K5103000) creating this part. To confirm the PCR, we ran an agarose gel electrophoresis and saw bands at ~3400 bp corresponding with Cry8Da, meaning the PCR procedure was successful. Additionally, we digested Cry8Da with HindIII and XhoI to create the staggered ends, ran another agarose gel, cut out the Cry8Da band from the gel, purified and used this for ligation in pYES2.




Figure 1. 0.8% Agarose gel electrophoresis of PRS-316 plasmid with Cry8Da and PYES2-Cry8Da digested with HindIII and XhoI. Ligation of Cry8Da in PRS-316 was unsuccessful as only bands observed were at ~5000 bp. Ligation of Cry8Da in pYES2 was successful as shown above. Faint lines at ~3400 bp correspond with Cry8Da and ~5800 bp with pYES2.

To confirm that the ligation worked, after transformation and success seeing colonies (see Part:BBa_K5103004 or Figure 2 below), we miniprepped and digested this plasmid again and saw bands corresponding with both Cry8Da and pYES2 plasmid backbone!



Figure 2. Transformant pYES2-Cry8Da in S. cerevisiae BY4742. Colonies were selected for using URA3 and uracil-deficient, selective media.

Furthermore, we inserted the sequence of Cry8Da into a protein folding software, AlphaFold, to determine the protein folding of our Cry8Da fragment and if it would interact with the ß-glucosidase receptor present in EAB midgut like the Cry8Da protein within Bt.[3,4]


Figure 3. Model of Cry8Da protein in a coiled structural motif using AlphaFold. Cry8Da is coloured using blue and green. The green part corresponds to the 54kDa portion of the protein. This part of the protein interacts the most with ß-glucosidase (pink).

Part:BBa_K5103004 represents the expression of BBa_K5103003 as being expressed in BBa_K5103002. Part:BBa_K5103004 does not meet assembly compatibility requirements because of it contains multiple restricted enzyme recognition sites originating from Part:BBa_K5103002 and it is incompatible with both RFC10 and RFC1000. BBa_K5103003 however is RFC1000 compatible.

References

[1] SnapGene. (n.d.) pYES2. Invitrogen. https://www.snapgene.com/plasmids/yeast_plasmids/pYES2
[2] ThermoFisher. (2024). PYES2 Yeast Expression Vector. ThermoFischer.com. https://www.thermofisher.com/order/catalog/product/V82520
[3] Shu, C., Yan, G., Huang, S., Geng, Y., Soberón, M., Bravo, A., Geng, L., & Zhang, J. (2020). Characterization of two novel Bacillus thuringiensis Cry8 toxins reveals differential specificity of protoxins or activated toxins against Chrysomeloidea coleopteran superfamily. Toxins, 12(10), 642. https://doi.org/10.3390/toxins12100642
[4] Bravo, A., Gill, S. S., & Soberón, M. (2007). Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 49(4), 423-435. https://doi.org/10.1016/j.toxicon.2006.11.022
[5] Yamaguchi, T., Bando, H., & Asano, S. (2013). Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica. Journal of Invertebrate Pathology, 113(2), 123-128. https://doi.org/10.1016/j.jip.2013.03.006
[6] Yamaguchi, T., Sahara, K., Bando, H., & Asano, S. (2010). Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle. Journal of Invertebrate Pathology, 105(3), 243-247. https://doi.org/10.1016/j.jip.2010.07.002
[7] Christiaens, J. F., Franco, L. M., Cools, T. L., De Meester, L., Michiels, J., Wenseleers, T., Hassan, B. A., Yaksi, E., & Verstrepen, K. J. (2014). The fungal aroma gene ATF1 promotes dispersal of yeast cells through insect vectors. Ecology and Evolution, 4(19), 3905-3919. https://doi.org/10.1016/j.celrep.2014.09.009
[8] Arbab, N., Grabosky, J. R., Leopold, R. (2022). Economic assessment of urban ash tree management options in New Jersey. Sustainability, 14(4), 2172. https://doi.org/10.3390/su14042172
[9] Gurkan, C., & Ellar, D. J. (2010). Expression of Bacillus thurigiensis Cyt2Aa1 toxin in Pichia pastoris using a synthetic gene construct. Biotechnology and Applied Biochemistry, 38(1), 25-33. https://doi.org/10.1042/BA20030017

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