Team:Wageningen UR/Description/Biocontainment

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Wageningen UR iGEM 2016

 

Biocontainment

Appropriate biocontainment measures form a significant part of the BeeT project's Safety aspect. 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.

Optogenetic 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.

The "kill" component of the kill switch, MazEF1, is a so-called toxin-antitoxin (TA) system. As the name implies, the system consists of two components: a toxin, MazF; and its complimentary antitoxin, MazE. The MazF protein functions as an endoribonuclease, cleaving mRNA and thereby inhibiting transcription, which is ultimately lethal for the cell. MazE forms a complex with MazF, preventing it from functioning. However, MazE is a more labile protein than MazF, degrading faster. For this reason, TA systems like MazEF are also known as addiction modules; the organism expressing the system becomes "addicted" to the antitoxin and is negatively affected should expression stop. In its native organisms, MazEF has a regulatory function. Under ordinary conditions, the proteins are co-expressed and the organism stays stable. However, under stressful conditions, such as nutrient starvation, this expression ceases. MazF is then free to cleave essential mRNAs, causing what is inferred to be a state of bacteriostasis.2 This process is reversible up until approximately 6 hours, the so-called Point of No Return. Much like programmed cell death (PCD) in multicellular organisms, this prevents excess growth the bacterial population cannot afford, improving viability.

The optogenetic kill switch is designed to exploit this mechanism. Rather than remaining active until a

Construction of genetic circuit

Both MazE and MazF have been registered as BioBricks. However, as of yet, neither are available. For this reason, we chose to isolate both genes directly from E. coli genomic DNA.

Cas9-based killswitch

Because we wanted to minimize the potential biosafety risks of BeeT, we wanted to have another approach to achieve biosafety next to the light kill switch. 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.

Using Cas9 to reinforce auxotrophy for a synthetic amino acid

To this end, we aimed to engineer Streptococcus pyogenes 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. nCas9 is a partially active Cas9 version that is able to make nicks in the DNA rather than double stranded breaks. However, it was shown that nCas9 is able to cause double strand breaks when two spacers are available to nick DNA on opposite strands in close proximity.

The switching between dCas9 and nCas9 depends on whether a BipA is incorporated into Cas9 (figure 1). 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.

Figure 1. Genetic circuit for switching between catalytically dead Cas9 and partially active (nickase form) Cas9 depending on the presence of a synthetic amino acid. aaRS = amino-acyl tRNA synthetase, SAA = synthetic amino acid, NAA = native amino acid, dCas9 = catalytically dead form of Cas9, nCas9 = nickase form of Cas9. (note: I will make an image in inkscape with the same composition that matches the other images).



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 to incorporate BipA was already available from the biocontainment strain1 (pEVOL-BipA). pEVOL-Asp was constructed to translate the TAG stopcodon to incorporate Aspartate instead.

To test translation of the TAG stopcodon and incorporation of BipA in Cas9, 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 and lower expression of dCas9-Ala10BipA. A faint band could be seen for dCas9-Ala10Asp, but since a control sample without any synthetic amino acid also displayed a (very) faint band, this result is not conclusive (figure 2).

Figure 2. SDS-PAGE of fractions after FPLC purification of Cas9 variants. Cas9 = 156 kDa. Red boxes indicate 150 kDa band of the ladder, red arrows indicates bands of the correct size corresponding to Cas9. The black arrow indicates elution with increasing concentration of Imidazole. a) Cas9. b) dCas9. c) dCas9-Ala10BipA. d) dCas9-Ala10Asp. e)dCas9-Ala10TAG, no synthetic amino acid. Ladder: Precision Plus protein ladder (Bio-Rad). CFE = Cell Free Extract.



In vitro Cas9 cleaving assays

To assess the functionality of our Cas9 variants, in vitro Cas9 cleavage assays were performed. An RFP PCR product of 4140 bp was chosen as the target for cleaving, which would generate one ~3100 bp fragment and one ~1040 bp fragment. All targets were chosen in close proximity; meaning all cleavage products would be roughly the same length, and targets on opposite DNA strands could generate a double strand break when incubated with dCas9-Ala10Asp (nCas9).

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 in all cases cleaved at least to some extend. As expected, dCas9 does not have this effect (one more assay, and I can have an indication that ala10BipA is also inactive). When two gRNAs that target two RFP on opposite strand size 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 nCas913 (which is basically the same as our dCas9-Ala10Asp) has some double stranded DNAse activity). Further testing is needed whether this caused the cleaving by dCas9-Ala10Asp in our case, or that it is an artefact of some sort (figure 3).

Figure 3. in vitro Cas9 activity assays with Cas9, dCas9 and dCas9-Ala10Asp. Substrate for cleaving is a PCR product including the gene encoding RFP, which is targeted at the N-terminal side, both on the template strand (guideRNA 2 and 4) and the non-template strand (gRNA 1). Size of the uncleaved PCR product is 4140 bp, cleaving generates a 3100 bp and a 1040 bp fragment. Ladder: 1kb (NEB).



In conclusion: in this part of our project we showed that an artificial amino acid can be incorporated in dCas9 in response to the TAG stopcodon (and that this version remains catalytically inactive). We also tried to incorporate Aspartate in response to the TAG stopcodon and while we have some indications that this restored nickase activity, further testing is needed to verify whether this is the case.

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. ↩

    2. Amitai, S., Yassin Y., Engelberg-Kulka, H. (2004) MazF-Mediated Cell Death in Escherichia coli: a Point of No Return. Journal of Bacteriology vol. 186 no. 24 8295-8300. ↩

    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 ↩

    13. 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.↩