Team:ETH Zurich/core/demonstrate

A Functional Toolbox for the Formation of Phage Libraries and the Selection of Best Variants

Phage therapy offers an alternative treatment for bacterial pathogens. Nowadays, it is mainly limited by two factors. First, only a low number of pathogens are treatable with naturally occuring phages. Second, screening for infectious phages from available databases is time consuming. We aim to overcome this problem by creating phage libraries with novel host specificities and building a bioreactor that rapidly selects the best variants. By showing experimentally the generation of phage libraries and the rapid selection of infectious phage variants, we prove that our approach works and can be used to create phages with novel affinities.

Tail Fiber Proteins Influence Host Specificity

The goal of our project is to create phage libraries that have novel host specificities. Host binding proteins are known to influence host specificity [1]<a style="color: #ffffff; text-decoration:none;" href="#biblio-demonstrate">Ando, H., Lemire, S., Pires, D. P., & Lu, T. K. (2015). Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell systems, 1(3), 187–196. doi:10.1016/j.cels.2015.08.013</a>. The host binding proteins of our model organism T7 are called tail fibers (Gp17). Regions that are not conserved amongst homologs of different phages with distinct host specificities are mainly present on the surface of the tip domain and are found in protein loops between secondary structures [2]<a style="color: #ffffff; text-decoration:none;" href="#biblio-demonstrate">Yehl, K., Lemire, S., Yang, A. C., Ando, H., Mimee, M., Torres, M. D. T., ... & Lu, T. K. (2019). Engineering phage host-range and suppressing bacterial resistance through phage tail fiber mutagenesis. Cell, 179(2), 459-469.</a>. This data supports the hypothesis that mutations in the non-conserved regions lead to altered host specificities. By randomizing the codons of four loops we aim to generate phage variants with novel binding specificities (Fig. 1). 

         <figure class="figure-center">
           <img src="T--ETH_Zurich--tailfiberzoom.png" alt="tail fiber zoom">
           <figcaption>Figure 1: Bacteriophage T7 binds to the bacterial surface with its tail fiber proteins. We identified unconserved protein loops that are likely to be important for host specificity. In our project we randomize those regions in order to form phage libraries with novel host specificities. </figcaption>
         </figure>

Generating Diversity in Four Selected Regions by PCA Assembly

As shown in figure 2, we successfully developed a method based on the principle of polymerase chain assembly that allows to create a DNA fragment that contains randomized base pairs in the designed protein loops. This fragment is introduced into the T7 genome with our genome editing toolbox in order to form phage libraries. 

         

         <figure class="figure-center">
           <img src="T--ETH_Zurich--loops.png" alt="Method to generate the randomized fragment">
           <figcaption>Figure 2: Method to Generate the Randomized DNA Fragment. The randomized fragment is constructed by polymerase cycling assembly using 60 bp long oligonucleotides with a 20 bp overlap. Degenerate oligos are used for the loop structures. The generated fragments are further amplified by PCR. Sequencing confirmed the successful randomization at the positions of interest.  Note that due to the synthesis errors, only 65 % of the assembled ordered oligos can be guaranteed to have the correct sequence. 
           </figcaption>
         </figure>

Genome Editing Toolbox

Creating Phage Genome Libraries with Randomized Base Pairs at the Designed Locations
Our <a class="a-link" href="https://2019.igem.org/Team:ETH_Zurich/Results">genome editing toolbox</a> allows for the randomization of the phage DNA at the designed position of interest. Figure 3 shows the successful introduction of novel DNA sequences in the protein loops. 

         <figure class="figure-center">
           <img src="T--ETH_Zurich--library_sequencing.png" alt="genome editing">
           <figcaption>Figure 3: Sequencing of Randomized T7 Genome. The sequencing results show that our genome editing toolbox is able to introduce the randomized DNA fragment into the position of interest.   
           </figcaption>
         </figure>


Formation of Functional Phages with Swapped Host Specificity
In order to test whether our system can form functional modified phages, the C-terminus of the T7 tail fiber protein was replaced with its T3 homolog. The swap of this region will lead to the change in host specificity and allows the newly formed T7/T3 hybrid phage to infect EcoR16, a bacterial strain that can be infected by T3 but not by T7. Figure 4 demonstrates that the swap in infectivity is effectively observed after changing the seqeunce with our genome editing toolbox and therefore confirms that functional modified phages can be formed with our method. 

         <figure class="figure-center">
           <img src="T--ETH_Zurich--swap.png" alt="swap">
           <figcaption>Figure 4: Infectivity Swap.  The swap of infectivity of the newly generated T7/T3 phage confirms that our system can be used for the formation of functional modified phages.    
           </figcaption>
         </figure>

Formation of Functional Phages with Novel Tail Fiber Protein Sequences
We showed that we are able to form functional modified T7/T3 hybrid phages and proceeded to forming phage libraries with the randomized phage genomes. In order to isolate single phages from the library, plaque assays with the phage library were performed on DH5α containing CRISPR-Cas targeting wild type T7. 7 single plaques were picked and the region of the genome containing the library insert was sequenced. 5 different sequences were found (Fig. 5), demonstrating that we have obtained a library of phages, including variants that retain their infectivity towards DH5α. 

         

         <figure class="figure-center">
           <img src="T--ETH_Zurich--phage-library-seq.png" alt="swap">
           <figcaption>Figure 5: Sequences of Plaques Originating From Our Phage Library  Library members were selected for by plating on cells with a CRISPR-Cas system targeting wild type T7 but not library members. On the bottom the sequence of T7 is shown as a reference.  
           </figcaption>
         </figure>

Rapid Selection of Infectious Phage Variants by Combining Theoretical Modeling with our Biorector

Our bioreactor assures optimal conditions for the rapid selection of the most infectious phage variants from the library. Our model is used to adjust reactor parameters in real time by continuously monitoring the OD in the reactor. We could show that infectious phage variants are effectively amplified by a facotor 103 after 2 hours of selection in our biorector (Fig. 6). 

         

         <figure class="figure-center">
           <img src="https://static.igem.org/mediawiki/2019/2/21/T--ETH_Zurich--hw-case_study_result.svg" alt="phage selection reactor">
           <figcaption>Figure 6: Selection of the Most Infectious Phage Variants in the Biorector. Spot assay (factor 10 dilution series) prior and after selection in the bioreactor show a factor 103 increase in the concentration of the infectious phage variants that can infect a host that has been engineered to be resistant against T7 phage containing the wild type version of the tail fiber protein with CRISPR-Cas.  
           </figcaption>
         </figure>


Selection of a Phage with Novel Host Specificity

Unfortunately, it was not possible to select a phage from our library that is able to infect a novel host such as EcoR16. Nevertheless, we demonstrated that all parts of our project work and that it is therefore only a matter of protocol optimization, time investment and upscaling of experimental setups until the first phage with novel host specifity can be selected form our library.


Bibliography

[1] Ando, H., Lemire, S., Pires, D. P., & Lu, T. K. (2015). Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell systems, 1(3), 187–196. doi:10.1016/j.cels.2015.08.013
[2] Yehl, K., Lemire, S., Yang, A. C., Ando, H., Mimee, M., Torres, M. D. T., ... & Lu, T. K. (2019). Engineering phage host-range and suppressing bacterial resistance through phage tail fiber mutagenesis. Cell, 179(2), 459-469.