iGEM 2021 — Team UNILausanne

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# Team:UNILausanne/Notebook

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# Lab Notebook

* [Introduction](#intro)
* [The Notebooks](#title1)

## Intro

Throughout the summer, each subproject held a laboratory notebook in order to keep track of the different achievements and to better visualize and plan the next steps. In this section, you can find the various lab notebooks.

## The Notebooks

### AFP Notebook

### Tailocins Notebook

### Phages Notebook

### Drylab Notebook

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# Team:UNILausanne/Engineering

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# Engineering Success

* [Overview](#intro)
* [Antifreeze Proteins](#title1)
* [Tailocins](#title2)
* [Phages](#title3)
* [Hardware / Software](#titleHS)
* [References](#references)

## Overview

With climate change, temperatures throughout the world are becoming more extreme and unpredictable, adversely impacting entire ecosystems. At a smaller scale, farming is particularly affected by these drastic weather fluctuations. For instance, in the Swiss region of Valais, we have observed repetitive freezing spells in the past few springs, causing devastating losses in crops, such as apricot trees. In order to solve this problem, we developed three complementary approaches and applied the engineering cycle (**Fig. 1**).

![Trulli](https://static.igem.org/mediawiki/2021/6/6b/T--UNILausanne--engineering_cycle.png)

**Figure 1 | Schematic representation of the engineering cycle.**

## Antifreeze Proteins

To reduce frost damage caused by ice crystals that destroy sensitive plant tissues during late spring freeze, we wanted to develop a solution containing antifreeze proteins (AFP). These proteins bind to ice crystals and thereby inhibit their growth. We did extensive research to learn more about how AFPs work and found out that they naturally exist in organisms living in cold temperatures. The proteins allow them to be resistant to freezing. Their activity can be characterized using either thermal hysteresis (TH) or ice recrystallization inhibition (IRI). TH describes the difference between the non-equilibrium freezing and melting temperatures [1]. IRI activity prevents the growth of larger ice crystals, which could damage plant tissues, at the expense of smaller ones [2]. Not all AFPs have the same activity. For this project, we therefore chose three antifreeze proteins, each one with a different activity. This allowed us to make comparisons and combine different AFPs to develop a solution efficient for our purpose. We chose to work with antifreeze proteins from *Rhagium inquisitor* (RiAFP), *Daucus carota* (DcAFP), and *Flavobacterium frigoris* PS1 (FfIBP).

First, we designed 6 new composite parts by inserting the three different AFPs into two vectors, pET-17b and pCold-I. The pET-17b plasmid contains a T7 promoter for regulated high-level expression of recombinant proteins. The pCold-I plasmid contains a cold-shock protein A (*cspA*) promoter for expression of proteins that are more difficult and cannot be expressed with the T7 system. We therefore compared expression of AFPs using each vector with the aim to optimize expression and synthesis of antifreeze proteins.

![Trulli](https://static.igem.org/mediawiki/2021/4/4c/T--UNILausanne--maps.png)

**Figure 2 | Plasmid maps of RiAFP, DcAFP, and FfIBP inserted into the pET-17b and pCold-I vectors.**

The various constructs (**Fig. 2**) were built and transformed into *Escherichia coli* BL21 (DE3) cells. We chose to work with *E. coli* BL21 (DE3) because it allows for high level expression and since the aim of our project is to develop a spray for apricot crops, large scale protein production would be necessary. *E. coli* is a commonly used organism and therefore easily available.

Initially, we first amplified the various AFP genes by PCR and linearized the plasmids by restriction enzyme digestion. Then, we assembled the various constructs by Gibson Assembly, transformed *E. coli* BL21 (DE3) cells by electroporation, and performed colony PCR to check if the genes were correctly assembled in the various constructs (**Fig. 3**). We obtained the expected band sizes and confirmed their sequence by sanger sequencing. We therefore concluded that we managed to successfully clone FfIBP, DcAFP, and RiAFP in *E. coli* BL21 (DE3) cells.

![Trulli](https://static.igem.org/mediawiki/parts/b/b0/T--UNILausanne--AFP_totalagarose.png)

**Figure 3 |** *Left to right: L – 1 kb DNA Ladder, 1 – pCold-I-DcAFP (1179 bp), 2 – pET-17b-DcAFP (1231 bp), 3 – pCold-I-FfIBP (927 bp) 4 – pET-17b-FfIBP (979 bp), 5 – pCold-I-RiAFP (588), 6 – pET-17b-RiAFP (640 bp)*

Next, the various AFPs were expressed in *E. coli* BL21 (DE3) using IPTG and then purified by His-tag affinity column and gel filtration. To test if the expression worked, we ran total cell SDS-PAGE (15%) of the different *E. coli* BL21 (DE3) cultures stained with Coomassie Blue.

We used two different vectors, pET-17b and pCold-I, to compare how the promoters influenced expression efficiency for both AFPs. The expression was successful for FfIBP and RiAFP in both pET-17b and pCold-I plasmids. We observed thicker bands that matched the weights of the different antifreeze proteins (FfIBP 28.4kDa, RiAFP 12.8 kDa). The pCold-I vector worked better for both RiAFP and FfIBP, and the bands on the gels appeared to be thicker for this plasmid (**Fig. 4** and **Fig. 5**). This can be explained by the fact that the natural expression of AFPs happens at cold temperatures, and any expression system adapted to these conditions will be more efficient. We induced expression at 15°C. At this temperature the 5’UTR of the mRNA from the pCold-I plasmid adopts a highly stable secondary structure and the TEE sequence then improves translation initiation of our AFP genes through a ribosome trapping process. The pET-17b plasmid does not have these features and is therefore less efficient. We can conclude that the pCold-I plasmid is a better system for expression of cold-stable proteins like AFPs.

![noooooooo](https://static.igem.org/mediawiki/parts/4/4c/T--UNILausanne--FfIBP_SDS2.png)

**Figure 4 | *SDS-PAGE electrophoresis before and after FfIBP purification.***  RiAFP was expressed in E. coli BL21 (DE3) using both pCold-I and pET-17b vectors. A) pCold-I-FfIBP: L – 10 to 250 kDa Protein Ladder, 1 – Bacterial lysate, 2 – Purified FfIBP. B) pET-17b-FfIBP: L – 10 to 250 kDa Protein Ladder, 3 – Bacterial lysate, 4 – Purified FfIBP.

![notgood](https://static.igem.org/mediawiki/parts/5/5f/T--UNILausanne--RiAFP_SDS.png)

**Figure 5 | *SDS-PAGE electrophoresis before and after RiAFP purification.*** RiAFP was expressed in E. coli BL21 (DE3) using both pCold-I and pET-17b vectors. Left to right: L - 10 to 250 kDa Protein Ladder, Bacterial Lysate: 1 - pET-17b-RiAFP, 2 - pCold-I-RiAFP. Purified Protein: 3 - pET-17b-RiAFP, 4 - pCold-I-RiAFP.

Once expression was successfully induced, we purified the AFPs and ran an SDS-PAGE (15%) of the purified recombinant proteins stained with Coomassie Blue. More information on this can be found [here.](https://2021.igem.org/Team%3AUNILausanne/Results)

The final test was the functionality of the proteins, which we did using two different assays. We measured the TH activity of the purified recombinant proteins and evaluated their effect on frost damage on plants. The details on these measurements can be found [here.](https://2021.igem.org/Team%3AUNILausanne/Measurement)

## Tailocins

Our main goal was to extract tailocins, phages-derived bacteriocins, to eliminate a bacterium present on apricot tree, *P. syringae* pv. *syringae*. This pathogen produces ice nucleation proteins to catalyze the formation of ice and therefore damage the plants’ membrane. Since we were not able to obtain the exact strains found on apricot trees, we therefore chose a closely related strain, *Pseudomonas syringae* pv. *syringae* B301D, as our target strain, since its genome sequence was available and this strain’s sensitivity to other *Pseudomonas* tailocins had already been characterized by Baltrus et al.[3]. Our strategy was therefore to produce tailocins from the strain *Pseudomonas syringae* pv. *aptata* DSM50252 which were shown to target *Pseudomonas syringae* pv. *syringae* B301D and proved that the produced tailocins have the ability to kill our target strains. To extract tailocins, we based our protocol on Baltrus et al.'s one with modifications suggested by Jordan Vacheron, from the University of Lausanne[3]. Briefly, we exposed our producing bacteria to mitomycin C, which triggered tailocins expression and cell lysis. We then purified tailocins from the lysis using PEG precipitation. Next, we tested their efficiency using an overlay essay. This method consists of serially diluting the purified tailocin solution and spotting the dilutions on a soft agar plate inoculated with our target strain.
With this assay, we have successfully proven that our target strain lyses in the presence of our two purified tailocins solutions.

![Trulli](https://static.igem.org/mediawiki/2021/e/eb/T--UNILausanne--results_tailocin_figure2_1.png)

**Figure 6 | Results of the overlay assay.**  A) From top to bottom: Tailocins extracted from bacteria exposed to 0 (A), 0.5 (B) or 3 (C) µg/mL mitomycin C as control. A drop of the Purification buffer alone (Tn50) on the black point. The buffer has no killing activity itself, as we can see it does not produce a plaque when we spot it on the plate.

Furthermore, we have proven that killing our target strain significantly reduces the freezing point of water. We compared two drops, one with our target strain and the tailocins and the other only with the target strain. Then we compared the freezing time using [FROZONE.](https://2021.igem.org/Team%3AUNILausanne/Hardware)The results prove that killing *Pseudomonas syringae* pv. *syringae* B301D, reduce the freezing time significantly.

![Trulli](https://static.igem.org/mediawiki/2021/6/63/T--UNILausanne--tailocin_graph.png)

**Figure 7 | Results of the FROZONE assay.**  Freezing time of water droplets containing *Pseudomonas syringae* pv. *syringae* B301D alone or treated with tailocins (Tailocin + *P. syringae* B301D). The tailocins treated droplets froze significantly more slowly than the ones containing only *Pseudomonas* (Welsch two sample t test: t = -4.42, df = 2.23, p-value = 0.04).

Having confirmed the efficiency of our tailocins approach in reducing *Pseudomonas syringae* pv. *syringae* B301D induced ice nucleation, we aimed at cloning the tailocins gene cluster in *E. coli*.

## Phages

The goal of this part of the project was to delete *InaZ*, the gene responsible for the production of Ice Nucleation Proteins (INP). INPs are harmful to fruit trees [4], since they allow the formation of ice crystals at a higher temperature compared to INP-free water, causing damage to plant epidermal cells, and thus disabling some of its physiological functions such as flowering [5]. So, by deleting the initial source of these proteins, in our case *InaZ*, we aim to disarm *P. syringae* pv. *syringae*, rendering it harmless to the plant. To achieve the deletion of this gene, we want to make use of the homologous recombination (HR) mechanism in the strain. The reason why we chose this repair mechanism is because the non-homologous end-joining (NHEJ) one appeared to be absent in this bacterial strain, contrarily to HR dedicated proteins [6]. To increase our chances to make the recombination happen, we decided to induce a DNA damage [7], notably a double-stranded break, into *InaZ* coding sequence.

The engineering procedure required the construction of a phagemid (i.e. a viral-derived plasmid) [8], the carrier of the two biological elements necessary for our goal. These two parts are a single-guide RNA (sgRNA), associated with the CRISPR/Cas9 cassette, which allows the DNA cutting machinery Cas9 to be directed towards a specific DNA sequence in the INP coding sequence. Three different sgRNAs were designed to target three different regions of *InaZ*, allowing us to select the most efficient in targeting and cutting this gene, but also the best to increase the chances of homologous recombination occurring.

The second element required for the construction of our engineered phagemid is called “Donor Sequence” and consists of a 2 kb sequence consisting of two regions homologous to the upstream and downstream sequences of *InaZ*. This double-stranded sequence provides the perfect template for the homologous recombination to occur. The *InaZ* coding sequence will eventually be deleted and replaced by the donor sequence in the *P. syringae* pv. *syringae*’s chromosome, as a result of the HR mechanism [9].

A schematic overview of this mechanism is represented here:

![Trulli](https://static.igem.org/mediawiki/2021/5/53/T--UNILausanne--Foto_per_WIKI--Engineering_success--iff83ONQ.jpeg)

**Figure 8 | Schematic representation of the mechanism allowing the replacement of the *InaZ* coding sequence by the donor sequence in the bacterial backbone.**

To trigger the mechanism shown in figure 8, the engineered phagemid needs to be efficiently delivered into *P. syringae* pv. *syringae*. This is provided by using a defective P1 phage that can carry our phagemid [10]. The resulting virions would eventually be harvested and put into our spray product.

The first step is to clone each of the three guide RNAs into a different plasmid unit containing a CRISPR/Cas9 system (pJH1) by using Golden Gate assembly. The insertion is made in the CRISPR sequence between the two BsaI sites [11].
We succeeded and obtained three different plasmids, each of them carrying one of the three sgRNA (**Fig. 9**).

The Golden Gate Assembly protocols can be found [here.](https://2021.igem.org/Team%3AUNILausanne/Experiments)

![Trulli](https://static.igem.org/mediawiki/2021/1/13/T--UNILausanne--Foto_per_WIKI--Results--sgRNA_1_copia-_FIgure_4.1.jpeg)
![Trulli](https://static.igem.org/mediawiki/2021/a/ad/T--UNILausanne--Foto_per_WIKI--Results--sgRNA_2_copia-_Figure_4.2.jpeg)
![Trulli](https://static.igem.org/mediawiki/2021/e/ea/T--UNILausanne--Foto_per_WIKI--Results--sgRNA_3_copia-_Figure_4.3_.jpeg)

**Figure 9 | Sequencing results aligned with pJH1 sequence in Snapgene.** On the left pJH1 carrying sgRNA 1, sgRNA 2 in the middle and sgRNA 3 on the right

The next step is to clone the donor sequence into the sgRNA-carrier pJH1 by using Gibson Assembly. We obtained our donor sequence by IDT and succeeded in cloning it into two of our 3 gRNA plasmids (**Fig. 10**).

The Gibson Assembly protocols can be found [here.](https://2021.igem.org/Team%3AUNILausanne/Experiments)
![Trulli](https://static.igem.org/mediawiki/2021/b/ba/T--UNILausanne--Foto_per_WIKI--Engineering_success--Donor_Sequence_.jpeg)

We decided to test the efficacy of our constructs in deleting *InaZ*. As a proof of Concept, we transformed two different plasmids carrying either sgRNA 1 or 2 and the donor sequence into *Pseudomonas syringae* pv. *syringae B301D*.

After transformation, the bacteria were plated into LB agar plates supplied with Kanamycin 50. The next day, we picked the colonies and checked the clones by performing colony PCRs to confirm the deletion of *InaZ*. Unfortunately, all grown colonies still carried *InaZ* in their genome, as shown in figure 11. The expected PCR product confirming *InaZ* deletion would have been around 2 kb, whereas a PCR product of about 6 kb would mean that *InaZ* is not deleted.

![Trulli](https://static.igem.org/mediawiki/2021/8/85/T--UNILausanne--results_phage_gel5.png)

There can be several things that might not have worked. First, we might need to test if Cas9 got expressed. We could perform a Western Blot to test Cas9 expression. Second, we could test the efficiency of the gRNAs. We could transform another plasmid containing the target region of the gRNAs and see if Cas9 induces a cut in this plasmid in *P. syringae*. Third, homologous recombination might have been low efficient in *P. syringae*, especially when the designed homology arms are not adjacent to the cut site. We could design a donor sequence carrying homologies adjacent to the cut site, which will not completely delete *InaZ* but at least disrupt it. Fourth, instead of one cut, we could cut *InaZ* twice by using two gRNAs targeting the start and end of the gene. This might facilitate the integration of the donor sequence by the HR repair mechanism, as the sequences next to the cut are directly homologous to the ones provided by the donor sequence. With these measures, we might be able to get our strategy work. In parallel, we tested two more strategies that look more promising.

## Hardware and Software

To perform some of our experiments, a precise control of the temperature was needed. We therefore created FROZONE, a precise cooling device that fits under a microscope, acting similarly to a Nanoliter Osmometer. The design of our machine was inspired by the [MicroIce LTD.](https://docs.google.com/document/d/1NyBdOtWBIU_gHp7boHjlQVsLQZPI1HuZc0qoaFAkeQM/edit) According to MicroIce, "A Nanoliter osmometer is a cooling stage mounted on an upright optical microscope. Cooling of the stage is achieved with the use of Peltier devices driven with a precision temperature controller."

Our nanoliter osmometer, FROZONE, was designed and created by us. The cooling of the device is achieved with a thermoelectric cooler controlled by a custom Python [software.](https://2021.igem.org/Team%3AUNILausanne/Software) We also designed a vacuum chamber to minimize the crystallization of ambient humidity on our samples. Put simply, we created a machine that precisely controls the temperature of a copper plate in order to measure various aspects of the solutions we produced.
The build of our machine was not successful on the first try - we had to make, test and improve many prototypes before being satisfied with our final product.

For more information about software click [here.](https://2021.igem.org/Team%3AUNILausanne/Software)

### Vacuum chamber

When we first designed our machine, we did not think to add a vacuum chamber around the copper plate on which we froze the samples. However, with this setup, we observed lots of undesired droplets forming on the plate due to the ambient humidity (**Fig. 12**).

### Top glass

We initially placed a single layer of glass over the copper plate to create the vacuum chamber, but condensation had a tendency to form on the outside of the glass. We therefore modified this glass to make it double layered, successfully preventing the formation of condensation. Between the two layers of glass, we placed silica gel beads to absorb any moisture.

### Light

The microscope we used to visualize the freezing of our solution drops on FROZONE’s copper plate had no top light, so we initially used a lamp to illuminate the machine. However, this LED lamp caused the heating of the plate, modifying this essential parameter. It also did not generate enough light, making it impossible for us to zoom in the images we captured. We instead decided to use an optical fiber light source to light up our copper plate. This light source was much more focused, allowing the proper illumination on our samples. This kind of lamp does not generate heat, so its use did not affect the temperature of our machine.

### Software - temperature control

At the beginning, we controlled the temperature on FROZONE through a graphical interface, on which we had added a slider - we needed to scroll this slider up and down to modify the temperature.
This was not the most efficient way to set the temperature on our machine, so we decided to replace the slider with an input box. This allowed us to enter the desired temperature with our keyboard. This mechanism worked well to set a temperature, but was limited in the fact that it was more difficult to gradually increase or diminish the temperature. We therefore designed a spinbox, allowing us to raise or diminish the temperature on our machine using the up and down arrows on our keyboard. Each time we press an upward or downward arrow on our keyboard, we can now raise or diminish the temperature of 0.1 °C respectively.

For more information about software click [here.](https://2021.igem.org/Team%3AUNILausanne/Software)

**References**

1. [1] A. K. Gruneberg et al., *Ice recrystallization inhibition activity varies with ice-binding protein type and does not correlate with thermal hysteresis*, In: Cryobiology, Vol. 99, pp. 28-39, April 2021 Available from: [https://doi.org/10.1016/j.cryobiol.2021.01.017](https://www.sciencedirect.com/science/article/pii/S0011224021000171)
2. [2] M. Chow‐Shi‐Yée et al., *Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells*, In: Protein Sci., Vol. 25, No. 5, May 2016 Available from: [/pmc/articles/PMC4838640/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838640/)
3. [3] D. A. Baltrus et al.,*Localized recombination drives diversification of killing spectra for phage-derived syringacins*, In: The ISME Journal, Vol. 13, pp. 237–249, 2019 Available from: <https://www.nature.com/articles/s41396-018-0261-3>
4. [4] S. E. Lindow, *The role of bacterial ice nucleation in frost injury to plants*, In: Annual Review Phytopathol, Vol. 21, pp. 363-384, 1983 Available from: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1065830/>
5. [5] S. J. Roeters et al., *Ice-nucleating proteins are activated by low temperatures to control the structure of interfacial water*, In: Nature Communications, Vol. 12, No. 1, pp. 1183-1192, 19 February 2021 Available from: <https://www.nature.com/articles/s41467-021-21349-3>
6. [6] B. Swingle et al., *Recombineering Using RecTE from Pseudomonas syringae*, In: Applied and Environmental Microbiology, Vol. 76, No. 15, 11 July 2010 Available from: <https://journals.asm.org/doi/10.1128/AEM.00911-10>
7. [7] P. J. G. Rauch et al., *The expression of the Acinetobacter calcoaceticus recA gene increases in response to DNA damage independently of RecA and of development of competence for natural transformation*, In: Microbiology, Vol. 142, Pt. 4, pp. 1025-1032, 1996 Available from: <https://pubmed.ncbi.nlm.nih.gov/8936328/>
8. [8] H. Qi et al., *Phagemid Vectors for Phage Display: Properties, Characteristics and Construction*, In: Journal of Molecular Biology, Vol. 417, No. 3, pp. 129-143, 30 March 2012 Available from: <https://pubmed.ncbi.nlm.nih.gov/22310045/>
9. [9] K. Sakaguchi et al., *A targeted gene knockout method using a newly constructed temperature-sensitive plasmid mediated homologous recombination in Bifidobacterium longum*, In: Applied Microbiology and Biotechnology, Vol. 95, pp. 499-509, 2012 Available from: <https://pubmed.ncbi.nlm.nih.gov/22639142/>
10. [10] J. T. Kittleson et al., *Scalable Plasmid Transfer using Engineered P1-based Phagemids*, In: ACS Synthetic Biology, Vol. 1, No. 12, pp. 583-589, 21 December 2012 Available from: <https://pubs.acs.org/doi/abs/10.1021/sb300054p>
11. [11] J. Ho et al., *The application of the CRISPR/Cas9 system in Pseudomonas syringae pv. actinidiae*, In: Journal of Medical Microbiology, Vol. 69, No. 3, pp. 478-486, March 2020 Available from: <https://pubmed.ncbi.nlm.nih.gov/31935181/>
12. Figure 8 created with [Biorender.com](https://biorender.com)

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# Experiments

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## The Protocols

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# Results

* [Overview](#intro)
* [Antifreeze Proteins](#title1)
  + [Plasmid Cloning](#subtitle1)
  + [Expression and Purification](#subtitle2)
  + [Assays](#assays)
* [Tailocins](#title2)
  + [Extraction](#subtitle3)
  + [Plasmid Cloning](#subtitle4)
  + [Testing](#test)
* [Phages](#title3)
  + [Golden Gate](#subtitle5)
  + [Gibson](#subtitle6)
* [References](#references)

## Overview

During spring season, fruit cultivators throughout Switzerland incur enormous losses due to frost each year. A widespread and renowned crop in the Suisse Romande region are apricots, which were particularly affected by the cold this year. The aim of our project is to develop three different approaches to tackle this problem. We focused on different aspects of this issue and either inhibited the formation of ice crystals directly by using antifreeze proteins (AFP) or found a way to inhibit the production of ice nucleation proteins by *Pseudomonas syringae syringae*, which will reduce the amount of ice crystals that are formed during the late spring freezes.

## Key Achievements

* We successfully constructed 6 new composite parts containing the various AFP genes.
* We expressed and purified RiAFP and DcAFP.
* We were able to determine the thermal hysteresis of RiAFP and DcAFP, and therefore proved that they are functional.
* We successfully demonstrated that the purified AFPs have a strong protective effect against frost damage on plants.
* We managed to extract tailocins from the killer strain and visualised them using electron microscopy.
* We confirmed the ability of our tailocins to kill our target strain thanks to an overlay assay.
* We were able to determine the effect of *P. syringae syringae* on freezing time.
* We proved that our tailocins affect freezing time of a solution containing *P. syringae syringae*.
* We successfully inserted all three sgRNAs into three different plasmids.
* We successfully inserted the donor sequence into two of the three plasmids carrying sgRNA.

## Antifreeze Proteins

The aim of this part of the project was to produce an antifreeze protein solution, which can bind to ice crystals during late spring freeze and therefore protect the crops from frost damage.

### Plasmid Cloning

Initially, we first amplified the various AFP genes by PCR and linearized the plasmids by restriction enzyme digestion. Then we assembled the various constructs by Gibson Assembly, transformed *E. coli* BL21 (DE3) cells by electroporation, and performed colony PCR to check if the genes were correctly assembled in the various constructs (Fig.1). We obtained the expected band sizes and confirmed their sequence by sanger sequencing. We therefore concluded that we managed to successfully clone FfIBP, DcAFP, and RiAFP in *E. coli* BL21 (DE3) cells.

![Trulli](https://static.igem.org/mediawiki/parts/b/b0/T--UNILausanne--AFP_totalagarose.png)

***Figure 1 | Colony PCR check.** Left to right: L – 1 kb DNA Ladder, 1 – pCold-I-DcAFP (1179 bp), 2 – pET-17b-DcAFP (1231 bp), 3 – pCold-I-FfIBP (927 bp) 4 – pET-17b-FfIBP (979 bp), 5 – pCold-I-RiAFP (588), 6 – pET-17b-RiAFP (640 bp)*

### Expression and Purification

After this, we induced expression of the various AFPs with IPTG and ran a total cell SDS-PAGE (15%) of the *E. coli* BL21 (DE3) cultures stained with Coomassie blue. We used two different vectors, pET-17b and pCold-I, to compare how the promoters influenced expression efficiency for both AFPs. The expression was successful for FfIBP and RiAFP in both pET-17b and pCold-I plasmids. We observed thicker bands that matched the weights of the different antifreeze proteins (FfIBP 28.4kDa, RiAFP 12.8 kDa). The pCold-I vector worked better for both RiAFP and FfIBP, and the bands on the gels appeared to be thicker for this plasmid (**Fig.2** and **Fig.3**). This can be explained by the fact that the natural expression of AFPs happens at cold temperatures, and any expression system adapted to these conditions will be more efficient. We induced expression at 15°C. At this temperature the 5’UTR of the mRNA from the pCold-I plasmid adopts a highly stable secondary structure and the TEE sequence then improves translation initiation of our AFP genes through a ribosome trapping process. The pET-17b plasmid does not have these features and is therefore less efficient. We can conclude that the pCold-I plasmid is a better system for expression of cold-stable proteins like AFPs.

![noooooooo](https://static.igem.org/mediawiki/parts/4/4c/T--UNILausanne--FfIBP_SDS2.png)

***Figure 2 | SDS-PAGE electrophoresis before and after FfIBP purification.** RiAFP was expressed in E. coli BL21 (DE3) using both pCold-I and pET-17b vectors. A) pCold-I-FfIBP: L – 10 to 250 kDa Protein Ladder, 1 – Bacterial lysate, 2 – Purified FfIBP. B) pET-17b-FfIBP: L – 10 to 250 kDa Protein Ladder, 3 – Bacterial lysate, 4 – Purified FfIBP.*

![notgood](https://static.igem.org/mediawiki/parts/5/5f/T--UNILausanne--RiAFP_SDS.png)

***Figure 3 | SDS-PAGE electrophoresis before and after RiAFP purification.** RiAFP was expressed in E. coli BL21 (DE3) using both pCold-I and pET-17b vectors. Left to right: L - 10 to 250 kDa Protein Ladder, Bacterial Lysate: 1 - pET-17b-RiAFP, 2 - pCold-I-RiAFP. Purified Protein: 3 - pET-17b-RiAFP, 4 - pCold-I-RiAFP.*

From these initial test purifications with magnetic beads we were able to conclude that the expression and purification of FfIBP and RiAFP worked. Therefore, we upscaled the protein purification and induced expression in a 1-liter culture. After that, we performed two purification steps: the first using the His-tag affinity column and the second a gel filtration. Then, we eluted the protein in various fractions, ran a SDS-PAGE (15%) of the fractions containing the purified protein stained with Coomassie blue, and finally pulled the fractions containing FfIBP (Fig.4). Finally, we measured the final concentration of our protein with the nanodrop and obtained 178µM or 4.53mg/mL.

![Trulli](https://static.igem.org/mediawiki/parts/2/21/T--UNILausanne--FfIBP_finalSDS.png)

***Figure 4 | SDS-PAGE electrophoresis after gel filtration purification of FfIBP**. FfIBP was eluted into multiple fractions. Left to right: L - Protein Ladder, 10 to 250 kDa, fractions B3 to B12.*

The upscaling for RiAFP did not work at first and we did not observe any bands that matched the weight of the protein on the total cell SDS-PAGE (15%) of *E. coli* cultures stained with Coomassie blue (data not shown). For the FfIBP purification, we used a baffled flask to grow the 1-liter culture for *E. coli* BL21 (DE3) expressing the construct. The edges of the flask maximize oxygen transfer to the culture media, but in some cases, they can also interfere with protein expression. Since we could not upscale the RiAFP expression using this method, we used a flask without baffles instead and succeeded that way. Then, we performed the same purification steps as we did for FfIBP purification. After gel filtration, we eluted RiAFP in various fractions, ran an SDS-PAGE (15%) of the fractions containing the purified protein stained with Coomassie blue, and finally pulled the fractions containing RiAFP (**Fig.5**). RiAFP does not contain any Tryptophan and Tyrosine residues in its sequence, hence, we could not measure its final concentration using a nanodrop and used the Bradford method to estimate it instead. In a standard reaction, 25µL of BSA concentration and 5µL of RiAFP concentration were mixed with 200µL of Bradford reagent. Absorbance was then measured at 595 nm. The calibration curve (**Fig.6**) is needed to calculate sample concentration. We obtained 0.75mg/mL or 58.94µM.

![noooooooo](https://static.igem.org/mediawiki/parts/4/46/T--UNILausanne--RiAFP_finalSDS.png)

***Figure 5 | SDS-PAGE electrophoresis after gel filtration purification of RiAFP.** RiAFP was eluted into multiple fractions. Left to right: L - Protein Ladder, 10 to 250 kDa; fractions B2 to B8.*

![notgood](https://static.igem.org/mediawiki/parts/4/4d/T--UNILausanne--RiAFP_Bradford.png)

***Figure 6 | Bradford assay standard curve of concentration versus absorbance.** The concentration of RiAFP (in mg/mL) was determined using the equation y=0.0729x-0.015 with an R2 value of 0.9988, where y is absorbance and x is concentration.*

The expression didn’t work for DcAFP in pET-17b. When we ran a total cell SDS-PAGE (15%) of *E. coli* cultures stained with Coomassie blue, we didn’t observe any thicker bands that would have corresponded to the protein. The root of the problem might be that the protein aggregate to form inclusion bodies. To remedy this problem, we attempted to optimize expression by using various concentrations of IPTG. Once more, we ran a total cell SDS-PAGE (15%) of *E. coli* cultures stained with Coomassie blue but did not observe thicker bands at the desired protein weight (**Fig.7**).

![Trulli](https://static.igem.org/mediawiki/parts/9/92/T--UNILausanne--DcAFP_exp.png)

***Figure 7 | SDS-PAGE electrophoresis of bacterial lysate of DcAFP expressed using the pET-17b plasmid.** Expression was induced for 24 hours at 15°C with various concentrations of IPTG. L –Protein Ladder, 10 to 250 kDa, 1 –0mM , 2 –0.01mM, 3 - 0.1mM , 4 - 0.2mM, 5 - 0.5mM*

Since we couldn’t optimize expression of DcAFP by adapting the concentration of IPTG, the next step would be to purify the protein from inclusion bodies. We adapted a protocol [1] to our DcAFP construct, which could be used in the future.

### Results of AFP TH Values

The measurements we performed on our AFP solutions were done in part with the machine we designed, FROZONE. This device, coupled with a software we created, allows the precise control of the temperature of a copper plate placed in a vacuum chamber. We placed a drop of our AFP solutions on the copper plates and visualised their freezing and melting temperatures using a microscope.
This allowed us to precisely determine the freezing and melting temperatures of our AFP solutions, allowing us in turn to determine the TH values of our proteins at specific concentrations. The values we obtained can be visualised in the following (Fig. 24.).

![3d model](https://static.igem.org/mediawiki/2021/0/07/T--UNILausanne--AFPs_TH.png)

***Figure 24 | Thermal hysteresis of RiAFP and FfIBP increase with their concentration.**Connected scatter plot graph showing the mean of biological triplicate +/- standard deviation. Thermal hysteresis is measured with two different AFPs, RiAFP and FfIBP, at different concentrations (µM).*

The data we obtained show that the thermal hysteresis of our proteins increases as their concentration in the solution increases.
From these results, we can conclude that our antifreeze proteins are both functional, as we have successfully measured their respective TH values at two distinct concentrations. With these results, we can also conclude that RiAFP is more effective than FfIBP at a concentration of 50µM.

### Result of Protective Effect of AFPs on Plants

The following graph shows the percentage of damage measured on the leaves of Arabidopsis plants that were incubated overnight at -5 °C in various solutions of AFPs or in our buffer solution. Our results show that plants incubated in the buffer alone are significantly more damaged than those incubated in the AFP solutions, therefore confirming the protective effect of our AFPs. We were not able to detect, however, a significant difference between the protection level provided by FfIBP, RiAFP or a mix of both AFPs.

![3d model](https://static.igem.org/mediawiki/2021/9/91/T--UNILausanne--FDT_GRAPH.png)

***Figure 25 | Our AFP solutions protect plants from frost damage.**The graph shows mean +/- standard deviation. Percentage of Damage on Arabidopsis thaliana leaves measured after overnight incubation in a climatic chamber at -5ºC. Plants were immersed in phosphate-buffered saline (PBS) liquid solution supplemented with two different AFPs: FfIBP [200 µM] (n=13) and RiAFP [50 µM] (n=17) and a mix of them (RiAFP + FfIBP) (n=15) at a concentration of 25 µM and 100 µM, respectively. These solutions were compared with the control in which the plants were immersed in the PBS buffer without any AFP (buffer) (n=17).
The damage ratio is significantly different between the buffer and all AFPs. ANOVA, \* significant at p<0.05.Damage ratio between the different AFPs are not significant.*

## Tailocins

In order to kill *Pseudomonas syringae*  pv. *syringae* B301D, our target strain similar to the frost-damage enhancing *Pseudomonas syringae* pv. *syringae* found on apricot trees, we are using tailocins naturally produced by *Pseudomonas syrinage* pv. *aptata* DSM50252.

### Tailocins Extraction

We successfully extracted tailocins from our killer strain, *P. syringae* DSM50252.
We first induced the production of tailocins in our killer strain using different concentrations of mitomycin C, *i.e.* 0.5 or 3 µg ml -1 (cf. tailocin extraction protocol (link)). Tailocin production can be indirectly monitored via the death of the producer bacteria, as tailocins synthesis results in their lysis. We could induce production of tailocins with as little as 0.5 µgml -1 mitomycin C, as both concentration tested led to cell lysis compared to our control flask without addition of mitomycin C (**Fig. 1**).

![Trulli](https://static.igem.org/mediawiki/2021/9/9a/T--UNILausanne--results_tailocin_figure1.png)

***Figure 8 | Mitomycin C induces tailocin production in  *P. syringae*  DSM50252.** Liquid cultures of *P. syringae* DSM50212 (killer strain) exposed to 0 (A), 0.5 (B) or 3 (C) µgml  -1  mitomycin C.*

Having induced the production of tailocins by our killer strain, we then set out to test their efficacy in killing our target strain  *P. syringae syringae* B301D. For this, we first proceeded to purify the tailocins from both the mitomycin-induced cell lysates and the non-induced culture as a negative control. We then used a soft agar overlay assay method (as described by Baltrus and Hockett) to assess the lysis of the target cells exposed to our purified tailocins.

![Trulli]( https://static.igem.org/mediawiki/2021/archive/e/eb/20211019071000%21T--UNILausanne--results_tailocin_figure2_1.png)
![Trulli]( https://static.igem.org/mediawiki/2021/e/ea/T--UNILausanne--overlay_assay.png)

***Figure 9 | Purified tailocins lyse P. syringae B301D.(A)**Photographs showing  *P. syringae*  B301D cell lawns exposed to serially diluted tailocins extracted from *P. syringae*  DSM5012 cultures exposed to 0, 0.5 or 3 µgmL-1 mitomycin C or purification buffer alone (Tn50). The black dot indicates where the sample was dropped. The buffer has no killing activity, as we can see that it does not produce a plaque when we spot it on the plate. **(B)**  Schematic representation of expected morphologies of lysis plaques induced by a serial dilution of tailocins (top) or phages (bottom).*

Our results indicate that we successfully purified tailocins from our killer strain and that these tailocins were able to kill our target strain  **(Fig. 2, A)**. Indeed, we observed clear concentration-dependent lysis of cells exposed to our diluted purified extract. The control solution, however, which we obtained by following the same extraction protocol but without inducing tailocin production with mitomycin C, showed some mild killing activity(**Fig. 2, A** ). This could indicate that some tailocins were produced even without mitomycin or that our killer strain produces another bacteriocin or antibiotic efficient against our target. Nevertheless, mitomycin C-induced lysates exhibit greater killing activities compared to the uninduced cultures, confirming that mitomycin C is essential to obtain significant tailocin yield. Additionally, we observed a higher killing activity in the 0.5 µg mL-1 mitomycin-induced extract than in the 3 µgmL -1 (**Fig. 2, A**), which suggests that the lower concentration led to a better yield of tailocins production.
As explained on the design page (link), our killer strain’s genome contains both a tailocin gene cluster and a prophage. As both can be induced by mitomycin C, we cannot exclude a priori that the killing activity is caused by the phage rather than the tailocins. Nevertheless, phages produce small individual plaques instead of big ones when they are diluted enough, whereas plaques produced by tailocins become gradually smaller and fainter as they are diluted (**Fig. 2, B**). As we do not observe individual plaques on our plates, it is most likely that the killing activity seen in our experiment is caused by tailocins. However, we cannot exclude that a small amount of phages is present and contributes to cell lysis. To prove that our tailocins only killed our target, further experiments should aim at deleting some of the phage’s genes from our strain’s genome.
As additional proof of the success of our tailocin purification, we performed negative staining of our tailocin extracts and visualized our samples by electron microscopy. This confirmed the presence of tailocins in our mitomycin C-induced extracts (**Fig. 3**), while no tailocins could be observed in our control sample (non-induced cells). Also, no phages could be seen in our samples, although more concentrated extracts could be required to observe them. By measuring the height and width of our tailocins, and obtained the following results: 168 nm long and 25 nm wide.

![Trulli](https://static.igem.org/mediawiki/2021/0/0a/T--UNILausanne--Tailocin_051.png)
![Trulli](https://static.igem.org/mediawiki/2021/7/7d/T--UNILausanne--Tailocin_052.png)
![Trulli](https://static.igem.org/mediawiki/2021/a/ae/T--UNILausanne--EM_measure.png)

***Figure 10 | Electron microscopy images of tailocin extracts.** From left to right : The first two images show tailocins extracted from  *P. syringae* DSM50252 when induced with 0.5 µg mL-1 of mitomycin C. Image on the right shows a tailocin found in the sample obtained from cultures with 3 µgmL-1 of mitomycin C.*

In parallel to obtaining tailocins directly from  *P. syringae* , we aimed at cloning the tailocoin gene cluster in a plasmid to express it heterologously in  *Escherichia coli* . As the cluster is relatively big (*i.e.* 14 kb), we first amplified it from  *P. syringae*  genomic DNA as four distinct fragments (**Fig 4**).

![Trulli]( https://static.igem.org/mediawiki/2021/6/66/T--UNILausanne--tailocin_PCR_with_an_A.png)
![Trulli]( https://static.igem.org/mediawiki/2021/3/3f/T--UNILausanne--results_tailocin_figure3_withB.png)

### Plasmid Cloning

Having obtained our different fragments, we then attempted to assemble them using Gibson assembly but we were unfortunately unsuccessful. After transformation of  *E. coli*  cells with our Gibson products, we obtained some colonies growing on selective media and none in the control (in which we performed a Gibson without one of the fragments). The absence of colonies on our control plate seemed to indicate that the assembly had worked. However, the colony PCRs we later performed revealed that our transformed bacteria carried only the empty backbone ( **Fig. 5** ). Due to lack of time, we were unfortunately not able to proceed further.

![Trulli]( https://static.igem.org/mediawiki/2021/7/7b/T--UNILausanne--result_tailocin_gisbon_plasmid.png)

### Versus

Our Versus assay was designed to test if the tailocins we produced successfully killed and unarmed *Pseudomonas syringae syringae*. For this assay, we tested a *P.syringae* solution at OD1 and a *P.syringae* solution at OD1, which we had previously incubated with our tailocin solution for an hour. We placed a drop of each of these solutions on our FROZONE machine, set at -15°C. We then measured the time it took for each of the drops to freeze. Figure 27 illustrates our results, obtained using a t-test.

![3d model](https://static.igem.org/mediawiki/2021/6/63/T--UNILausanne--tailocin_graph.png)

We can see that there is a significant difference between the time it took for the *P.syringae* solution to freeze versus the time it took for the same solution but previously incubated with tailocins to freeze. As the tailocins incubation is the only variable between the two solutions, we can conclude that these protein complexes allow the reduction of *P.syringae*’s ice nucleation activity.

### Measuring the Time Before Freezing - *P. syringae* Solution

*Pseudomonas syringae syringae*  (PSS) accelerates and facilitates the formation of ice crystals. Theoretically, adding *P.syringae* to a solution at a sub-zero temperature should shorten the time it takes for this solution to freeze.
To test this hypothesis, we used our FROZONE machine to perform the TTF assay. This assay measures the time it takes for a drop of solution to freeze at -15°C. The results of this assay, illustrated in Figure 26, show that it takes an average of 300 seconds for a 10µL drop of buffer to freeze at -15°C whereas it takes an average of 102 seconds for a solution containing an OD of 0.7 of *P. syringae* to freeze in these conditions. We also compared this data to the freezing time of a solution containing an OD 0.7 of *E. coli*, which takes an average of 1500 seconds to freeze. We tested two other concentrations of *P. syringae* to try and determine when the presence of the bacteria started to have an effect on the time before freezing. From these measurements, we established that even at very low concentrations, the bacteria have a drastic effect on the time it takes for a drop of solution to freeze.
This assay allowed us to confirm that *P. syringae* drastically reduces the Time before freezing, regardless of the concentrations tested.

![3d model](https://static.igem.org/mediawiki/2021/8/88/T--UNILausanne--effect_of_PSS.png)

## Phages

In this part of the project we primarily focused on engineering a plasmid responsible for delivering a CRISPR-Cas9 construct with a sgRNA targeting the genomic site of *InaZ*, the gene coding for the Ice Nucleation Protein. We tested three different sgRNAs in parallel (each sgRNA on a different plasmid) targeting different positions of *InaZ*.
In addition, as our purpose was to take advantage of the innate homologous recombination process of *Pseudomonas syringae*, we also wanted to insert a Donor Sequence into our plasmid, consisting of a 2 kb homology region allowing such mechanism to take place. These two elements would allow us to prove the concept that such an intervention could trigger the deletion of *InaZ*.
In a second step, we would use the same techniques used to engineer the plasmid in order to engineer the final carrier consisting of a phagemid, allowing us to generate an engineered defective phage capable of infecting *Pseudomonas syringae*, eventually causing the deletion of *InaZ*.

All the protocols that we used to get our results can be found [here.](https://2021.igem.org/Team%3AUNILausanne/Experiments)

### Golden Gate Assembly (for sgRNA insertion)

We used a Golden Gate Assembly to clone the three different sgRNAs into pJH1 (*i.e.* our vector).
We first annealed the three pairs of complementary oligos which gave us the three different sgRNAs ready to be cloned by Golden Gate Assembly into pJH1 opened by BsaI-HFv2.

The actual Golden Gate Assembly reaction was hosted by a thermal cycler. We then transformed electrocompetent NEB5alpha with the Golden Gate reaction product, giving us the results shown in figure 13:

![Trulli](https://static.igem.org/mediawiki/2021/5/54/T--UNILausanne--Foto_per_WIKI--Results--Figure_1_.jpeg)

***Figure 13 | Transformed *E. coli NEB5alpha.*** On the right, the plate containing NEB5alpha bacteria transformed with pJH1 carrying sgRNA 1 is shown, one half (upper one) undergoing initial dilution in HEPES and the other (bottom one) without undergoing initial dilution. In the center (bottom plate) the plate containing bacteria transformed with pJH1 carrying sgRNA 2 is shown, following the same disposition as the plate on the right. The upper plate contains bacteria transformed with pJH1 carrying sgRNA 3, with the same disposition as the other two plates. On the left, the plate containing bacteria that did not undergo transformation is shown, representing a control*

From here we decided to adhere to the following workflow:

![Trulli](https://static.igem.org/mediawiki/2021/8/8f/T--UNILausanne--Foto_per_WIKI--Results--Figure_2_.jpeg)

***Figure 14 | Representation of the followed workflow.**  Starting from plated transformed bacteria to extraction and amplification of the specific fragment (the right branch) as well as the plasmid extraction (the left branch)*

First, we performed a colony PCR followed by a gel electrophoresis to check the presence and size of the amplified fragment. One of the two primers used during colony PCR is priming in the sgRNA insertion, meaning that if we observe a band, the presence of the guide RNA is confirmed, while its absence would be the result of a lack of sgRNA insertion.
The electrophoresis gel is shown in figure 15:

![Trulli]( https://static.igem.org/mediawiki/2021/5/51/T--UNILausanne--results_phage_gel4.png)

***Figure 15 | Electrophoresis results of the sgRNA insertion.**  Colony PCR after Golden Gate Assembly. The first lane on the left named “Ladder” corresponds to the molecular-weight size marker. Lanes numbered 1-14 correspond to single colonies tested. Wells 1-10 and 13-14 are from the insertion of the sgRNAs, while wells 11-12 were from the insertion of the donor sequence (not presented here)*

All 12 colonies tested show a bright band at the expected molecular size of approximately 900 bp, which means that in those cases the sgRNA insert is probably present. We purified the PCR product and sent it for sequencing.

We also made a miniprep to extract the engineered pJH1. The sequencing analysis results are shown in figure 16:

![Trulli](https://static.igem.org/mediawiki/2021/1/13/T--UNILausanne--Foto_per_WIKI--Results--sgRNA_1_copia-_FIgure_4.1.jpeg)
![Trulli](https://static.igem.org/mediawiki/2021/a/ad/T--UNILausanne--Foto_per_WIKI--Results--sgRNA_2_copia-_Figure_4.2.jpeg)
![Trulli](https://static.igem.org/mediawiki/2021/e/ea/T--UNILausanne--Foto_per_WIKI--Results--sgRNA_3_copia-_Figure_4.3_.jpeg)

***Figure 16 | Sequencing results aligned with pJH1 sequence in Snapgene.** On the left pJH1 carrying sgRNA 1, sgRNA 2 in the middle and sgRNA 3 on the right*

These results confirm that all three sgRNAs had been efficiently integrated into pJH1 (each sgRNA in a different plasmid entity).

### Gibson Assembly (for Donor Sequence insertion):

We also used Gibson Assembly to clone into pJH1 (*i.e.* our vector) the donor sequence, carrier of the homology regions responsible for the homologous recombination event occurring in *Pseudomonas syringae*.
We firstly ordered the donor sequence from IDT (Integrated DNA Technologies company) and dissolved it upon arrival.

Once the donor sequence was prepared for further manipulations, the first procedure that took place in this part of the project was the amplification of such sequence via PCR in order to have a sufficient concentration of it to perform a more efficient cloning procedure.
A verification of the amplified fragment was provided by a gel electrophoresis test, where the results are presented in figure 17:

![Trulli]( https://static.igem.org/mediawiki/2021/9/95/T--UNILausanne--results_phages_gel2.png)

***Figure 17 | Electrophoresis results of the donor sequence amplification.**  The first lane on the left named “Ladder” corresponds to the molecular-weight size marker. The right lane, named “PCR prod.”, hosts the PCR amplification product. The red circle defines the molecular size of interest (*i.e.* 2 kb)*

The length of the donor sequence is 2 kb, which is the molecular size where the gel in figure 17 shows the brightest and thickest band, confirming the correct amplification of this sequence.
We digested the backbone into which an insert sequence is supposed to be cloned. In our case, the backbone opening is achieved by a double digestion, where we used two different restriction enzymes (NheI-HF and SphI-HF) to avoid relegation of the resulting sticky ends.
To confirm the efficiency of the digestion we loaded the digested samples on a gel.

![Trulli]( https://static.igem.org/mediawiki/2021/b/b5/T--UNILausanne--results_phages_gel3.png)

***Figure 18 | Electrophoresis results of the pJH1 digestion.**  The molecular-weight size marker corresponds to the left well, named “Ladder”. The well named “pJH1” hosts the original plasmid (without undergoing digestion), the same as the one used as backbone during the double digestion. The wells 1-3 correspond to three digested pJH1, all of them carrying a different sgRNA (either 1, 2 or 3)*

From figure 18, it is easily noticeable that the results are the expected ones, namely three bands at a molecular size near 8 kb appeared, which correspond to the linearised open plasmid. In addition, we observed three fainter bands at the bottom of the gel, which reflect the 337 bp sequences cut out.

After having efficiently amplified the donor sequence and properly digested the backbone, the Gibson Assembly was performed in order to insert the 2 kb (*i.e.*  the donor sequence) into the 8 kb sequence (*i.e.* the open pJH1).
We then transformed the Gibson Assembly products into electrocompetent cells (*E. coli NEB5alpha*).
The plating results are displayed in figure 19:

![Trulli](https://static.igem.org/mediawiki/2021/8/86/T--UNILausanne--Foto_per_WIKI--Results--Insertion_of_Donor_Sequence-_Figure_7_.jpeg)

***Figure 19 | Plate results.** On the left, one half (upper one) of the plate containing NEB5alpha bacteria transformed with pJH1 carrying sgRNA 1 and the donor sequence, while the bottom half hosts bacteria transformed with pJH1 carrying sgRNA 3 and the donor sequence. On the right, the upper half of the plate contains NEB5alpha transformed with pJH1 carrying sgRNA 2 and the donor sequence, while the bottom half contains the control, NEB5alpha that did not undergo transformation with pJH1*

Next, we tested 12 clones by colony PCR and ran a gel to check the presence and characteristics of the amplified fragment, directly suggesting which colony presented the donor sequence and which did not.

The electrophoresis gel is shown in figure 20.

![Trulli](https://static.igem.org/mediawiki/2021/8/85/T--UNILausanne--results_gel_phages1.png )

***Figure 20 | Electrophoresis results of the donor sequence insertion.**  Colony PCR to confirm insertion of the donor sequence. The molecular-weight size marker corresponds to the left well, named “Ladder”. Wells 1-12 correspond to each single colony out of all that grew onto the LBA+Kan50 plates. All wells host colonies that underwent Gibson Assembly in order to insert the donor sequence*

If the donor sequence is integrated, a band at a molecular size of 2’105 bp is expected, whereas if a band at 442 bp molecular size is visible, it means that the insertion did not occur properly and that the Gibson Assembly did not work for those colonies. As it is noticeable in figure 20, we obtained six colonies (*i.e.* 2-5, 8 and probably 9) with the correct insert. These PCR products were then sent for sequencing.

![Trulli](https://static.igem.org/mediawiki/2021/f/f4/T--UNILausanne--Foto_per_WIKI--Results--Donor_Sequence_copia-_FIgure_9_.jpeg)

***Figure 21 | Sequencing results aligned with pJH1 sequence carrying the donor sequence insertion in Snapgene.***

As we can see in figure 21, the sequences confirmed that the team successfully inserted the donor sequence into pJH1. However, from the sequencing analysis results, as already suggested from the electrophoresis results, it turned out that one out of the three different types of pJH1 (one per each sgRNA type) (i.g. pJH1 bearing sgRNA 3) did not integrate the donor sequence. Nevertheless, we pursued our assays with pJH1 bearing either sgRNA 1 or sgRNA 2.

Once we obtained our engineered plasmid bearing both sgRNA and the donor sequence, we properly prepared *Pseudomonas syringae* pv. *syringae B301D* for transformation via electroporation.
To test the efficacy of our construct in deleting *InaZ*, we transformed the bacteria with three lines of plasmids, two of which bore sgRNA 1 while the other carried sgRNA 2.The bacteria were then plated onto LB agar plates supplied with Kanamycin 50.
The resulting plates are shown in figure 22:

![Trulli](https://static.igem.org/mediawiki/2021/a/a9/T--UNILausanne--Foto_per_WIKI--Results--Transformed_Pss-_Figure_10_.jpeg)

***Figure 22 | Plate results.** On the left the plate containing bacteria transformed with pJH1 bearing sgRNA 1.3 (upper half) and sgRNA 1.5 (bottom half), while on the right there is the plate containing bacteria transformed with pJH1 carrying sgRNA 2.1 (bottom half) and a control containing bacteria that underwent electroporation without any plasmid*

We did colony PCRs of the grown colonies by using two primers specific to the bacterial genome to confirm the *InaZ* deletion followed by running a gel. From the size of the amplified fragment, we can directly say whether the deletion was efficiently induced or not.
The electrophoresis results are shown in figure 23:

![Trulli](https://static.igem.org/mediawiki/2021/8/85/T--UNILausanne--results_phage_gel5.png )

***Figure 23 | Electrophoresis results of the colony PCR to confirm the deletion of InaZ.** The molecular-weight size marker corresponds to the left well, named “Lad.”. Wells 1-17 correspond to each single grown colony. Well 18 hosts the template control, where no DNA was added to the PCR reaction mix. Well 19 represents another control where the bacteria did not undergo transformation with our construct*

Unfortunately, all grown colonies still carried *InaZ* in their genome. In fact, the expected PCR product confirming *InaZ* deletion would have shown a band at around 2 kb, whereas, as shown in figure 23, all our PCR products have a band at about 6 kb, which means that *InaZ* is not deleted.

As we did not manage to delete *InaZ* from the bacterial genome, additional attempts would have been made if there was more time. Besides, small changes to the protocol could be tried, for example by either shortening the recovery time after electroporation or changing the temperature at which this process occurs. However, the reason we did not achieve our goal could also come from a problem in our construct. First, we might need to test if Cas9 got expressed. We could perform a Western Blot to test Cas9 expression. Second, we could test the efficiency of the sgRNAs. We could transform another plasmid containing the target region of the sgRNAs and see if Cas9 induces a cut in this plasmid in *Pseudomonas syringae*. Third, the homologous recombination might have been low efficient in *Pseudomonas syringae*, especially when the designed homology arms are not adjacent to the cut site. We could design a donor sequence carrying homologies adjacent to the cut site, which will not completely delete *InaZ* but at least disrupt it. In addition, instead of one cut, we could cut *InaZ* twice by using two gRNAs targeting the start and the end of the gene. This might facilitate the integration of the donor sequence by the HR repair mechanism, as the sequences next to the cut are directly homologous to the ones provided by the donor sequence.

**References**

1. [1] D. Q. Zhang et al., *Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants*, In: Protein Expression and Purification, Vol. 35, No. 2, pp. 257-263, June 2004  Available from: <https://www.sciencedirect.com/science/article/pii/S1046592804000907>
2. [2] K. L. Hockett et al., *Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds*, In: Journal of Visualized Experiments, Vol. 119, 2017 Available from: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352255/>
3. Tailocins : Figures 2,4,5 created with [Biorender.com](https://biorender.com)
4. Phages : Figure 2 created with [Biorender.com](https://biorender.com)

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