Our designed biopesticide PHOCUS is based on engineered bacteriophages that contain DNA encoding for molecules that are toxic to locusts, such as the crystal toxin Cry7Ca1 and RNA interference (RNAi) precursors. Throughout our project, we went through several iterations of the Engineering-Design cycle in different examples (see Design and Results). This engineering cycle consists of the following steps: Research, Imagine, Design, Test, Learn, Improve (Figure 1). The steps of the process are repeated to improve the final product.
We have used iterations of this cycle in order to perform phage engineering and isolation of our desired engineered phage from a mixture with the wild-type phage. We first performed an engineering cycle regarding the phage engineering process and screening methods that we would use. We performed a second cycle to improve the screening conditions and method, and needed a third cycle to optimize it. Finally, our last cycle was focused on using CRISPR-Cas9 to select the engineered phage.
Design cycle 1: Phage engineering process and screening
Phage characteristics and engineering methods
The killing principle of our designed biopesticide was based on the toxic action of the Cry7Ca1 toxin as well as the RNA interference response to dsRNA. During the initial phase of our project, we thought about a method for delivering these molecules, and identified bacteriophages as a suitable option (For more detailed information see Design):
- After phage infection, bacteriophages hijack the bacterial transcription/translation system to redirect all resources to the production of new phage particles.
- By using phages we take advantage of the fact that the bacteria are already present in the gut.
- In the locust gut, the phages are expected to produce high levels of toxin.
Therefore, bacteriophages should be engineered to contain the DNA encoding for these molecules, which will be produced by host bacteria in the locust gut after infection by the phage. We also performed research in the following topics to shape our design:
- Locust gut bacteria: The bacterial community composition of the desert locust gut has been studied thoroughly to investigate the interactions between the locust and locust gut bacteria [1, 2, 3, 4]. A recent study shows that gregarious, field-collected, desert locusts maintain a core population of the bacterial Enterobacter genus .
- Lytic/lysogenic bacteriophages: Bacteriophages can infect bacteria following two different life cycles: the lytic and the lysogenic cycles . In the lytic cycle, bacteriophages inject their DNA in the host bacteria upon infection. This DNA is then transcribed and translated by the host's bacterial metabolic machinery after which the host produced new phages. Eventually the host produces endolysin, causing the cells to burst and the release of the replicated bacteriophages (Figure 2) . In the lysogenic cycle, after phage infection, the phage integrates its genome into the host DNA. The phages genetic material replicates together with that of the host .
- Bacteriophage engineering methods: we looked into different techniques that could be used. A useful summary of these techniques can be found in the review written by Pires et al. .
- Bacteriophage genetic material organisation: Gene expression of phages occurs in three stages, the early stage, middle stage and late stage . Bacteriophages contain essential and non-essential genes .
Model phage and how to engineer it
After performing literature research about the locust gut microbiota, the different types of bacteriophages and the possible engineering methods, we identified the requirements for the design of our engineered bacteriophages. These also include the traits related to the performance of our product.
These characteristics led us to design our experiments for engineering our model phage, responding to the criteria described below:
- Target bacteria: Enterobacter genus. We used Escherichia coli (E. coli) BL21 (DE3) as our host model organism. Microbiota from the locust gut is classified as Biosafety level 2, therefore we used a host model from the same genus.
- Type of phage: lytic phage. This characteristic is linked to the requirements of enough toxin production and fast action mode. Lytic phages lyse the host bacteria after replication, therefore releasing also the recombinant molecules produced. These virulent phages also infect a great number of bacteria, thereby producing high amounts of toxin. We will use the lytic T7 bacteriophage as this is classified as Biosafety level 1.
- Bacteriophage engineering method: Bacteriophage Recombineering of Electroporated DNA (BRED) . This method has been described to be the most efficient phage engineering method , and has already been successfully applied to engineer the T7 phage .
- Insert position: Due to the limited capsid space in the T7 bacteriophage, we decided to replace a non-essential gene instead of inserting our desired gene as an additional one. as the space in the phage capsid is limited [Dr. Franklin L. Nobrega, personal interview]. Another reason for replacing non-essential genes is that it increases the likelihood that the phage works after it has been engineered, as the phage still contains its essential genes. We hypothesised that by expressing early/middle genes toxin expression would be maximized. Early genes are expressed in an earlier stage thus we thought this would correspond to a higher level of protein. Therefore, three non-essential T7 genes were considered: 0.6A (early gene), 1.1 (early gene) and 4.3 (middle gene) . By comparing these different locations we also wanted to determine the different expression levels to find the best position to insert our desired gene. We also tried to find the optimal position for homologous gene expression computationally with help of the UT Austin iGEM team. For more information visit our Partnership page.
Replacing a non-essential gene of the T7 bacteriophage using BRED
During this phase we selected a promising solution from all our ideas. We linked our design requirements for phage engineering and came up with our final idea: “Replacing a non-essential gene of the T7 bacteriophage using BRED”
In order to decide which of the non-essential genes 0.6A, 1.1 and 4.3 should be replaced, we designed a proof of concept where the three non-essential genes 0.6A, 1.1 and 4.3 genes would be replaced by an enhanced GFP del6 (229) (GFP) using BRED (Figure 3). GFP is commonly used as a robust fluorescent reporter. Therefore, it would be useful to assess the feasibility of the gene insertion for the production of the toxic molecule. Furthermore, with such a reporter, we could also determine at which of the tested positions the toxic molecule is expressed the most.
DNA template amplification and BRED
In order to engineer the T7 phage in the three defined positions, we used BRED. The electroporated DNA for recombination was obtained by amplification of the eGFP from a plasmid by PCR, and was later incorporated by electroporation in E. coli BL21 (DE3) cells infected with the T7 phage (Figure 4). Our goal was to obtain plaques by PCR. Then, by detecting eGFP with a UV lamp we could select the engineered phages. Unfortunately, we did not see any individual plaques, as all the cells were lysed .
Screening conditions and method
From this first attempt, we realised that the conditions used did not seem appropriate to obtain individual plaques . We also thought that checking for fluorescence from the plaques by using a UV lamp could lead to false negatives due to lack of fluorescence. Therefore, we thought of different screening conditions and methods that we could use.
Design cycle 2: Screening conditions and method
Screening conditions and method
Screening conditions: In order to determine which was the better dilution to see individual plaques over time, we thought about performing plaque forming unit (PFU) experiments from the sample obtained after performing BRED. This approach consists of plating different phage dilutions to determine the optimal one to obtain individual plaques.
Screening method: in order to screen for engineered phages, we designed flanking primers that bind outside the replaced gene (Figure 5). With this, we could determine by PCR whether the screened plaque contained the GFP gene or the native T7 gene.
Repeating the (improved) experiment
We repeated the experiments after our two improvements. We obtained single plaques with a 10^8 phage dilution (Figure 6). Unfortunately, after PCR screening of these plaques, we could not find any phages that contained GFP, as all the PCR only showed amplification corresponding to the wild-type phage.
Identifying phage detection problems
As we failed to detect our engineered phages, we thought that there were two possible problems:
- The primers used for the PCR detection were very specific to the GFP insert . Thus, with a very low concentration of engineered phages, the wild-type phages might have overruled during the PCR.
- The efficiency of our engineering is lower than the expected 10% . Thus we need to do many PCRs to find the engineered bacteriophage.
Design cycle 3: Obtaining engineered phages
Improving the primers and screening method
Following the engineering principles we improved our design with two additional adjustments:
Repeating the (improved) experiment
We ordered the new primers and repeated our screening experiment using the newly designed primers binding to the GFP and the flanking regions, as well as the flanking primers themselves. We did a pool phage PCR and obtained positive results, showing that there was at least one engineered phage in our pool of phages (Figure 7).
Our results confirmed that we had obtained engineered phages . However, they were mixed with the wild-type ones. To isolate the phage we realized we needed to remove the wild-type phages from the heterogeneous phage mixtures.
Design cycle 4: Using CRISPR-Cas9 to select the engineered phage
Researching phage isolation methods
We researched methods on how to isolate the engineered phages and outcompete the wild-type T7 phages. We found that we could implement a CRISPR-Cas system for targeting the genes that we wanted to replace in the wild-type T7 phage. This method has been already reported by R. Kiro  as a successful strategy when engineering the T7 phage.
Designing single-guide RNA
We designed the sequences that would be used as single-guide RNA (sgRNA) targeting the 0.6A, 1.1 and 4.3 T7 phage genes. We used the pKDsgRNA-p15 plasmid as a backbone to insert our designed single-guide RNA (sgRNA). This plasmid will be co-expressed in E. coli BL21 (DE3) together with a plasmid carrying Cas9 (pCas9-CR4), and induced to degrade the T7 wild-type DNA upon infection.
Repeating the (improved) experiment
In the lab we built the plasmids containing each of the sgRNA plasmid by Gibson Isothermal Assembly. We repeated our phage experiments using E. coli BL21 (DE3) containing our CRISPR-Cas9 plasmid and our sgRNA plasmid targeting the T7 wild-type phages for these experiments. Unfortunately, after doing multiple rounds of screening and re-plating of the positive colonies, we were not able to isolate them. Future research should focus on isolating the engineered phages. To obtain engineered phages we advise to do more rounds of screening and re-plating of positive colonies.
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