Team:Wageningen UR/Results/Alternative Phage Engineering

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Alternative Phage Engineering

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Introduction

As an alternative to the yeast-based genome assembly and TXTL approach, a novel approach using recombination and CRISPR-Cas was used to engineer phage lambda. This method is based on recent work on E. coli phage T4 and listeria phages [1, 2]. Parallel to this, we worked on hybrid phages, which rely on plasmid-based expression of the fusion protein. These can be used to test the chitin binding properties of the engineered phage.

Figure 1: An overview of the recombination and counterselection methods

Approach

The phage lambda engineering method consists of a recombination and a counterselection step. Methods in which CRISPR-Cas cutting is combined with rescue by recombination have been shown to be effective. [1, 2] Our method separates the recombination and the selection in two different E. coli strains and two different experiments. The first strain harbors two plasmids, one contains the lambda red system for recombination and the other one a recombination module to obtain recombined phages. This strain is infected with phage lambda to obtain a recombinant phage lysate. The second E. coli strain contains a Cas12a counterselection system to select against phages that have not recombined. Cas12a will recognize protospacers on the lambda genome and cut the phages that did not recombine. Phages that did recombine will escape the counterselection and form pickable plaques.

Recombination System

The recombination of phages is carried out in E. coli DH10B containing the lambda red system, which increases recombination, and a plasmid containing the fusion protein flanked by 200 bp homology regions. The fusion protein is gpD, which is a phage lambda capsid protein, fused to either the chitin binding domain of Chitinase A1, chitin binding domain HAD, chitin binding protein PD1764 or GFP. These fusions are all connected by the same flexible linker (CGSGSGSG). The flanking homology regions are designed in such a way that the insert fusion protein is built in directly behind the original capsid protein, as can be seen in figure 2. The choice was made for 200 bp homology regions as a good compromise between ease of cloning and high recombination efficiency [3]. The recombinant phages are generated by infecting the cells carrying the recombination system with phage lambda. This generates a lysate, from which the phages can be purified.

  • Parts arrow_downward

    The insertion constructs can be found as parts in the registry.

    Table 1: An overview of the parts used in Alternative Phage Engineering.
    Part Biobrick Code
    Lambda gpD - Chitinase A1 recombination module BBa_K3286277
    Lambda gpD - GFP recombination module BBa_K3286278
    Lambda gpD - HAD recombination module BBa_K3286279
    Lambda gpD - PD1764 recombination module BBa_K3286280
Figure 2: A map of the phage Lambda genome, showing the gpD region and the primer pairs used
Figure 3: A map of the gpD region of the phage Lambda genome, including the homology regions from the recombination module
Counterselection Method

The counterselection is based on Cas12a. This endonuclease cuts double stranded DNA (dsDNA) based on recognition of a 5’- TTN PAM and a 20 bp protospacer. As shown in figure 3, three different spacers target the region that is replaced by successful recombination. Multiple spacers are used to prevent low efficiency by an underperforming spacer. Counterselection DH10B E. coli cells carry a plasmid coding for Cas12a and a plasmid carrying one of these spacers.

Counterselection is carried out by infecting the counterselection cells with the phage stock from the recombination step. Recombinant phages escape the counterselection as the protospacer is removed by the recombination. Phages that can escape the counterselection will form plaques that can be screened through PCR. For the counterselection all three spacers are used individually. To decrease the chance of phages escaping selection we also test a combination of the three spacers by doing a plaque assay with mixed bacterial cultures. The mix of spacers should only show plaques for phages that can escape all three spacers. This makes false positives less likely and reduces screening workload.

Figure 4: An overview of the spacers used for the counterselection system
Hybrid Phage

To be able to quickly test the chitin binding of the phages, we worked on the hybrid phage system. By expressing the capsid fusion on a plasmid and infecting cells with phages or by producing phages using a Transcription Translation (TXTL) mixture, phages can be created that have a mix of WT and fusion proteins on their capsid. The phages carrying fusion proteins can then be used in chitin binding assays to determine the effectivity of the chitin binding. The hybrid phage system was constructed for both phage lambda and phage T7, as production of T7 using TXTL has already been shown [4]. The hybrid phage expression plasmid consists out of either lambda gpD or T7 capsid protein P10 fused by a flexible linker (CGSGSGSG) to the different chitin binding proteins (Chitinase A1, HAD, PD1764) or GFP. The gpD fusions are under the control of the Anderson J23116 promoter and the P10 fusions are under control of the T7 promoter. The J23116 is a weak Anderson promoter at 16% strength (relative to J23100). This choice was made to avoid toxicity problems during cloning after earlier problems with J23100. For P10 the choice was made for the T7 promoter as it was already in front of the gene on the T7 genome and could thus be cloned in easily. Furthermore, the promoter is highly active when T7 infects the cell, giving expression exactly when it is required.

Protein model

Using protein modelling we created a 3D model of the gpD to GFP fusion including a flexible linker (CGSGSGSG), which can be seen in figure 5. In this model, it is observable that the gpD protein can correctly fold and is not sterically hindered by the fused domain. The linker creates a clear separation between gpD and GFP. This model gives us an indication that GFP could be correctly displayed on the phage capsid.

Figure 5: 3D model of the gpD to GFP fusion protein. In blue, the gpD protein, in red the linker and in green the Gfp protein.

Results

All the plasmids needed for the aforementioned experiment were successfully constructed, except for T7 P10+ChitinaseA, T7 P10+GFP and multiplexing counterselection spacer. These constructs suffered from mutations and were abandoned due to lack of time.

The recombination step was carried out according to the aforementioned design. To compensate for potentially low level of recombinants, the counterselection step was carried out at a Multiplicity Of Infection (MOI) of 1.5. The resulting plates did not show growth of E. coli. We hypothesized that the phage concentration was so high that it allowed for many escape mutants or that it overwhelmed the counterselection system. To correct for this, we repeated the counterselection with dilutions of 10^-3, 10^-4 and 10^-5 of the recombinant phage stock and performed spot assays with WT phage on the selection cells. As can be seen in figure 4, the resulting plates still showed almost complete lysis of E. coli, while the spot assays in figure 5 show no resistance of the cells. At this point we had to assume that either our counterselection system was broken, which had been successfully tested on GFP and GFP before, or that there was something wrong with our phage stock.

Figure 4: A 10-4 dilution of phage lambda stock counterselected by a mix of the three spacers shows a clear plate
Figure 5: This plate shows what a full plate looks like
Figure 6: Phages produced on E. coli carrying the GFP recombination module diluted 10-5 counterselected by spacer 1 show a clear plate

Before the experiment started, the phage genome had been isolated using phenol chloroform method and had been tested using digestion, with restriction enzymes EcoRI and PstI, and PCR to confirm the identity of the phage. Whilst the digestion did not show bands except for a band above the ladder of which the size could not be determined PCR of the gpD gene did give the expected band. The PCR product of the gpD gene was sent for sequencing and matched the sequence of phage lambda perfectly. Based on this it was assumed that we were working with phage lambda. Still, the new results, combined with a BLAST search of the gpD gene, which indicated that some E. coli strains carry the gpD gene on their genome, prompted a renewed investigation.

To confirm that the phage used was indeed phage lambda, another PCR was performed. In this PCR the phage stock was directly used as template. As a positive control the phage lambda genome was used, while E. coli strains DH10B and LE392 were used as negative control. The gpD gene, the PL region and PR+PRM region were targeted by the used primers. The PCR was carried out using OneTaq and Q5 DNA polymerases. For OneTaq the PCR was performed with and without DMSO, Q5 was used with DMSO only. The agarose gel, which is shown in figure 5, gave no bands if the phage was used as template. E. coli DH10B showed clear bands for gpD in the reaction with added DMSO. E. coli LE392, showed vague bands for gpD with DMSO. The phage lambda genome positive control shows expected bands. This result shows that there likely is a copy of gpD on the E. coli genome, while the PRM and PL regions are missing from the E. coli genome. The clear and correctly sized bands for E. coli DH10B show that the gpD gene is likely present on the E. coli genome and cast doubt on the validity of the earlier sequencing results. The presence of the gpD gene also explains earlier trouble during cloning of the spacers, which suffered from very low cloning efficiency. Self-targeting of the spacers could have caused this.

Figure 7: The agarose gel image of the phage lambda verification PCR. Orange emphasizes the bands for gpD (487 bp), green those for PL (429 bp) and purple those for PRM+PR (330 bp). The markers used are the NEB 1kb and 100 bp DNA ladders.

As the PCR removed our evidence for phage lambda, we looked critically at the TEM image we produced of our phage stock and compared it to TEM images of other E. coli bacteriophages. From TEM images we conclude that the phage in our stock is likely to be phage T4 or a closely related phage.

Figure 8: An image of the T4-like phage that was present in the phage stock

Many characteristics of the phage pictured in figure 6 point towards a T4-like phage and not phage lambda. The head of phage T4 is rectangular in shape, while the phage lambda head is round. The tail of phage T4 is relatively short compared to the head, while phage lambda has a long tail. At the T4 tail has a plate at the end, while the phage lambda tail ends pointed. Measurements of the dimension confirm that the phage is like T4. Average width and length of the head of imaged phages are 75 nM and 103 nM, in comparison to 86 nM and 120 nM for phage T4 [5]. This is somewhat smaller, which could be caused by the measurement method and damage to the phages, as the variation between imaged phages is significant.

T4 contamination can also explain earlier results. After genome isolation from the phage stock digestion using restriction enzymes was attempted. This did not result in restriction fragments that could be visualized on gel. The assumption was made that there was something wrong with the phage genome isolation method. However, phage T4 modifies its genome through methylation and glucosylation, which protects it against endonucleases and explains the lack or digestion [6].

Conclusion

Unfortunately, lack of time and lack of phage lambda meant that we were not able to complete testing of the lambda engineering method and the hybrid phage method. As such, it is not possible to draw conclusions on the viability of the methods for producing chitin binding phages. Another attempt can be made, using verified phage lambda, when there is time.

  • Materials and methods arrow_downward

    Strains and vectors

    • E. coli DH10B was used for cloning of the different constructs used.
    • pSB1C3 (chloramphenicol resistance) was used as a backbone for the recombination insert and the hybrid phage system.
    • pBeloBAC11, carrying a kanamycin resistance instead of a chloramphenicol resistance, carries the lambda red recombination system. This plasmid maintains only a single copy per cell. Because of the low copy number, 2/5 of standard kanamycin concentrations should be used.
    • Cas12a is carried on a pACYC backbone with a chloramphenicol resistance. The spacer plasmid has an ampicillin resistance.
    • The phage lambda genome was bought from Sigma Aldrich, sold as methylated genome from Escherichia coli host strain W3110.  
    • The lambda gpD gene and the homology regions were amplified from bought phage lambda genome using Q5 polymerase.
    • T7 P10 gene was amplified from isolated T7 genome using Q5 polymerase.

    Phages

    Phage lambda, more on the content of this in the results and discussion, and phage T7 were taken from a phage stock created by Marnix Vlot and stored at 4 °C in our lab.

    Protocols

    Protocols for genome isolation, Q5 PCR, Gibson assembly of constructs and transformation can be found here. PCR verification of phage lambda was performed using a protocol which is suitable to both Q5 and OneTaq polymerase. Reactions with DMSO contained 0.7 µL DMSO on 25 µL reactions. From the phage stock 5 µL was used as template. From the phage lambda genome 10 ng was used as template. For DH10B and LE392, 5 uL of MQ water with dissolved culture was used as a template. For the gpD primers with phage stock as template a temperature gradient PCR (from 61 °C to 62 °C) was performed, to give the maximum chance of successfully repeating the earlier positive result. For all PCRs the following protocol was used:

    Table A1: PCR protocol
    Temperature (°C) Time Cycle
    98 °C 5 minutes
    98 °C 5 minutes 35 cycles
    Primer specific Tm 30 seconds 35 cycles
    72 °C 30 seconds 35 cycles
    72 °C 2 min
  • References arrow_downward
    1. Tao, P., Wu, X., Tang, W. C., Zhu, J., & Rao, V. (2017). Engineering of bacteriophage T4 genome using CRISPR-Cas9. ACS synthetic biology, 6(10), 1952-1961.
    2. Hupfeld, M., Trasanidou, D., Ramazzini, L., Klumpp, J., Loessner, M. J., & Kilcher, S. (2018). A functional type II-A CRISPR–Cas system from Listeria enables efficient genome editing of large non-integrating bacteriophage. Nucleic acids research, 46(13), 6920-6933.
    3. Bassalo, M. C., Garst, A. D., Halweg-Edwards, A. L., Grau, W. C., Domaille, D. W., Mutalik, V. K., ... & Gill, R. T. (2016). Rapid and efficient one-step metabolic pathway integration in E. coli. ACS synthetic biology, 5(7), 561-568.
    4. Shin, J., Jardine, P., & Noireaux, V. (2012). Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS synthetic biology, 1(9), 408-413.
    5. Black, L. W., & Rao, V. B. (2012). Structure, assembly, and DNA packaging of the bacteriophage T4 head. In Advances in virus research (Vol. 82, pp. 119-153). Academic Press.
    6. Karam, J. D., Drake, J. W., Kreuzer, K. N., Mosig, G., Hall, D. H., Eiserling, F. A., Black, L. W., Spicer, E. K., Kutter, E., Carlson, K., and Miller, E. S. (1994) Molecular biology of bacteriophage T4, American Society for Microbiology, Washington, DC.