Team:Wageningen UR/Description/Biocontainment

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Wageningen UR iGEM 2016

 

Biocontainment

BeeT is intended to be used outside the lab, in beehives, where it will be in close contact with nature. As we cannot be sure about the effect on existing ecosystems if BeeT would be released in the environment, it must be engineered to die if it leaves the beehive. Our first measure to achieve this is a light-induced kill switch that relies on the balance of a bacterial toxin and an antitoxin that are expressed simultaneously. In the dark beehive, the system is unaffected. In the presence of blue light, a component of sunlight, the balance is disturbed in favour of the bacterial toxin. This will kill the bacterium.

As an additional safety measure, BeeT relies on the presence of a synthetic amino acid that is to be applied to the beehive. In the presence of the synthetic amino acid, catalytically dead Cas9 (dCas9) is produced that has the synthetic amino acid incorporated, at the expense of catalytically active Cas9. If BeeT escapes from the hive, the synthetic amino acid is no longer available, and dCas9 can no longer be formed. Instead, catalytically active Cas9 is produced which cuts the BeeT genome as well as any heterologous DNA that is present, thereby killing the bacterium and preventing horizontal gene transfer.

Light Kill Switch

Introduction

The optogenetic kill switch is the unification of two different genetic systems: pDusk, an artificially-created light-sensing promoter system; and mazEF, a toxin-antitoxin system native to E. coli.

MazEF, on the other hand, []. As the name implies, the system consists of two components: the toxin, mazF; and the antitoxin, mazE. In its native organisms, mazEF has a regulatory function. Under stressful conditions

The optogenetic kill switch, therefore, is designed to work as follows.

Construction of genetic circuit

Cas9-based killswitch

Introduction

Because we could image that using …, we wanted to have another approach to achieving biocontainment in parallel. An idea that came up was using a bacterial strain developed by Mandell and colleagues1 as a chassis for BeeT. This strain can be confined to a certain area because it is auxotrophic for a synthetic amino acid, para-L-biphenylalanine (BipA). Several essential proteins of this bacterial strain were engineered to function only when BipA is incorporated in the active site, leading to death of the bacterium when the synthetic amino acid is not available. In our case, BipA is applied to the beehive, so BeeT cannot survive when it escapes in the environment, or when the beekeepers ceases to supply BipA.
A drawback of this system is that, even though the GMO dies as soon as it is deprived of the synthetic amino acid, it’s heterologous DNA will remain in the environment. Since DNA is rather stable given the right circumstances2, it can be taken up by other bacteria through horizontal gene transfer3,4. In our project, we sought to complement the biocontainment strain by creating a switch to destroy heterologous DNA, depending on the presence of BipA. Additionally, the switch can be used to further strengthen the auxotrophy for the SAA by cutting genomic DNA as well. To this end, we aimed to engineer Streptococcus pyrogenes Cas9 to switch from a catalytically dead form (dCas9)5 to an active nickase form (nCas9)6 as soon as the bacteria leave the hive. This switching depends on whether a BipA is incorporated into Cas9. Two residues known to be essential for DNA cleaving were targeted: Asp10 and His840. Additionally, one of these residues is changed to alanine (as targeting only one AA residue will make a switch between fully active Cas9 and nCas9). To incorporate the BipA, the codon for the native AA is replaced by the TAG stop codon. To translate the TAG codon, an amino-acyl-RNA-synthetase (aaRS) and a tRNA that recognizes the TAG codon (tRNACUA) that are specific for BipA will be provide7. To make translation of the stop codon more efficient, we will use a genetically recoded strain that lacks any TAG stop codons as well as release factor 18.

BipA will be loaded on the tRNACUA as long as the E. coli stays in the hive, where BipA is supplied. This results in the formation of dCas9. When E. coli escapes from the hive, no charged tRNACUAs are available, the Cas9 transcript cannot be translated and dCas9 is not formed.

To switch from expression of dCas9 to nCas9, dCas9 is used to repress transcription9,10 of another aaRS and tRNACUA that is charged with a natural amino acid, for example aspartate11. When the organism is deprived of BipA, the repression is released. With the tRNACUA(Asp) available, the Cas9 transcript can be translated again. However, this time Aspartate is incorporated in Cas9 instead of BipA, resulting in the formation of nCas9 rather than dCas9. The nCas9 is able to cut any DNA for which two suitable spacers are offered, including all heterologous DNA.

Cloning and expression of Cas9 variants

Catalytically active Cas9 was PCR taken from the iGEM registry (BBa_K1218011), while dCas9 was pdCas9 was a gift from Luciano Marraffini (Addgene plasmid # 46569)10. Using mutagenesis PCR, Ala10TAG and Ala840TAG mutations were introduced separately in the dCas9 construct. A vector to translate the TAG stopcodon was already available from the biocontainment strain1 (pEVOL-BipA). pEVOL-Asp was constructed to translate the TAG stopcodon to Aspartate.
To text translation of the TAG stopcodon and incorporation of BipA, the expresso system for rhamnose induced expression from Lucigen was used12. Bacterial cultures transformed with Cas9-expresso constructs, and in case of a mutant Cas9, either with pEVOL-BipA or pEVOL-Asp. SDS-PAGE of the FPLC purified samples showed clear expression of both Cas9 and dCas9, lower expression of dCas9-Ala10BipA and very low (if at all) expression of dCas9-Ala10Asp.

In vitro Cas9 cleaving assays

To assess the functionality of our Cas9 variants, in vitro Cas9 cleavage assays were performed. From the assays, it can be concluded that our purified Cas9 is functional using several gRNAs targeting a different part of RFP, as the linear target was cleaved at least to some extend. As expected, dCas9 does not have this effect, nor has dCas9-Ala10BipA. When two gRNAs that target two spots of RFP are offered to dCas9-Ala10Asp, it is expected to see some cleaving caused by the two nicks in close proximity. Indeed, some cleaving was observed. However, also some cleaving was observed when offering only one gRNA. It has been shown that the His840Ala version of nCas9 (which is basically the same as our dCas9-Ala10Asp) has some double stranded DNAse activity (ref). Further testing is needed whether this is the case for our Cas9-mutant, or that it is an artefact.

References

     

    References for optogenetic kill switch

    1. Engelberg-Kulka, H., Hazan, R., Amitai, S. (2005). mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. Journal of Cell Science 118, 4327-4332. ↩

    References for Cas9-based kill switch

    1. Mandell, D. J., Lajoie, M. J., Mee, M. T., Takeuchi, R., Kuznetsov, G., Norville, J. E., ... & Church, G. M. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature, 518(7537), 55-60. ↩

    2. Romanowski, G., Lorenz, M. G., Sayler, G., & Wackernagel, W. (1992). Persistence of free plasmid DNA in soil monitored by various methods, including a transformation assay. Applied and Environmental Microbiology,58(9), 3012-3019. ↩

    3. Thomas, C. M., & Nielsen, K. M. (2005). Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nature reviews microbiology, 3(9), 711-721. ↩

    4. Smillie, C. S., Smith, M. B., Friedman, J., Cordero, O. X., David, L. A., & Alm, E. J. (2011). Ecology drives a global network of gene exchange connecting the human microbiome. Nature, 480(7376), 241-244. ↩

    5. Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., ... & Church, G. M. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature biotechnology, 31(9), 833-838. ↩

    6. Shen, B., Zhang, W., Zhang, J., Zhou, J., Wang, J., Chen, L., ... & Skarnes, W. C. (2014). Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nature methods, 11(4), 399-402. ↩

    7. Xie, J., Liu, W., & Schultz, P. G. (2007). A genetically encoded bidentate, Metal‐Binding amino acid. Angewandte Chemie, 119(48), 9399-9402. ↩

    8. Lajoie, M. J., Rovner, A. J., Goodman, D. B., Aerni, H. R., Haimovich, A. D., Kuznetsov, G., ... & Rohland, N. (2013). Genomically recoded organisms expand biological functions. Science, 342(6156), 357-360. ↩

    9. Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173-1183. ↩

    10. Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic acids research, 41(15), 7429-7437. ↩

    11. Pastrnak, M., Magliery, T. J., & Schultz, P. G. (2000). A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair for evolving an organism with an expanded genetic code. Helvetica Chimica Acta, 83(9), 2277-2286. ↩

    12. Expresso® Rhamnose Cloning & Protein Expression System ↩