Part:BBa_K2643011

Mosaic Ends-flanked Kanamycin cassette and RFP

ME-KanR-ME

Usage and Biology

Usage

This part is a composite of the default RFP insert and a kanamycin resistance cassette. The kanamycin resistance cassette is flanked by Mosaic Ends (MEs) to be recognized by the Tn5 transposase. The transposase is then capable of looping it out of the donor DNA and integrating it into another locus. For sole plasmid propagation, there are no specific host strain requirements, other than being compatible with the antibiotic marker and replication origin located on the vector. For functionality testing, the plasmid needs to be harbored by a host that also harbors Tn5 transposase activity.

Biology

Transposons are mobile DNA sequences capable of changing location within a genome. For this, a transposon contains the full expression cassette for the transposition machinery, referred to as a transposase. The transposon itself is flanked with recognition sequences, so-called Mosaic Ends, which can be recognized by the transposase. This way, transposases hold both the ability to recognize the transposon they were originally transcribed from, as well as the ability to pick up this entire transposon and integrate it at an alternative site . This integration happens in a rather random manner. Our methodology makes use of this semi-specific transposon activity, and aims to make it more targeted by complementing it with CRISPR-mediated guidance. To this extent, we have fused Tn5 transposase with dxCas9, of which the latter is a catalytically inactive but programmable endonuclease. The compatible gRNA supplied is expected to guide the complex to a certain locus and concentrate Tn5 activity to this locus.

Characterization

Introduction

In our project, we aim to targetedly alter a local DNA sequence of interest. Apart from in vitro testing, we have designed and experimented with an in vivo approach. This approach would enable higher throughput screens of the functionality of our methodology and quicker selection of better constructs, based on blue/white screenings and antibiotic selections. The approach taken in the in vivo screen involves single-step targeted genome editing in E.coli, in order to demonstrate the activity of our fusion construct. Putatively, this single-step genome editing can be used by itself as a tool in synthetic biology.

Strain construction

This part has been constructed by means of restriction ligation cloning. The ME-flanked kanamycin resistance cassette was cloned as an insert next to (downstream, in the reverse direction) the default RFP insert in pSB1C3.

When transforming the ligate into E. coli DH5α and selecting on medium complemented with both chloramphenicol and kanamycin, we observed formation of red colonies. This indicates that the kanamycin cassette is intact and functional, as well as the RFP cassette. Colony PCR using the biobrick verification primers VR and VF2 indeed yielded the 2454bp amplicon, for all 16 screened colonies (figure 1). The sequence verification confirmed both sequences were intact and oriented as desired. DH5α was not able to grow on LB+Kan+Cam, and demonstrates the negative control.

Figure 1.Gel electrophoresis results from colony PCR on construction of this part. Colony 4 and 6 were used for subsequent plasmid isolation and sequence verification.

Procedure

To visualize in vivo whether a donor DNA sequence is indeed integrated close to a target sequence, we built a strain that harbors the dxCas9-Tn5 fusion protein, as well as a lacZ gRNA expression cassette, both on the same episomal vector. IPTG-Induction of protein expression enables immediate loading of the fusion construct with the gRNA. An additional requirement for activity is a donor DNA sequence flanked by MEs, which can be recognized by the Tn5 transposase domain for cut and paste integration near the gRNA target. This disrupts the lacZ gene and renders catalytically inactive beta-galactosidase, which can be visualized by blue/white screening with X-gal (figure 2).

Figure 2.Overview of the single-step genome editing mechanism in the in vivo assay. (A) The fusion construct is loaded with a gRNA that targets lacZ and recognizes ME flanked kanamycin resistance cassette for (B) picking it up. (C) CRISPR-mediation drives the loaded complex to the genomic lacZ locus, where the transposase domain (D) integrates the donor DNA. (E) The host strain contains an integrated copy of kanamycin resistance, while the lacZ gene is disrupted.

In our laboratory experiments, the donor DNA sequence was a kanamycin resistance expression cassette, flanked with MEs. Integration of this linear fragment into the lacZ locus would thereby confer the ability to grow on medium containing kanamycin, while still making use of the blue/white screen (note the selection medium contains kanamycin, chloramphenicol, IPTG and X-gal). In presence of kanamycin, cells should only be able to grow if the supplied donor DNA sequence is integrated into the genome. For non-targeted integration, intact lacZ confers chromogenic catalytic functionality when growing on X-gal, resulting in a blue phenotype . For targeted integration, lacZ is disrupted, resulting in loss of chromogenic capability and thus in a white phenotype. This composite part purposely contains both RFP and kanamycin, in order to distinguish surviving colonies based on whether genomic integration occurred, or whether the plasmid is simply propagated. The latter is a false positive, expected to confer a red color. In combination with blue product formation by intact LacZ, this is expected to result in a purple phenotype. Table 1 gives an overview of the expected possibilities.

Table 1. Overview of expected phenotypes in the in vivo assay.

Genomic Integration	Template with RFP cotransformed?	Growth on LB+Cam+Kan?	lacZ disrupted?	Colony phenotype*
On-target	no	yes	yes	white
On-target	yes	yes	yes	red
Off-target	no	yes	no	blue
Off-target	no	no	no	purple
None	no	no	no	n/a
None	yes	yes	no	purple

*phenotype when growing on LB+Cam+Kan+IPTG+X-gal

The characterization of this part is closely linked to the characterization of BBa_K2643010, which is a composite part containing the sequence for gRNA production. Several experiments have been done to show their compatibility and functionality. Transformation efficiency of linear fragments was optimized by using electroporation instead of heat-shock transformation. We tested the following conditions:

• Electroporating 200ng kanamycin CDS flanked with MEs
• Electroporating 200ng kanamycin expression cassette without MEs
• Electroporating 200ng kanamycin expression cassette with MEs (+/-5bp)*
• Electroporating 500ng kanamycin expression cassette without MEs
• Electroporating 500ng kanamycin expression cassette with MEs (+/-5bp)*
• Electroporating 800ng kanamycin expression cassette with MEs (+/-1kb)*
• Electroporating 800ng kanamycin expression cassette without MEs (+/-1kb)*
• Electroporating 50ng of pSB1K3**

*+/- gives the length of the spacer between the end of the linear fragment and the start of the closest ME. Prior to making them electrocompetent, the cells were already induced. Longer spacers were thought to make the ME flanks less prone to exonuclease degradation.
** this positive control confirmed that it is possible for the host to harbor both antibiotic markers and be selected for both simultaneously.

Results & Conclusion

The use of proper controls is crucial in this assay, especially to exclude false positives. Table 2 gives an overview of the results obtained for these controls. These controls confirm that BL21DE3 indeed contains intact and functional lacZ, judging by the blue phenotype. Furthermore, the parental strain is resistant to chloramphenicol and neither to kanamycin. Harboring the fusion construct and the gRNA targeting lacZ does not influence the functionality of the lacZ, judging by the remaining blue phenotype.

Table 2. Overview of observed phenotypes of the controls taken along in the in vivo assay.

Strain	DNA	LB-medium supplements	Observed phenotype
DH5α	-	X-gal + IPTG	white
BL21DE3	-	X-gal + IPTG	blue
BL21DE3	-	Cam	No growth
BL21DE3	-	Kan	No growth
BL21DE3	pACYCdxCas9Tn5gRNA	Cam + X-gal + IPTG	blue
BL21DE3	pACYCdxCas9Tn5gRNA + pSB1K3	Cam + Kan + X-gal + IPTG	purple
BL21DE3	pSB1K3	Kan + X-gal + IPTG	purple

When transforming this part into E. coli DH5α and selecting on medium complemented with chloramphenicol and kanamycin, we observed formation of red colonies. This indicates that the kanamycin cassette is intact and functional, as well as the RFP cassette. The sequence verification confirmed both sequences were intact and oriented as desired. DH5α was not able to grow on LB+Kan+Cam, and demonstrates the negative control.

The MEs were also sequence verified and confirmed to be located as designed, but we have not been able to demonstrate their in vivo functionality. We were not able to show that the MEs were recognized and that the donor DNA was integrated, neither in a targeted nor a non-targeted manner. Surviving colonies demonstrated development of purple phenotype, sometimes requiring several days (figure 3), while going through an interesting metamorphosis of color changes. BL21DE3 itself has a blue phenotype on X-gal+IPTG plates (table 2). Based on the executed controls, it is highly probable that a purple phenotype is due to the presence of the original RFP-containing template plasmid used for creating the linear donor DNA sequence. The RFP cassette was kept on the plasmid to observe this deviant purple color to identify growing false positives. These colonies are surviving in presence of kanamycin, because this template plasmid is propagated and harbors an intact and functional kanamycin resistance expression cassette.

Figure 3.Effect of incubation time on chromogenic in vivo assays. These LB+Cam+Kan+X-gal+IPTG plates demonstrate growth of BL21DE3+pACYCdxCas9Tn5gRNA strains that are electroporated with donor DNA. (A) 1 day after electroporation with linear ME flanked kanamycin resistance cassette, blue colony formation was observed, whereas (B) a week later red phenotype has developed strongly. (C) 1 day after transformation with ME flanked kanamycin resistance cassette in a plasmid, blue colony formation was observed, whereas (D) a week later purple phenotype has developed strongly

We optimized diagnostic genomic PCR reactions for verification of putative integration of the kanamycin fragment in/near the lacZ locus (figure 4). When designing and optimizing these genomic PCR reactions, we favoured PCR reactions that always result in amplicons, regardless of integration. Such anticipating primer design helps evaluating the results and judging whether genomic PCR on a picked colony worked in the first place, and then evaluate whether targeted integration took place. When integration occurs, the amplicon size increases. The negative control used for these genomic PCRs was purified isolated gDNA of the BL21DE3 parent strain. Alternatively, we were planning on whole genome sequencing of individual colonies, in the case of encountering blue or white colonies, to verify where and how many times the donor DNA would have been integrated, and whether this activity was indeed concentrated near the original target sequence.

Figure 4.Overview of relative binding sites used in diagnostic genomic PCRs. Used primer combinations: IV007&IV019; IV020&IV008; IV007&IV022 and IV021&IV008.

These genomic PCRs have demonstrated that integration did not take place near the desired target site. The negative control used for these genomic PCRs was purified isolated gDNA of the BL21DE3 parent strain. Even transformation of donor DNA into a competent strain harboring only Tn5 did not result in colonies, suggesting a problem related to the transposase effector function of the fusion construct.

Source

The kanamycin resistance part of the composite biobrick sequence was isolated from plasmid pSB1K3. The RFP part (BBa_J04450) was already present in the iGEM backbone .

Safety

This part does not pose any danger for safe work in BSL-1 laboratory work spaces. The expression of the fusion construct is regulated with IPTG, and the Tn5 activity is controlled by supplying a Mosaic End flanked donor DNA sequence. In our case, this is a kanamycin resistance marker, which is a common sequence used in BSL-1 lab spaces. This composite part purposely contains both RFP and kanamycin, in order to distinguish surviving colonies based on whether genomic integration occurred, or whether the plasmid is simply propagated. The latter is a false positive, expected to confer a red color. In combination with blue product formation by intact lacZ, this is expected to result in a purple phenotype. Since our experimental approach makes use of T7 polymerase expression systems, we needed a lacZ-containing strain that also harbors T7 expression machinery, driving us to work with E. coli BL21(DE3). The strain’s complete genome sequence was used as a template to choose a convenient gRNA target site in lacZ, and for excluding presence of other Mosaic Ends in the genome.