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Team:Montpellier/Experiments

Team:Montpellier - 2020.igem.org

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Team:Montpellier

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EXPERIMENTS



Experiments

This year, due to the exceptional circumstances of the COVID-19 crisis, only 2 students from the team could access the laboratory for a single month. This severely limited the productivity of our lab work.

We thus decided to focus our efforts on a reasonable objective: characterizing the infection of a bacterial population by a genetically modified phage. Additionally, building a modified phage, with antibiotic resistance and a fluorescent reporter (here, sfGFP) is a prerequisite to easily manipulate and characterize phages modified with oncolytic genes. We chose to use the well-characterized Escherichia coli, a natural inhabitant of the intestinal microbiota, some strains of which have also been used to colonize tumors. We chose to work with the M13 coliphage for several reasons: i) it is an established genetic tool (e.g. phage display); ii) it is responsible for chronic infection that does not kill bacteria; iii) as a filamentous phage, its packaging capacity is not restricted and large fragments of DNA can be inserted into its genome; and iv) it only infects E. coli strains expressing its receptors born by the F episome (limiting possible contamination to other experiments).


Bacterial strains:

  • E. coli DH5-α: used for cloning and plasmid production.
  • E. coli XL1-BLUE: carries the F factor as an episome (F+) and thus expresses the pili necessary for M13 susceptibility

Phage:

  • M13: filamentous coliphage, enters susceptible bacteria after binding to their F pili. As a chronic phage, it does not kill infected bacteria. This property is desirable in the framework of our project because it might improve the continued production of our proteins of interest, as well as increase the persistence of the new functions introduced in the microbiota. It is also used for phage display and DNA origami.

The circular, double-stranded replicative form of the M13 genome has been amongst the first genetic vehicles used for molecular cloning. In 1977, the 𝛼 fragment of the β-galactosidase gene and its upstream regulatory region from E. coli’s Lac operon was inserted at a permissive intergenic location without impairing phage function [1]. This construction was turned into a full-fledged cloning vector, the classic M13mp18, by the insertion of an in-frame multiple cloning site that enabled white/blue screens [2]. This construction, and derivatives such as p7560, was later used to produce large quantities of single-stranded DNA (isolated from the packaged genome) for the field of DNA origami [3].

Although they enable the production of fully functional phages, the focus of these vectors was to replicate and transfer cloned DNA rather than follow infectious processes. As such, they do not bear any antibiotic resistance or reporter gene.

To facilitate the study of phage infection, we decided to build an M13 derivative carrying the kanamycin resistance gene and the super folder green fluorescence protein reporter gene (sfGFP) at the permissive locus described above. To achieve this, we inserted a fragment carrying the KanR and sfGFP expression cassettes isolated from plasmid pFAB217 [4] into the native M13 genome amplified from p7560. This construct, which we named pM13-sfGFP-Kan, supports the production of functional phages; infected XL1-Blue cells can be selected by Kanamycin and monitored at the single-cell and population level by fluorescence measurements after induction by IPTG.


p7560: A derivative of the M13mp18 strain bacteriophage, it is a lac-phage vector commonly used for DNA origami folding reactions but it stills contain all the genes allowing the phage multiplication.

pFAB217: A synthetic plasmid carrying the gene resistance for kanamycin and the gene for the GFP which are both under the control of the pTac promoter.

A - Preparation of genetic materials

We first sought to obtain a clean DNA extract of the p7560 phage vector and pFAB217 plasmid.

We prepared a batch of chemically competent DH5-α E. coli. We tested their transformation efficiency by heat-shock at 42°C using a pUC19 plasmid with known concentration.

For pFAB217, we transformed our competent cells, selected transformants on LB-Agar plates containing Kanamycin, and miniprepped an overnight culture started from one of the colonies.

For p7560, we had access to purified phage particles packaging the vector, as well as a single-stranded circular form of the vector purified from these phage particles (a gift from Allan Mills, CBS). During its replication, the M13 phage produces many double-stranded circular copies of its genome that resemble a regular plasmid.

To isolate the double-stranded version of p7560, we first infected a population of exponentially growing XL1-Blue cells with the purified phage without agitation for 3 hours. We then grew that population overnight and performed a miniprep. This yielded too little material for us to work with. Next heat-shocked our competent DH5-α cells with the single-stranded form of p7560. Since the transformants cannot be selected (there is any selective marker on p7560) and may suffer from a growth defect imposed by the phage, we grew the cells overnight in a large volume and performed a maxiprep. This procedure yielded enough double-stranded phage material for us to work with.


B - Cloning of a M13-KanR-sfGFP construct

To maximize our chances of success in building the recombinant phage vector, we used several cloning strategies: 1- restriction/ligation, 2- Golden Gate assembly and 3- Gibson Assembly.



Figure 1: pM13-sfGFP-Kan v1 VS pM13-sfGFP-Kan v2


Figure 2: Cloning Workflow Comparison and Results. Both restriction/ligation and Golden Gate failed, but Gibson Assembly was successful.

1. Restriction / Ligation

Both the pFAB217 plasmid and the p7560 phage vector bear a unique BamH1 restriction site at positions compatible with our construction objective. This would readily permit cloning of the entire plasmid into the phage vector to create a phagemid. This construct could be used for initial tests and later be amended by inverse PCR to remove the plasmid’s origin of replication if necessary. While we were waiting for the delivery of primers necessary for the other two strategies, we decided to give this cloning a try.

We digested the two vectors by BamH1-HF (NEB) for 1h at 37°C, and further dephosphorylated the product from pFAB217 using CIP (NEB) for 1h at 37°C to prevent downstream self-ligation. We then incubated the products from these reactions mixed at 1:1 and 4:1 pFAB217/p7560 ratio with concentrated T4 DNA ligase for 20 min at room temperature. We transformed the ligation product into our competent DH5-α strain and plated the cells on LB agar supplemented with kanamycin (50 µg/mL). The next day, we identified 2 fluorescent colonies (expressing GFP) that had grown.

We picked and grew these two colonies overnight and extracted their plasmids by miniprep. To verify these candidate plasmids, we carried a BamH1 digestion and ran the product on an agarose gel along with digested and native versions of the original plasmids. We expected 2 fragments corresponding to the linearized p7560 and pFAB217. To our dismay, the size of our digested candidate was identical to pFAB217 (Figure 3).



Figure 3: BamHI restriction profile of our colonies, compared to controls. Lanes 1 and 2: candidate plasmids extracted from our colonies and digested with BamHI. Lane 3: p7560, digested with BamHI; Lane 4: p7560, non digested. Lane 5: pFAB217, digested by BamHI; Lane 6: pFab217, non digested. Both candidates have the same size as digested pFAB217.

We thus concluded that this first cloning experiment failed: the only seemingly positive (GFP+) colonies that we obtained were false positives, likely arising from undigested or incompletely dephosphorylated pFAB217. In line with this, we noticed that our first minipreps were contaminated with genomic DNA, which could have titrated the enzymes.

However, this was a “quick-and-dirty” experiment that we performed while waiting for our Golden Gate and Gibson Assembly primers to be delivered. We did not have the time to redo the procedure before our primers arrived, and decided to move on to our real objectives.


2. Golden Gate Assembly

Golden Gate Assembly is a seamless, one pot cloning strategy that uses the capacity of Type IIs restriction enzymes to cut outside of their restriction site [5]. The most used Type IIs enzyme for this purpose is BsaI, which recognizes the non-palindromic sequence GGTCTC to produce a 4-nucleotide long 5’ overhang one nucleotide after the recognition sequence.





In Golden Gate cloning, BsaI is used in conjunction with a DNA ligase, and the input DNA are designed in such a way that upon ligation, the intended construct does not bear the restriction site (e.g. the left part in the above reaction is a leftover of the construct’s assembly). Through cycles of digestion followed by ligation, the desired product rises in frequency in the reaction, making it a powerful and efficient cloning method.

We designed the primers in such a way that:

  • KanR and sfGFP expression cassettes can be amplified on a single amplicon for pFAB217 (thus excluding its p15a origin of replication)
  • The exact native genome of M13 is amplified from p7560, at the exclusion of any sequence introduced with the β-galactosidase cassette and subsequent modifications (see above)
  • BsaI sites with compatible overhangs are added at the extremity of the two amplicons

Assembly of these amplicons would lead to the native M13 genome with a selective and reporter marker inserted at a known permissive locus.


Primers design for Golden Gate
FW p7560 GG: aaaaGGTCTCaCCTAttggttaaaaaatgagctgatttaacaaaaatttaatg
RV p7560 GG: aaaaGGTCTCaCCGAaatcggcaaaatcccttataaatc

FW pFAB217 GG: aaaaGGTCTCaTAGGcaaatgtagcacctgaagtcagc
RV pFAB217 GG: aaaaGGTCTCaTCGGgagtcagtgagcgaggaagc

Note: The uppercase sequence corresponds to the sequence recognized and cut by BsaI enzyme. Since BsaI is a type II restriction enzyme it will cut close to its recognition sequence (5’GGTCTCa 3’) at the sequence written in uppercase toward the 3’ end. The lower case sequence downstream of the cut site corresponds to the priming sequence, per se, that binds specifically to the target DNA sequence.


We performed PCR on pFAB217 and p7560 using primers specifically designed for Golden Gate Assembly. After checking that we obtained amplicons of the expected size on agarose gels, we added DpnI to the PCR mixtures to digest the template plasmids. The products of these reactions were purified (QIAquick PCR Purification Kit, Qiagen) to remove enzymes and excess primers. We then measured the fragment concentrations using a Nanodrop. While we readily obtained a sufficient quantity of the desired amplicon from pFAB217, we needed to repeat the procedure and pool the products of several reactions to get enough genetic material from p7560.

We concurrently tested 9 conditions for the Golden Gate assembly, performing all combinations of 3 numbers of digestion-ligation cycles (40, 80, and 100) by 3 ratios of p7560/pFAB217 (1:1, 1:2, 1:3). We then separately transformed our chemically competent DH5-α cells with the resulting products and plated them on LB agar with kanamycin.

The next day, most plates were covered with a mat of grown bacteria. This is indicative of an absence of selection for the transformant. We might have made an error with the quantity of added antibiotic or destroyed it by adding it to LB-Agar that was still too hot. On two plates, however, we could identify isolated colonies on a region of the plates. These colonies were not fluorescent, indicating that they were in fact not transformed with the expected construct.

Without much hope, we nonetheless picked 3 colonies to perform colony PCR with the primers for p7560 designed for Golden Gate. Unsurprisingly, an agarose gel for these PCR products showed a complete absence of amplification from these colonies.



Figure 4: Agarose gel of the colony PCR of 3 colonies obtained after the transformation with Golden Gate attempt. Lanes 9, 10 and 11: products of colony PCR from 3 different colonies amplified with the Golden Gate primers for p7560. Lane 12: pFAB217 amplified by PCR with Golden Gate primers (positive control); Lane 13: p7560 single stranded amplified by PCR with Golden Gate primers (positive control). No band of the expected size could be seen for the three candidates shown.

3. Gibson Assembly

Instead of performing the Golden Gate cloning again, we tried to clone pM13-sfGFP-Kan using Gibson Assembly for which we also had designed primers. This cloning technique consists of using primers to add cohesive ends (overlaps) between the insert and the vector. While the reaction occurs, an exonuclease chews back the 5’ ends of each fragment, thereby giving “stricky ends” that will be able to anneal. Finally, a DNA polymerase fills the gap that was previously created and a ligase ligates them. We designed oligonucleotides with priming sequences identical to those used for Golden Gate preappended with the cohesive sequences.


Primers design for Golden Assembly
FW p7560 GG: cctattggttaaaaaatgagCTGATTTAACAAAAATTTAATG
RV p7560 GG: ccgaaatcggcaaaatccctTATAAATCAAA

FW pFAB217 GG: ctcattttttaaccaataggCAAATGTAGCACCTGAAGTCAGC
RV pFAB217 GG: agggattttgccgatttcggGAGTCAGTGAGCGAGGAAGC


Amplicons were produced as explained in the previous section. As for Golden Gate assembly, we had to pool several PCR products to get enough material for the p7560 amplicon.

We combined the resulting DNA fragments at ratio 1:3 to perform Gibson Assembly. We used a reaction with the product from p7560 as a negative control. We checked the result of the Gibson Assembly on an agarose gel (Figure 5).



Figure 5: Gel analysis of 2 Gibson Assembly reactions. Lanes 1 and 2 contain the products of the performed Gibson Assembly. We observe 3 bands in each lane. The size of the topmost band (highlighted by red arrows) corresponds to that of expect reaction’s product (around 8.5 kb). The middle band corresponds to p7560 (around 6.5 kb). The bottom band corresponds to pFAB217 (around 2.5 kb).

As we could detect a faint band at the expected size, we transformed the products in competent DH5-α cells and selected on LB agar plates with kanamycin. The next day, we picked 20 colonies to perform a colony PCR test using the primers for p7560. The expected size for positive clones was 8.5kb. Figure 6 shows that most colonies were positive, indicating the Gibson Assembly worked beautifully.



Figure 6: Gel analysis of 20 colony PCRs. Lanes 1-11 and 13-21 contain the products of colony PCR with the primers of p7560 for 20 different colonies. Lanes 12 and 22 are positive controls with p7560 single stranded DNA amplified by PCR with Gibson Assembly primers.

C - Test phage infection

After obtaining the recombinant phage vector pM13-sfGFP-Kan, we endeavored to purify phage particles to verify that we could indeed infect bacteria that would become resistant to kanamycin and produce GFP.

To that end, we concurrently transformed candidate pM13-sfGFP-Kan isolated from different colonies into XL1-BLUE cells and grew them overnight in liquid LB supplemented by Kanamycin (50 µg/mL). The next day, we centrifuged the cultures, collected their respective supernatants, and purified the phages particles inside through precipitation with PEG-NaCl (20% (wt:vol) PEG 8000; 2,5M NaCl). Phages isolated in the pellet from that precipitation were resuspended in STE buffer (100 mM NaCl; 10 mM Tris-Cl, pH 8.0; 1 mM EDTA). We could approximately quantify the phage titer by calculating 0.8 * A(280 nm), as measured by nanodrop. Although that quantification indicated that we had successfully produced phages, we could not pursue the infection phase for lack of time in the lab.


Conclusion:

During our one month in the wet lab, we successfully constructed a recombinant M13 phage vector carrying a selected marker and a fluorescent reporter. We were also able to produce phage particles packing that vector.

Our plan is to follow up on these initial experiments during the iGEM 2021 competition.

We will be ready to use our reporter phage to characterize the phage’s infectious parameters, as well as its burden on the cell. These measurements will be used to parameterize our mathematical model.

We will also use our vector as a basis to construct derivatives carrying anti-cancer devices with various cytotoxic and immunomodulatory genes, as described in the section oncolytic genes. We will then characterize their activity in bacteria (protein production) and on cancer cells (cultured in the presence of infected bacteria).



Figure 7: Phagent Designing Plan


Protocols

Bacteria-specific protocols



Phage-specific protocols



Molecular biology protocols



Protocols for analysis






References

[1] Sanger, F., Nicklen, S., & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America, 74(12), 5463–5467. https://doi.org/10.1073/pnas.74.12.5463
[2] Yanisch-Perron, C., Vieira, J., & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene, 33(1), 103–119. https://doi.org/10.1016/0378-1119(85)90120-9
[3] Zahid, M., Kim, B., Hussain, R., Amin, R., & Park, S. H. (2013). DNA nanotechnology: a future perspective. Nanoscale research letters, 8(1), 119. https://doi.org/10.1186/1556-276X-8-119
[4] Mutalik, V. K., Guimaraes, J. C., Cambray, G., Mai, Q. A., Christoffersen, M. J., Martin, L., Yu, A., Lam, C., Rodriguez, C., Bennett, G., Keasling, J. D., Endy, D., & Arkin, A. P. (2013). Quantitative estimation of activity and quality for collections of functional genetic elements. Nature Methods, 10(4), 347-353. https://doi.org/10.1038/nmeth.2403
[5] Engler, C., Kandzia, R., & Marillonnet, S. (2008). A one pot, one step, precision cloning method with high throughput capability. PloS one, 3(11), e3647. https://doi.org/10.1371/journal.pone.0003647