iGEM 2020 — Team TUDelft

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:TUDelft/Notebook

PHOCUS

[![](https://static.igem.org/mediawiki/2020/b/b3/T--TUDelft--Logo_TUDelft.png)](https://2020.igem.org/Team%3ATUDelft)

Team

[Team Members](https://2020.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2020.igem.org/Team%3ATUDelft/Collaborations)
[Team Bonding](https://2020.igem.org/Team%3ATUDelft/Team_Bonding)

Project

[Project Description](https://2020.igem.org/Team%3ATUDelft/Description)
[Contribution](https://2020.igem.org/Team%3ATUDelft/Contribution)
[Design](https://2020.igem.org/Team%3ATUDelft/Design)
[Engineering Success](https://2020.igem.org/Team%3ATUDelft/Engineering)
[Partnership](https://2020.igem.org/Team%3ATUDelft/Partnership)
[Sustainability](https://2020.igem.org/Team%3ATUDelft/Sustainable)

Lab

[Experimental Details](https://2020.igem.org/Team%3ATUDelft/Experiments)
[Notebook](https://2020.igem.org/Team%3ATUDelft/Notebook)
[Results](https://2020.igem.org/Team%3ATUDelft/Results)

Models

[Overview](https://2020.igem.org/Team%3ATUDelft/Model)
[Phage infection and toxin production](https://2020.igem.org/Team%3ATUDelft/Model/Toxin_Production)
[2D biofilm simulation](https://2020.igem.org/Team%3ATUDelft/Model/Biofilm_Simulation)

Parts

[Overview](https://2020.igem.org/Team%3ATUDelft/Part_Collection)
[Basic parts](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section1)
[Composite parts](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section2)
[Part collection](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section3)

Human Practices

[Human Practices](https://2020.igem.org/Team%3ATUDelft/Human_Practices)
[Integrated Human Practices](https://2020.igem.org/Team%3ATUDelft/Human_Practices/iHP)
[Education & Engagement](https://2020.igem.org/Team%3ATUDelft/Education)
[Inclusion](https://2020.igem.org/Team%3ATUDelft/Inclusion)
[Proposed Implementation](https://2020.igem.org/Team%3ATUDelft/Implementation)
[Safety](https://2020.igem.org/Team%3ATUDelft/Safety)

Entrepreneurship

[Overview](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship)
[Customer](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Customer)
[Product](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Product)
[Company](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Company)
[Business Plan](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Business_Plan)

Acknowledgements

[Attributions](https://2020.igem.org/Team%3ATUDelft/Attributions)
[Crowdfunding](https://2020.igem.org/Team%3ATUDelft/Crowdfunding)
[Sponsors](https://2020.igem.org/Team%3ATUDelft/Sponsors)

Achievements

[Judging Form](https://igem.org/2020_Judging_Form?id=3407)
[Achievements](https://2020.igem.org/Team%3ATUDelft/Achievements)
[Awards and Nominations](https://2020.igem.org/Team%3ATUDelft/Awards)

#

# Notebook

* [General](https://2020.igem.org/Team%3ATUDelft/Notebook)
* [Bacteriophages](https://2020.igem.org/Team%3ATUDelft/Notebook/Bacteriophages)
* [Production and characterization of Cry7Ca1](https://2020.igem.org/Team%3ATUDelft/Notebook/Cry)
* [RNAi](https://2020.igem.org/Team%3ATUDelft/Notebook/RNAi)
* [BPUL laccase and Decarboxylase](https://2020.igem.org/Team%3ATUDelft/Notebook/BPUL)

## General

Week 24

Week 25

#### LAB TRAINING

Week 26

Week 27

### 2/7

###### Iris and Maartje:

Ampicillin stock

To obtain a 100 mg/ml Ampicillin stock, 500 mg Ampicillin was dissolved in 5 ml milliQ water and filter sterilized using a 45µm filter.

Week 28

### 8/7

###### GFP:

* Colonies grew on plate o/n.
* 10ml cultures with one colony picked from the plate [10ml cultures]
* Control (-) by inoculating with a sterile tip LB (no Ab).

### 9/7

###### GFP:

* Colonies grew on plate o/n.
* 10ml cultures with one colony picked from the plate [10ml cultures]
* Control (-) by inoculating with a sterile tip LB (no Ab).

### 13/7

###### Alicia

Plates of LB agar containing either Chloramphenicol or Ampicillin were prepared following the [[agar plate protocol]](https://2020.igem.org/Team%3ATUDelft/Experiments#general).

### 14/7

###### GFP:

2 x 10ml cultures [[Liquid starter culture]](https://2020.igem.org/Team%3ATUDelft/Experiments#general) by inoculating from the cultures stored in the fridge.

### 15/7

###### GFP:

* Cultures grew sucessfully.
* 3 minipreps per each culture [[Plasmid DNA isolation]](https://2020.igem.org/Team%3ATUDelft/Experiments#DNA).

Week 29

Week 30

Week 31

### 27/7

###### GFP:

Cultures grew successfully, no growth was seen in negative controls, indicating a successful manipulation.

### 28/7

###### GFP:

Cultures grew successfully, no growth was seen in negative controls, indicating a successful manipulation.
3x Minipreps [[Plasmid DNA isolation]](https://2020.igem.org/Team%3ATUDelft/Experiments#DNA) were done, resulting in ratios of absorbance A260/A280 below 1.8, which indicates protein contamination.

### 29/7

###### GFP:

2x 10ml cultures of GFP from colony 1 [[Liquid starter culture]](https://2020.igem.org/Team%3ATUDelft/Experiments#general) were cultured again overnight to ensure good quality stock of plasmids without contamination.

### 30/7

###### GFP:

2 x 10ml cultures [[Liquid starter culture]](https://2020.igem.org/Team%3ATUDelft/Experiments#general) by inoculating from the cultures stored in the fridge.

### 31/7

###### GFP:

Phage insert was prepared to be transformed into BL21 for phage engineering. PCR using KOD Xtreme [[KOD PCR]](https://2020.igem.org/Team%3ATUDelft/Experiments#DNA) was used to amplify with primers GFP\_1.1\_Fw & Rv, GFP\_0.6\_Fw & Rv, GFP\_4.3\_Fw & Rv using eGFP plasmid as a template from minipreps made yesterday. The 50 µl PCR products were loaded into a 1% agarose gel with Sybr [[AGAROSE GEL]](https://2020.igem.org/Team%3ATUDelft/Experiments#general).

Results indicate the expected band size amplification. A gel-purification was performed to escise the bands obtaining 85 ng/µl (GFP\_insert\_0.6), 110 ng/µl (GFP\_insert\_1.1), 93 ng/µl (GFP\_insert\_4.3) [[GEL PURIFICATION]](https://2020.igem.org/Team%3ATUDelft/Experiments#DNA).

Week 32

Week 33

Week 34

Week 35

Week 36

### 4/9

###### Alicia:

Plates of LB agar containing either Chloramphenicol, kanamycin or Ampicillin were prepared following the [[agar plate protocol]](https://2020.igem.org/Team%3ATUDelft/Experiments#general).

Week 37

### 10/9

###### Gabriela

Plate reader calibration for fluorescence, done following iGEM protocol:
![](https://static.igem.org/mediawiki/2020/6/65/T--TUDelft--Fluorescein_st_curve.png)

Week 38

Week 39

Week 40

Week 41

Week 42

## Special thanks to our sponsors!

[![TU Delft](https://static.igem.org/mediawiki/2020/5/55/T--TUDelft--TUDelft_logo.png "TU Delft")](http://tudelft.nl/en)
[![](https://static.igem.org/mediawiki/2020/f/f1/T--TUDelft--BT_Logo.jpeg "Biotechnology")](https://www.tudelft.nl/en/faculty-of-applied-sciences/about-faculty/departments/biotechnology/)
[![TU Delft Bionanoscience Department](https://static.igem.org/mediawiki/2020/1/17/T--TUDelft--BN_Logo.jpg "Bionanoscience")](https://www.tudelft.nl/en/faculty-of-applied-sciences/about-faculty/departments/bionanoscience/)
[![Faculty of Applied Sciences](https://static.igem.org/mediawiki/2020/f/ff/T--TUDelft--AS_logo.png "Faculty of Applied Sciences")](https://www.tudelft.nl/en/faculty-of-applied-sciences/)
[![TU Delft Chemical Engineering](https://static.igem.org/mediawiki/2020/b/b8/T--TUDelft--Chemicalengineering_logo.png "TU Delft Chemical Engineering")](https://www.tudelft.nl/en/faculty-of-applied-sciences/about-faculty/departments/chemical-engineering/)
[![TU Delft Bioengineering Institute](https://static.igem.org/mediawiki/2020/5/58/T--TUDelft--Bioengineering_Logo.png "TU Delft Bioengineering Institute")](https://www.tudelft.nl/bioengineering/)
[![Delft Health Initiative](https://static.igem.org/mediawiki/2020/5/5c/T--TUDelft--HealthInitiativeLogo.png "Delft Health Initiative")](https://www.tudelft.nl/health/)
[![STUD](https://static.igem.org/mediawiki/2020/6/69/T--TUDelft--STUDLogo.png "STUD")](https://www.stud.nl)
[![Simonis](https://static.igem.org/mediawiki/2020/7/75/T--TUDelft--SimonisLogo.png "Simonis")](https://www.simonisbv.nl/)
[![Simonis](https://static.igem.org/mediawiki/2020/6/6c/T--TUDelft--ErasmusLogo.jpg "Erasmus MC")](https://www.erasmusmc.nl/)
[![Fast Funds](https://static.igem.org/mediawiki/2020/9/95/T--TUDelft--FastLogo.png "Fast Funds")](www.tudelft.nl/fast)
[![ArborBiosciences](https://static.igem.org/mediawiki/2020/3/33/T--TUDelft--Arbor_Logo.jpg "ArborBiosciences")](www.arborbiosci.com)
[![De Bink](https://static.igem.org/mediawiki/2020/a/a4/T--TUDelft--De_Bink_Logo.jpg "De Bink")](https://bink.nl/)
[![Promega](https://static.igem.org/mediawiki/2020/f/f5/T--TUDelft--Promega_Logo.png "Promega")](https://promega.com/)
[![SnapGene](https://static.igem.org/mediawiki/2020/7/72/T--TUDelft--Snapgene_Logo.png "Snapgene")](http://www.snapgene.com)
[![NEB](https://static.igem.org/mediawiki/2020/9/92/T--TUDelft--NEB_Logo.jpg "NEB")](https://www.neb.com)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:TUDelft/Engineering

PHOCUS

[![](https://static.igem.org/mediawiki/2020/b/b3/T--TUDelft--Logo_TUDelft.png)](https://2020.igem.org/Team%3ATUDelft)

Team

[Team Members](https://2020.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2020.igem.org/Team%3ATUDelft/Collaborations)
[Team Bonding](https://2020.igem.org/Team%3ATUDelft/Team_Bonding)

Project

[Project Description](https://2020.igem.org/Team%3ATUDelft/Description)
[Contribution](https://2020.igem.org/Team%3ATUDelft/Contribution)
[Design](https://2020.igem.org/Team%3ATUDelft/Design)
[Engineering Success](https://2020.igem.org/Team%3ATUDelft/Engineering)
[Partnership](https://2020.igem.org/Team%3ATUDelft/Partnership)
[Sustainability](https://2020.igem.org/Team%3ATUDelft/Sustainable)

Lab

[Experimental Details](https://2020.igem.org/Team%3ATUDelft/Experiments)
[Notebook](https://2020.igem.org/Team%3ATUDelft/Notebook)
[Results](https://2020.igem.org/Team%3ATUDelft/Results)

Models

[Overview](https://2020.igem.org/Team%3ATUDelft/Model)
[Phage infection and toxin production](https://2020.igem.org/Team%3ATUDelft/Model/Toxin_Production)
[2D biofilm simulation](https://2020.igem.org/Team%3ATUDelft/Model/Biofilm_Simulation)

Parts

[Overview](https://2020.igem.org/Team%3ATUDelft/Part_Collection)
[Basic parts](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section1)
[Composite parts](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section2)
[Part collection](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section3)

Human Practices

[Human Practices](https://2020.igem.org/Team%3ATUDelft/Human_Practices)
[Integrated Human Practices](https://2020.igem.org/Team%3ATUDelft/Human_Practices/iHP)
[Education & Engagement](https://2020.igem.org/Team%3ATUDelft/Education)
[Inclusion](https://2020.igem.org/Team%3ATUDelft/Inclusion)
[Proposed Implementation](https://2020.igem.org/Team%3ATUDelft/Implementation)
[Safety](https://2020.igem.org/Team%3ATUDelft/Safety)

Entrepreneurship

[Overview](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship)
[Customer](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Customer)
[Product](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Product)
[Company](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Company)
[Business Plan](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Business_Plan)

Acknowledgements

[Attributions](https://2020.igem.org/Team%3ATUDelft/Attributions)
[Crowdfunding](https://2020.igem.org/Team%3ATUDelft/Crowdfunding)
[Sponsors](https://2020.igem.org/Team%3ATUDelft/Sponsors)

Achievements

[Judging Form](https://igem.org/2020_Judging_Form?id=3407)
[Achievements](https://2020.igem.org/Team%3ATUDelft/Achievements)
[Awards and Nominations](https://2020.igem.org/Team%3ATUDelft/Awards)

#

# Engineering success

* [Design Cycle 1](#section1)
* [Design Cycle 2](#section2)
* [Design Cycle 3](#section3)
* [Design Cycle 4](#section4)

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](https://2020.igem.org/Team%3ATUDelft/Design) and [Results](https://2020.igem.org/Team%3ATUDelft/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.

![](https://static.igem.org/mediawiki/2020/b/bd/T--TUDelft--Design-cycle.png)

*Figure 1. The seven stages of the engineering design cycle.*

We have successfully used the Engineering-Design cycle in our project for:

* [Creating engineered phages](https://2020.igem.org/Team%3ATUDelft/Part_Collection)
* [Creating and characterising new parts](https://2020.igem.org/Team%3ATUDelft/Results)

**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

![](https://static.igem.org/mediawiki/2020/4/4b/T--TUDelft--EC_Research.png)

#### Phage characteristics and engineering methods

![](https://static.igem.org/mediawiki/2020/c/c7/T--TUDelft--ArrowDown2.png)

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](https://2020.igem.org/Team%3ATUDelft/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]](#reference). A recent study shows that gregarious, field-collected, desert locusts maintain a core population of the bacterial *Enterobacter* genus [[5]](#reference).
* **Lytic/lysogenic bacteriophages:**  Bacteriophages can infect bacteria following two different life cycles: the lytic and the lysogenic cycles [[6]](#reference). 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**) [[6]](#reference). 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 [[7]](#reference).

![](https://static.igem.org/mediawiki/2020/9/96/T--TUDelft--Lytic_phage.png)

*Figure 2. The lytic life cycle of bacteriophages.*

* **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. [[9]](#reference).
* **Bacteriophage genetic material organisation:**  Gene expression of phages occurs in three stages, the early stage, middle stage and late stage [[10]](#reference). Bacteriophages contain essential and non-essential genes [[11]](#reference).

![](https://static.igem.org/mediawiki/2020/d/d3/T--TUDelft--EC_Imagine.png)

#### Model phage and how to engineer it

![](https://static.igem.org/mediawiki/2020/c/c7/T--TUDelft--ArrowDown2.png)

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)** [[12]](#reference). This method has been described to be the most efficient phage engineering method [[9]](#reference), and has already been successfully applied to engineer the T7 phage [[13]](#reference).
* **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)**  [[11]](#reference). 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](https://2020.igem.org/Team%3ATUDelft/Partnership).

![](https://static.igem.org/mediawiki/2020/b/b4/T--TUDelft--EC_Design.png)

#### Replacing a non-essential gene of the T7 bacteriophage using BRED

![](https://static.igem.org/mediawiki/2020/c/c7/T--TUDelft--ArrowDown2.png)

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.

![](https://static.igem.org/mediawiki/2020/2/21/T--TUDelft--GFP_phage_engineering.png)

**Figure 3. Scheme representing amplification of the GFP gene with primers containing homologous regions to the T7 flanking parts of the target genes. Homologous regions are represented in red.**

![](https://static.igem.org/mediawiki/2020/b/b6/T--TUDelft--EC_Test.png)
![](https://static.igem.org/mediawiki/2020/d/d0/T--TUDelft--EC_Build.png)

#### DNA template amplification and BRED

![](https://static.igem.org/mediawiki/2020/c/c7/T--TUDelft--ArrowDown2.png)

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** .

![](https://static.igem.org/mediawiki/2020/3/31/T--TUDelft--BRED.png)

*Figure 4. BRED. Double-stranded DNA (dsDNA) containing homologous regions to the bacteriophage are electroporated into cells containing bacteriophages. Recombination results in recombinant bacteriophages, containing the dsDNA fragment [[14]](#reference).*

![](https://static.igem.org/mediawiki/2020/3/35/T--TUDelft--EC_Learn.png)

#### Screening conditions and method

![](https://static.igem.org/mediawiki/2020/1/11/T--TUDelft--Arrowdown4.png)

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

![](https://static.igem.org/mediawiki/2020/0/06/T--TUDelft--Improve.png)

#### Screening conditions and method

![](https://static.igem.org/mediawiki/2020/c/c7/T--TUDelft--ArrowDown2.png)

**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.

![](https://static.igem.org/mediawiki/2020/0/04/T--TUDelft--Engin_Figure5.png)

*Figure 5. Primers designed to hybridize to the regions outside the replaced gene.*

![](https://static.igem.org/mediawiki/2020/b/b6/T--TUDelft--EC_Test.png)

#### Repeating the (improved) experiment

![](https://static.igem.org/mediawiki/2020/c/c7/T--TUDelft--ArrowDown2.png)

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.

![](https://static.igem.org/mediawiki/2020/0/02/T--TUDelft--Engin.s_plaques.png)

*Figure 6. Plaques resulting from a PFU of 10^8.*

![](https://static.igem.org/mediawiki/2020/3/35/T--TUDelft--EC_Learn.png)

#### Identifying phage detection problems

![](https://static.igem.org/mediawiki/2020/c/c7/T--TUDelft--ArrowDown2.png)

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%**  [[9]](#reference). Thus we need to do many PCRs to find the engineered bacteriophage.

## Design cycle 3: Obtaining engineered phages

![](https://static.igem.org/mediawiki/2020/3/35/T--TUDelft--EC_Learn.png)

#### Improving the primers and screening method

![](https://static.igem.org/mediawiki/2020/1/11/T--TUDelft--Arrowdown4.png)

Following the engineering principles we improved our design with two additional adjustments:- **We designed new primers**  that bind the GFP and the flanking regions.
- As the screening method with one PCR per plaque did not show results, **we screened the whole sample obtained by BRED**, as advised by Dr. Franklin L. Nobrega.

![](https://static.igem.org/mediawiki/2020/d/d0/T--TUDelft--EC_Build.png)
![](https://static.igem.org/mediawiki/2020/b/b6/T--TUDelft--EC_Test.png)

#### Repeating the (improved) experiment

![](https://static.igem.org/mediawiki/2020/c/c7/T--TUDelft--ArrowDown2.png)

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**).

![](https://static.igem.org/mediawiki/2020/2/29/T--TUDelft--Engin.S_PCRresult.png)

*Figure 7. Results of BRED targeting different non-essential genes (0.6A, 1.1, 4.3), amplification of the entire lysate obtained after electroporation.*

![](https://static.igem.org/mediawiki/2020/3/35/T--TUDelft--EC_Learn.png)

#### Phage isolation

![](https://static.igem.org/mediawiki/2020/1/11/T--TUDelft--Arrowdown4.png)

**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

![](https://static.igem.org/mediawiki/2020/4/4b/T--TUDelft--EC_Research.png)
![](https://static.igem.org/mediawiki/2020/d/d3/T--TUDelft--EC_Imagine.png)

#### Researching phage isolation methods

![](https://static.igem.org/mediawiki/2020/1/11/T--TUDelft--Arrowdown4.png)

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 [[15]](#reference) as a successful strategy when engineering the T7 phage.

![](https://static.igem.org/mediawiki/2020/b/b4/T--TUDelft--EC_Design.png)

#### Designing single-guide RNA

![](https://static.igem.org/mediawiki/2020/1/11/T--TUDelft--Arrowdown4.png)

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.

![](https://static.igem.org/mediawiki/2020/d/d0/T--TUDelft--EC_Build.png)
![](https://static.igem.org/mediawiki/2020/b/b6/T--TUDelft--EC_Test.png)

#### Repeating the (improved) experiment

![](https://static.igem.org/mediawiki/2020/1/11/T--TUDelft--Arrowdown4.png)

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.

#### If you want to learn more about other aspects of our engineering journey have a look at our [Design](https://2020.igem.org/Team%3ATUDelft/Design) and [Results](https://2020.igem.org/Team%3ATUDelft/Results) page!

References

1. Dillon, R. J., & Charnley, A. K. (1995). Chemical barriers to gut infection in the desert locust: In Vivo production of antimicrobial phenols associated with the bacterium Pantoea agglomerans. Journal of Invertebrate Pathology, 66(1), 72–75. https://doi.org/10.1006/jipa.1995.1063
2. Dillon, R., & Charnley, K. (2002). Mutualism between the desert locust Schistocerca gregaria and its gut microbiota. In Research in Microbiology (Vol. 153, Issue 8, pp. 503–509). Elsevier Masson. https://doi.org/10.1016/S0923-2508(02)01361-X
3. Dillon, R. J., Vennard, C. T., Buckling, A., & Charnley, A. K. (2005). Diversity of locust gut bacteria protects against pathogen invasion. Ecology Letters, 8(12), 1291–1298. https://doi.org/10.1111/j.1461-0248.2005.00828.x
4. Dillon, R. J., Vennard, C. T., & Charnley, A. K. (2002). A note: Gut bacteria produce components of a locust cohesion pheromone. Journal of Applied Microbiology, 92(4), 759–763. https://doi.org/10.1046/j.1365-2672.2002.01581.x
5. Lavy, O., Gophna, U., Gefen, E., & Ayali, A. (2019). The Effect of Density-Dependent Phase on the Locust Gut Bacterial Composition. Frontiers in Microbiology, 9(JAN), 3020. https://doi.org/10.3389/fmicb.2018.03020
6. Salmond, G. P. C., & Fineran, P. C. (2015). A century of the phage: Past, present and future. In Nature Reviews Microbiology (Vol. 13, Issue 12, pp. 777–786). Nature Publishing Group. https://doi.org/10.1038/nrmicro3564
7. Paul, J. H., & Jiang, S. C. (2001). Lysogeny and transduction. Methods in Microbiology, 30, 105–125. https://doi.org/10.1016/s0580-9517(01)30042-9
8. Figure 1: The phage replication cycle. | Nature Reviews Microbiology. (n.d.). Retrieved October 24, 2020, from https://www.nature.com/articles/nrmicro2315/figures/1
9. Pires, D. P., Cleto, S., Sillankorva, S., Azeredo, J., & Lu, T. K. (2016). Genetically Engineered Phages: a Review of Advances over the Last Decade. Microbiology and Molecular Biology Reviews, 80(3), 523–543. https://doi.org/10.1128/mmbr.00069-15
10. Guzina, J., & Djordjevic, M. (2015). Inferring bacteriophage infection strategies from genome sequence: Analysis of bacteriophage 7-11 and related phages. BMC Evolutionary Biology, 15(Suppl 1), S1. https://doi.org/10.1186/1471-2148-15-S1-S1
11. Abedon, S. T., The Bacteriophages - Google Books. (n.d.). Retrieved October 24, 2020, from https://books.google.nl/books?id=hYcRDAAAQBAJ&pg=PA281&lpg=PA281&dq=gp10b+t7+early+gene&source=bl&ots=urt\_oqnZvJ&sig=ACfU3U0gSCrxAIsW12oKcn68oUgtGtQEQA&hl=nl&sa=X&ved=2ahUKEwiJ5LDRi-PpAhUOKuwKHbgjA\_QQ6AEwAHoECAoQAQ#v=onepage&q=gp10b&f=false
12. Marinelli, L. J., Piuri, M., Swigoňová, Z., Balachandran, A., Oldfield, L. M., van Kessel, J. C., & Hatfull, G. F. (2008). BRED: A simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS ONE, 3(12). https://doi.org/10.1371/journal.pone.0003957
13. Nobrega, F. L., Costa, A. R., Santos, J. F., Siliakus, M. F., Lent, J. W. M. Van, Kengen, S. W. M., Azeredo, J., & Kluskens, L. D. (2016). Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine. Scientific Reports, 6. https://doi.org/10.1038/srep39235
14. Chen, Y., Batra, H., Dong, J., Chen, C., Rao, V. B., & Tao, P. (2019). Genetic engineering of bacteriophages against infectious diseases. In Frontiers in Microbiology (Vol. 10, Issue MAY, p. 954). Frontiers Media S.A. https://doi.org/10.3389/fmicb.2019.00954
15. Kiro, R., Shitrit, D., & Qimron, U. (2014). Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system. RNA Biology, 11(1), 42–44. https://doi.org/10.4161/rna.27766

## Special thanks to our sponsors!

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# Team:TUDelft/Experiments

PHOCUS

[![](https://static.igem.org/mediawiki/2020/b/b3/T--TUDelft--Logo_TUDelft.png)](https://2020.igem.org/Team%3ATUDelft)

Team

[Team Members](https://2020.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2020.igem.org/Team%3ATUDelft/Collaborations)
[Team Bonding](https://2020.igem.org/Team%3ATUDelft/Team_Bonding)

Project

[Project Description](https://2020.igem.org/Team%3ATUDelft/Description)
[Contribution](https://2020.igem.org/Team%3ATUDelft/Contribution)
[Design](https://2020.igem.org/Team%3ATUDelft/Design)
[Engineering Success](https://2020.igem.org/Team%3ATUDelft/Engineering)
[Partnership](https://2020.igem.org/Team%3ATUDelft/Partnership)
[Sustainability](https://2020.igem.org/Team%3ATUDelft/Sustainable)

Lab

[Experimental Details](https://2020.igem.org/Team%3ATUDelft/Experiments)
[Notebook](https://2020.igem.org/Team%3ATUDelft/Notebook)
[Results](https://2020.igem.org/Team%3ATUDelft/Results)

Models

[Overview](https://2020.igem.org/Team%3ATUDelft/Model)
[Phage infection and toxin production](https://2020.igem.org/Team%3ATUDelft/Model/Toxin_Production)
[2D biofilm simulation](https://2020.igem.org/Team%3ATUDelft/Model/Biofilm_Simulation)

Parts

[Overview](https://2020.igem.org/Team%3ATUDelft/Part_Collection)
[Basic parts](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section1)
[Composite parts](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section2)
[Part collection](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section3)

Human Practices

[Human Practices](https://2020.igem.org/Team%3ATUDelft/Human_Practices)
[Integrated Human Practices](https://2020.igem.org/Team%3ATUDelft/Human_Practices/iHP)
[Education & Engagement](https://2020.igem.org/Team%3ATUDelft/Education)
[Inclusion](https://2020.igem.org/Team%3ATUDelft/Inclusion)
[Proposed Implementation](https://2020.igem.org/Team%3ATUDelft/Implementation)
[Safety](https://2020.igem.org/Team%3ATUDelft/Safety)

Entrepreneurship

[Overview](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship)
[Customer](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Customer)
[Product](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Product)
[Company](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Company)
[Business Plan](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Business_Plan)

Acknowledgements

[Attributions](https://2020.igem.org/Team%3ATUDelft/Attributions)
[Crowdfunding](https://2020.igem.org/Team%3ATUDelft/Crowdfunding)
[Sponsors](https://2020.igem.org/Team%3ATUDelft/Sponsors)

Achievements

[Judging Form](https://igem.org/2020_Judging_Form?id=3407)
[Achievements](https://2020.igem.org/Team%3ATUDelft/Achievements)
[Awards and Nominations](https://2020.igem.org/Team%3ATUDelft/Awards)

#

# Experimental details

* [General](#general)
* [DNA](#DNA)
* [Proteins](#proteins)
* [Phages](#phages)
* [RNA](#RNA)

## General

Primers

[List of primers](https://static.igem.org/mediawiki/2020/4/45/T--TUDelft--PHOCUS_Primers.pdf)

Agar plate

1. Dissolve 14.24 g of LB-agar in 400 mL of milli-Q.
2. Autoclave the LB-agar solution.
3. Cool the agar down to room temperature. To facilitate the process, you can hold the bottle under the cold water tap.
4. Add the correct amount of the correct antibiotic. For example, for our antibiotic stock solutions we used 1 µL of antibiotic solution/mL of LB-agar.
5. Under sterile conditions, pour the LB-agar in empty Petri dishes.
6. Leave the Petri dishes near the flame with the lid partially off for approximately 5 minutes so the agar can solidify.

Antibiotic stock solution

For 1000X antibiotic stock solution:

1. Weigh X grams of the specific antibiotic of interest.
2. Resolve in 95% ethanol (Chloramphenicol) or MilliQ (other antibiotics)

Glycerol stocks

Bacterial stock with glycerol

1. Incubate ~50 µl cells in 5 ml LB and selection marker overnight
2. Pipette 1,5 ml in eppendorf and spin down the bacterial culture
3. Repeat step 2 till enough pellet has been formed (about 3ml of bacterial culture)
4. Resuspend the pellet in 0.9 ml liquid bacterial culture
5. Add the liquid bacterial culture from eppendorf to cryotube
6. Add 0.9 ml 50% glycerol to the cryo tube and mix well
7. Store at -80°C

Liquid medium

LB medium preparation

1. Dissolve Luria Broth powder in water according to instructions by the manufacturer.
2. Heat sterilize (121 °C) the medium in the autoclave.
3. After cooling down, add the required antibiotic in sterile conditions.
*NOTE: With our antibiotic stock solutions of 1000x we used 1µL of antibiotic solution per mL of LB-agar.*4. Store the medium at 4 °C if complemented with antibiotics.

Liquid starter culture

1. Label as many 15mL Falcon tubes and mark well: date, content of tube including organism and insert, name.
2. In sterile conditions from now on, pipette 5ml - 10mL of liquid medium (LB or SOC) for each starter culture.
*NOTE: Supply with relevant antibiotics.*3. Pick a colony with the inoculation loop and swirl it in the starter culture to inoculate.
4. Grow the liquid cultures at 37 °C overnight, shaking at 250rpm.)

Making electrocompetent cells

1. Thaw your cells on ice
2. Streak the cells on a plate with the corresponding antibiotics and let it grow overnight at 37 °C
3. Inoculate 30 mL LB with picked colonies from the plate in a 50 mL Greiner tube.
4. Grow till an OD of ~0.5
5. Centrifuge for 10 minutes at 4 °C at 3900 rpm.
6. Discard the supernatant and dissolve pellet in 20 mL cold milli-Q.
*NOTE: make sure all the cells are resuspended!*7. Centrifuge for 10 minutes at 4 °C at 3900 rpm.
8. Discard the supernatant and dissolve pellet in 20 mL cold milli-Q.
9. Centrifuge for 10 minutes at 4 °C at 3900 rpm.
10. Discard supernatant and resuspend in 200 µL glycerol
11. Prepare aliquots of 50 µL
12. Either transform straight away or store at -80 °C

Primer working stock

Based on protocol given by [Integrated DNA Technologies (IDT)](https://eu.idtdna.com/pages).

1. Centrifuge the tube containing the primer for 3−5 seconds (>3,000 x g) to pellet the material to the bottom of the tube.
2. Dissolve the DNA material in sterile MilliQ according to the supplier IDT. This creates a 100µM stock solution.
3. Heat the primer stock solution to 65 °C for 20 minutes.
4. Centrifuge the primer stock solution at maximum speed (~17,000 x g) for 2 minutes.
5. Prepare a 10X diluted work solution (10µM) by dilution with sterile MilliQ.
6. Store both Stock and Work solutions at -20 °C.

Sequencing samples

1. Prepare sample in 1.5mL tube; mix DNA template and primer: 5μL template + 5μL primer in each tube or well. Concentrations of sample vary depending on type of sample. See table:

2. | Sample type | Concentration [ng/µl] | Minimum volume [µl] |
   | --- | --- | --- |
   | Plasmid | 100 | 5 |
   | Purified PCR product | <300bp → 10-15, 300-700bp → 25-50, >700bp → 50 | 5 |
   | Primer | 60 | 5 |
3. Label (EZ-seq label)
4. Submit sample

## DNA

DNA concentration determination

Using NanoDrop UV-VIS Spectrophotometer.

1. Press the button for dsDNA to measure the concentration of double-stranded DNA in your samples.
2. Clean the measurement surface with a piece of tissue and ethanol.
3. Use 1-1.5 µL of sterile milli-Q as a blank.
4. Clean the measurement surface with a piece of tissue and water.
5. Use 1-1.5 µL of sample to measure its concentration.
*NOTE: It is best to measure the same sample in triplo and use the average value.*6. If you have multiple samples, clean the measurement surface in between measurements.

DNA electrophoresis

1. Prepare TAE buffer: take the 10X concentrated TAE from the chemicals cabinet and dilute it 10 times with milli-Q. For 500 mL, add 50 mL to 450 mL of dH2O.
2. Weigh agarose for a 1% gel. For 200 mL, 2 g of agarose is necessary.
3. Mix the TAE solution with the agarose and heat the solution (in a microwave) until it is completely dissolved.
4. Add SYBR Safe to the gel mould. For a small gel (40 mL of the prepared TAE/agarose mixture) add 4 µL of SYBR Safe; for a large gel (100 mL) add 10 µL of SYBR Safe.
5. Pour the solution into the mould, making sure there are no bubbles and that the SYBR Safe is completely mixed. Add a comb to create wells for the samples. Let it solidify (approx. 20 minutes).
6. Transfer the gel to the electrophoresis cell minding the arrow that indicates the direction of DNA migration. Remove the combs and cover it with TAE.
7. Prepare the electrophoresis samples (on parafilm); 1 µL of Nucleic Acid Loading Buffer per 5 µL of sample.
8. Load the molecular weight marker (ladder) in the first well (check the appropriate volume for each marker, generally 5 µL works fine) and load 5-10 µL of the samples in the other wells, according to the order in your lab journal.
*NOTE: Do not contaminate the loading buffer and ladder with SYBR Safe! Do not touch it while wearing a glove.*9. Connect the cables following the colour code and run at 100-130 V for 30-60 min.

DNA gel purification & PCR cleanup (Promega Wizard kit)

1. Excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge.
2. Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and
3. Incubate at 50–65°C until the gel slice is completely dissolved.
4. Insert SV Minicolumn into Collection Tube.
5. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn
6. assembly. Incubate at room temperature for 1 minute.
7. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
8. Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g
   for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
9. Repeat Step 9 with 500μl Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
10. Empty the Collection Tube and centrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

Genome extraction

Golden Gate assembly

1. Set up 25 µl assembly reactions as follows:

   | Reagent | Quantity |
   | pGGA Destination plasmid, 75 ng/μL | 1μL |
   | 24 precloned inserts cloned into pMiniT 2.0, 100ng/μl each | 0.75μl each (18μL total) |
   | T4 DNA ligase buffer (10X) | 2.5 μL |
   | T4 DNA ligase, 1000 U/μL | 0.5 μL |
   | Bsal-HFv2, 20U/μL | 1.5 μL |
   | Nuclease-free water | 1.5 μL |
   - Mix gently by pipetting up and down 4 times.
   - Briefly microcentrifuge (1 sec.) to bring material to the bottom of tube.
   - Transfer to thermocycler and program as follows: (5 min 37°C → 5 min 16°C) x 30 cycles followed by 5 min 60°C. If reactions are done overnight, add a 4°C terminal hold to the protocol, but repeat the final 5 min 60°C step the next day before the transformations.
   - Perform transformation .

Ligation

1. Thaw the ligase buffer on ice, to prevent damaging the ATP.
2. Prepare a sample as follows:

   | Component | Volume (µL) |
   | --- | --- |
   | 10X Ligase buffer | 2 |
   | T4 DNA Ligase | 1 |
   | DNA vector | x (~100ng) |
   | DNA fragment | Y\* |
   | MilliQ | Z |
   | Total | 20 |
3. Incubate for at least one hour at 4 °C. Optimally, incubate overnight at 4 °C.

NEBuilder HiFi DNA assembly

1. The following on ice:

   |  | Recomended amounts |  |
   | --- | --- | --- |
   |  | Assembly of 2-3 fragments | Assembly of 4-6 fragments |
   | Vector:insert ratio | 1:2 | 1:1 |
   | Fragments | 0.03-0.2pmol (XµL)) | 0.2-0.5 pmol (XµL) |
   | NEBuildier HiFi DNA assembly Master mix | 10µL | 10µL |
   | Dionized water | 10-X µL | 10-X µL |
   | Total | 20µl | 20µl |
2. Incubate at 50 degrees C for 15 minutes for 2-3 segments; one hour for 4-6 fragment
3. Store at -20 degrees C.

PCR (GoTaq)

1. Add the following components in a PCR tube:

| Component | Volume (µl) |
| --- | --- |
| PCR grade water | X |
| 5x Green GoTaq buffer | 10 |
| PCR Nucleotide Mix, 10mM each | 1 |
| 1µM Primer FW | 1 |
| 1µM Primer RV | 1.5 |
| GoTaq DNA Polymerase (5.0 U/µL) | 0.25 |
| TemplateDNA (<0.5µg/50µl) | Y |
| Total | 50 |

2. Perform PCR with thermocycle:

| Step | Temperature (°C) | Time | #cycles |
| --- | --- | --- | --- |
| Initial denaturation | 95 | 2 minutes | 1 |
| Denaturation | 95 | 30 sec | 25-35 |
| Annealing | 42-65 (based on Tm) | 30 sec |  |
| Extension | 72 | 1 minute/kb |  |
| Final extension | 72 | 5 | 1 |
| Hold | 12 | ∞ |  |

PCR (KOD Xtreme™)

1. Add the following components for a KOD Xtreme™ PCR in a PCR tube:

| Component | Volume (µl) |
| --- | --- |
| PCR grade water | X |
| 2x KOD Xtreme™ PCR Buffer | 25 |
| 2 mM dNTPs | 10 |
| 10pmol/µL Primer FW | 1.5 |
| 10pmol/µL Primer RV | 1.5 |
| KOD Xtreme™ Hot Start DNA Polymerase (1.0 U/µL) | 1 |
| Genomic DNA (200ng) | Y |
| Plasmid DNA (50 ng) | Z |
| Total | 50 |

2. Perform PCR with three-step thermocycle:

| Step | Temperature (°C) | Time |
| --- | --- | --- |
| Initial denaturation | 94 | 2 minutes |
| Denaturation | 98 | 10 sec |
| Annealing | Tm minus 5 | 30 sec |
| Extension | 68 | 1 minute/kb |
| Hold | 12 | ∞ |

PCR (Q5 High-fidelity 2x)

1. Add the following components in a PCR tube:

| Component | Volume (µl) |
| --- | --- |
| PCR grade water | X |
| Q5 High-Fidelity 2x Master Mix | 25 |
| 10µM Primer FW | 2.5 |
| 10µM Primer RV | 2.5 |
| TemplateDNA (< 1,000ng) | Y |
| Total | 50 |

2. Perform PCR with thermocycle:
3. Perform PCR with three-step thermocycle:

| Step | Temperature (°C) | Time | #cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 seconds | 5-10 seconds |
| Denaturation | 98 | 5-10 sec | 25-35 |
| Annealing | 50-72 (based on Tm) | 10-30 sec |  |
| Extension | 72 | 20-30 seconds/kb |  |
| Final Extension | 72 | 2 minutes | 1 |
| Hold | 12 | ∞ |  |

PCR&DNA clean-up (NEB Monarch)

1. Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex.

| Sample type | Ratio of binding; buffer:sample |
| --- | --- |
| dsDNA < 2 kb (plasmids, gDNA) | 2:1 |
| dsDNA &st; 2 kb (some amplicons, fragments) | 5:1 |
| ssDNA\* < 200 nt | 7:1 |

2. Insert column into collection tube and load sample onto column.
3. Spin for 1 minute, then discard flow-through.
4. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
5. Repeat step 4.
6. Transfer column to a clean 1.5 ml microfuge tube.
7. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix.
8. Wait for 1 minute, then spin for 1 minute to elute DNA.
9. Label tube and store at -20 degrees C

Plasmid DNA isolation (Promega PureYield)

#### Prepare Lysate

1. Transfer 1.5mL of bacterial culture to sterile 1.5 ml microcentrifuge tube(s). Centrifuge at max speed for 5 minutes. Discard supernatant.
2. Add additional 1.5mL and repeat step 1.
3. Add 600µL sterile water to the pellet and resuspend.
4. Add 100 µL of Cell Lysis Buffer (blue), and mix by inverting the tube 6 times.
5. Add 350 µl of cold (4–8 °C) Neutralization Solution, and mix thoroughly by inverting.
6. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
7. Transfer the supernatant (~900 µl) to a PureYield™ Minicolumn without disturbing the cell debris pellet.
8. Place the minicolumn into a Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds.
9. Discard the flowthrough, and place the minicolumn into the same Collection Tube.

#### Wash

10. Add 200 µl of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds
11. Add 400 µl of Column Wash Solution (CWC) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.

#### Elute

12. Transfer the minicolumn to a clean 1.5 ml microcentrifuge tube, then add 30 µl of sterile milli-Q directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
13. Centrifuge for 15 seconds to elute the plasmid DNA.
14. Determine plasmid DNA concentration. Store at -20 °C.

Restriction

1. Decide on which restriction enzyme(s) to cut with. Check online (NEB) what buffer the enzyme(s) work(s) in . For most of the restriction enzymes, the CutSmart buffer 10X can be used.
2. Prepare a 20µL sample as follows:

| Component | Volume (µL) |
| --- | --- |
| 10X CutSmart buffer (NEB) | 2 |
| Fragment (~1-2μg) | X |
| Restriction Enzyme(s) | 1 (per enzyme) |
| MilliQ | Y |
| Total | 20 |

3. Incubate for 4 hours at 37 °C.
4. Inactivate the restriction enzyme(s) by heating to 65 °C for 10 minutes.

Transformation of chemically competent cells

1. Aliquot competent cells into 50 µL. Thaw on ice for 10-15 min.
2. Add 1-100ng plasmid DNA (1-5µL) to the 50 µL of competent cells. Mix by flicking the tube 4-5 times. Incubate on ice for 30 minutes.
3. Heat shock at 42 °C for exactly 10 seconds. Then place on ice for 5 minutes.
4. Add 350µL of fresh, warmed (approx. 37 °C) LB or SOC-medium.
5. Incubate at 37°C with shaking for 1 hour.
6. Plate 50µL of the cells out on pre-warmed (approx. 37 °C) LB-agar plates with the correct antibiotic to select. Remaining liquid can be plated on another plate.
7. Incubate at 37 °C overnight. Alternatively, you can incubate over the weekend, leaving the plate on the bench.

Transformation of electrocompetent cells

1. Thaw 20 ul of electrocompetent cells on ice
2. Add 1-2 ul of DNA to the cells and incubate on ice +/- 1 min
3. Transfer the cell-DNA mixture to the ice-cold electroporation cuvette
4. Put the cuvette in the electroporator and press start (settings: 2500 V).
*NOTE: if the lid does not fit, put parafilm instead.*5. Immediately add 950 ul of SOC medium and pipette up and down
6. Transfer to an eppendorf tube and incubate for 1-1.5 h at 37ºC 800 rpm.
7. Plate the cells on LB plates with the required antibiotic:
   * 50 µl of cells
   * Spin down the rest 3 min at max rpm, discard supernatant and plate.
8. Incubate the plates at 37ºC overnight.

## Proteins

HiLink™ Spin Protein Purification System (Promega)

Preparation of FasBreak™ reagent/DNase I solution

1. Add 80µl of Nuclease-Free Water to the vial of DNase I.
2. Mix completely to dissolve the powder.
3. Remove the DNase solution from the vial and add it to 1ml of Nuclease-Free Water. Mix well.
4. Prepare FasBreak™ reagent/DNase I solution by mixing 5.8µl DNase I solution with 64.2µl FastBreak™ reagent, 10X. This is the quantity for one reaction.

Centrifugation protocol

5. Pipet 700µl of bacterial culture into a 1.5ml microcentrifuge tube. Add 70µl of the FastBreak™ Reagent/DNase I solution to the culture.
6. Transfer 75µl of the HisLink™ Resin from the settled resin bed to the 1.5ml microcentrifuge tube (pipet slowly and deep into the settled resin).
7. Incubate the sample and resin for 30 minutes on a shaking platform.
8. Place a Spin Column onto a Collection Tube (or a new 1.5ml microcentrifuge tube). Transfer the lysate and resin from the original 1.5ml microcentrifuge tube to the spin column.
9. Centrifuge the spin column with the collection tube for 5 seconds or until the liquid clears the spin column.
10. Place the spin column back onto the collection tube. Add 500µl of HisLink™ Binding/Wash Buffer to the spin column, then cap the spin column. Centrifuge for 5 seconds or until the Binding/Wash Buffer clears the spin column. Discard the flowthrough. Repeat for a total of two washes.
11. Take the spin column off the collection tube and wipe the base of the spin column with a clean absorbent paper towel to remove any excess HisLink™ Binding/Wash Buffer.
12. Place the spin column onto a new 1.5ml microcentrifuge tube. Add 200µl of HisLink™ Elution Buffer. Cap the spin column and tap or flick it several times to resuspend the resin. Wait 3 minutes.
13. Centrifuge the spin column and microcentrifuge tube at 14,000rpm for 1 minute to collect the eluted protein.

Protein expression

All work was performed within a sterile field created by a bunsen burner flame.

1. Grow culture containing (plasmid) DNA coding for protein of interest as described in liquid starter culture protocol.
2. Measure the OD600 to confirm growth
3. Refresh starter culture in 150mL LB with required antibiotics and let the culture grow until an OD600 of 0.4-0.6. Induce protein expression by adding IPTG.
4. Grow the cells for 16 hours or 18 °C and 180rpm rotation. Sample every hour if needed - check OD600 standardize sample to OD600 of 1.
5. Harvest the liquid culture by centrifugation at max speed for 15 minutes.
6. Store pellet in -20°C

SDS-PAGE precast gels

1. Add 5 µL sample buffer (SDS) to 20 µL protein sample.
2. Heat at 95 °C for 10 min.
3. Load 5µL boiled sample to precast gel in electrophoresis cassette. Gel is covered in TAE buffer.
4. Run Gel at 150 V for 50 min
5. Remove gel from plastic casing, wash with milliQ. Repeat wash process.
6. Pour coomassie stain over the gel and leave gel shaking for 10 min.
7. Discard stain, cover gel in demi-water and leave to destain for a couple of hours.

Western blot

#### Prepare buffers for Western Blotting:

1. Transfer buffer (keep at room temperature) with end concentrations of:
   * 25mM Tris base
   * 102mM glycine
   * 20%methanol
   * 0.05% SDS
2. 1x TBS-T
   * 10mM Tris pH 7.5
   * 150mM NaCl
   * 0.1% Tween-20: 100mL pre-made 10xTBS + 1mL Tween-20
3. Anti-His-HRP antibody (10mL Clonetech)
   * 1x TBS-T
   * 5% milk (2.5g skim milk powder with 50mL 1x TBS-T)

#### Run SDS-PAGE gel

#### Transfer protein from gel to membrane (semi-dry-blotting):

1. Soak 2x filter paper and gel in transfer buffer
2. Soak PVDF membrane in 100% methanol, followed by soaking it in water, then transfer buffer; alternatively, use cellulose membrane soaked in transfer buffer
3. Stack: filter bottom paper, PVDFmembrane, gel (from SDS-PAGE; facing down!), filter paper on top
4. Roll out bubbles with glass pipette
5. Attach top electrode plate
6. Transfer 40min at 15V

#### Develop blot with Supersignal West pico kit (Pierce)

1. Block the blot with Anti-His-HRP antibody buffer for one hour to overnight. Discard
2. Incubate the blot with primary antibody (His) diluted 1:5000 (10µL in 50mL) in TBS-T and 5% milk.
3. Wash four times 10 minutes with 1xTBS-T on an orbital shaker at room temperature.
4. Incubate the blot with secondary antibody (HRP) diluted 1:100000 in TBS-T for 45 minutes at room temperature. Discard.
5. Wash four times 10 minutes with 1xTBS-T on an orbital shaker at room temperature.
6. Prepare Working Solution by mixing equal parts of the Stable Peroxide Solution and the Luminol/Enhancer Solution. Use 0.1 mL Working Solution per cm2 of membrane.
7. Incubate the blot in Working Solution for 5 minutes.
8. Remove the blot from Working Solution and drain excess reagent.
9. Place the blot in clear plastic wrap or sheet protector and remove bubbles.
10. Expose the blot to X-ray or imaging system.

## Phages

BRED

#### Part 1. Construction of recombineering substrates: Preparing double-stranded DNA (dsDNA) (gene of interest + overhangs) by performing KOD Xtreme PCR

#### Part 2. Preparation of recombineering competent *E.coli* BL21 cells: Add plasmid

1. Grow *E.coli* BL21 cells in LB medium to mid-log phase in 100 ml LB: OD600 between 0.4 and 0.6
2. Harvested by centrifugation (3000 × g, 4 °C, 15 min).
3. Make cells electrocompetent:
   * Concentrate culture 500-fold. Wash three times with ice-cold sterile 10% (v/v) glycerol, and suspended in a final volume of 80–300 μL.
4. Electroporate 100–500 ng of pKD46 into aliquots of 20 μL electrocompetent cells at 1.8 kV and 25 pF using a Gene Pulser XCell Microbial System (BioRad).
5. Recover cells with SOC and incubate for 1–2 h at 37 °C, 200 rpm.
6. Spread cells in LB agar plates with Ampicillin (LB-Amp) and incubated overnight at 30 °C.
7. Pick transformed *E. coli* BL21::pKD46 colonies and grow in fresh LB-Amp medium for a few hours.
8. Do plasmid extraction (NucleoSpin® Plasmid (NoLid) kit, Macherey-Nagel) and digestion with the restriction enzyme BamHI-HF (New England Biolabs) to confirm the presence of pKD46.

#### Preparation of electrocompetent *E.coli* BL21(DE3)::pKD46 cells with T7

1. Pick a colony of BL21(DE3)::pKD46 cells and grow them in 5-10 ml LB-amp O/N at 30°C
2. Grow BL21(DE3)::pKD46 in 100mL LB-amp medium till early log-phase (OD600 ±0.4) at 30°C
3. Induce the expression of lambda Red by pKD46 for 30 min with L-arabinose (0.1 % final concentration)
4. Infect the cells with T7 at a multiplicity of infection (MOI) of 1-3 for the eclipse time (10 min.)
*MOI = (phage volume x phage titer)/ amount of cells* 5. Divide the culture over 4x50 ml tubes and centrifuge at 4°C for 15 minutes at 3000 x g. Discard the supernatant.
6. Resuspend all the pellets in a total of 20 ml ice-cold 10% glycerol and centrifuge again for 15 minutes. Discard the supernatant.
7. Repeat the washing step with 10 and 5 ml ice-cold 10% glycerol.
8. Resuspend the pellet in 500µL ice-cold 10% glycerol and divide in aliquots of 100µl

#### Electroporation and recovery

1. Add 100-500 ng of the dsDNA substrate to 100µL of the electrocompetent cells at 1.8 kV and 25 pF. Check time constant → should be >18 ms (19-21 ms is best)
2. Recover the cells in 900 µl SOC medium for 1–2 h at 37 °C, 200 rpm.
3. Mix the suspension with 3 mL soft agar and 100µL of an *E.coli* BL21(DE3) culture.
4. Pour this mixture onto LB plates and incubate overnight at 37 °C

#### Recovery and confirmation of mutant phages

1. Pick 18-25 individual plaques into 100µl phage buffer;
2. Incubate at room temperature for 1-2 hr or at 4°C overnight; store plaques at 4°C.
3. Use 1 µl of each plaque to use as a template for PCR with the flanking primers or primers specific to your insert.
4. Use WT phage DNA (5-10 ng) as a control
5. When you have found a positive plaque, serial dilute this plaque until 10^-7 in phage buffer and infect 300 µL BL21(DE3) with 10µL of each dilution. Add 2-3 ml top-agar and pour it over a bottom LB layer.
6. Incubate overnight, pick at least 18 secondary plaques and screen by PCR. In addition, dissolve the plate with more than 1000 plaques in 3-5 ml phage buffer, incubate for at least 1 hr, collect the buffer in a sterile tube. Also screen this lysate for engineered phages. Repeat step 5 and 6 until you obtain a pure sample with engineered phages, confirmed by PCR using flanking primers.
7. When a pure mutant plaque is confirmed, replate and obtain a lysate from the plate containing a web pattern. Filter sterilize the lysate.

#### Confirmation PCR

1. Follow the PCR protocol for GoTaq polymerase with 35 cycles.

#### Preparation of phage buffer

1. Prepare 5M NaCl by dissolving 29.22g NaCl in 100ml Milli Q water and 0.2um filter solution into a sterile container.
2. Prepare 1M Tris-Cl, pH7.4, by dissolving 7.882g Tris-HCl in 90ml Milli Q water, adjusting pH to 7.4 with 1N NaOH, bringing final volume to 50ml and filter sterilizing through a 0.2um filter into a sterile container.
3. Prepare 1M MgSO4 by dissolving 12.3g MgSO4-7H2O in 50ml Milli Q water and filter sterilize through 0.2um filter into sterile container
4. Mix together 3.0ml 5M NaCl, 4.0ml 1M Tris-Cl, pH 7.4, and 1.0ml 1M MgSO4 in 92ml sterile Milli Q water in a sterile container.
5. Filter solution through a 0.02um Anotop syringe filter into a sterile container.
6. Final phage buffer will be 150mM NaCl, 40mM Tris-Cl, pH 7.4, 10mM MgSO4.

Bacteriophage selection method

Goal: The goal of these experiments is to select the engineered phages from the pool with wild type and engineered phages. For this CRISPR Cas9 will be used.

#### In Vitro gRNA design:

1. Design primers for cloning your protospacer (target) into the pKDsgRNA plasmid. The protospacer must precede a PAM site of NGG on either strand.
   * Add this sequence to the 3’ end of the forward primer - gttttagagctagaaatagcaag
   * Add this sequence to the 3’ end of the reverse primer - gtgctcagtatctctatcactga

#### pKDsgRNA plasmid construction:

1. Do two PCR reactions.
   * To amplify part 1: use your designed F primer with this primer: tttataacctccttagagctcga
   * To amplify part 2: use your designed R primer with this primer: ccaattgtccatattgcatca

#### Co-transformation of Cas9 plasmid and pKDsgRNA

1. Add at least 10ng of each plasmid to 20 µl electrocompetent cells.
   * Mix plasmids well in advance.
2. Follow the protocol for transformation of electrocompetent cells. Recover the cells in SOC medium at 30 degrees C
3. Plate the cells on LB plates with a selection marker spectinomycin (Spec) and chloramphenicol (Cam) at 30 degrees C.

#### Selection of engineered phages

1. Grow three colonies overnight in LB-Spec/Cam (60 µg/ml and 35µg/ml) at 30C
2. Put the bacteria to grow in LB-Spec/Cam 30 µg/ml and 17.5 µg/ml final concentration at 30C until OD600=0.5
3. At OD = 0.5 , add arabinose (0.75% final concentration) and aTC (100 ng/ml), to induce λ-Red recombinase and pCas9. Incubate for an additional 30 minutes.
4. Make serial dilutions of the phage stock solution containing engineered phages.
5. Plate 10µl of each dilution with 100µl bacteria in LB top-agar supplemented with arabinose (0.75% final concentration) and aTC (100 ng/ml) on LB-Spec/Cam bottom layer.
6. Incubate overnight at 30C.
7. Pick the plaques and screen for the mutation by PCR.

Phage infectivity assay

Aim: To evaluate potential changes in the mutant’s phage infectivity.
The latent period, eclipse time and burst size of the phages can be determined from the one step growth curve as described by Ellis and Delbrück (1939) [1].

Equipment

* 0.6% top agar
* LB agar plates
* 37℃ stove
* 37℃ shaker
* BL21(DE3)
* T7 wild type
* Phage buffer (1x PBS, 1mM MgCl2, 1mM MgSO4

#### Protocol

Day1:

* Put BL21(DE3) to grow O/N at 37℃ 150 rpm

Day2:

* Grow 2 ml BL21(DE3) in 210 ml LB at 37℃, 150 rpm until an OD600 of 0.35.
* Divide the culture over two erlenmeyers, make sure to have in each erlenmeyer exactly 100 ml culture.

Do the following steps in duplo (in each erlenmeyer with 100 ml culture)

* Add wild type T7 or mutant phages at an MOI of 0.001. I.e. add 10µl of phage stock of 10^10 pfu/ml.
* Incubate for 5 minutes at 37℃ 150 rpm
* Take a sample, measure OD600 and immediately filter it with a 0.22 or 0.45 µm filter and ten times serial dilute in phage buffer until 10^-8
  + This will be your t=0 sample and adsorption control
* Take a sample every 5 minutes for 90 minutes, measure OD600 and ten times serial dilute your sample in a phage buffer until 10^8.
* Incubate the dilutions at RT for 1-2 hours and store them at 4℃

Day 3:

* Aliquot 38 times 3 ml of 0.6% top agar, keep it warm so the agar does not solidify.
* Add 300µl of fresh BL21(DE3) to each tube, mix well and immediately pour it over an LB agar plate
* Incubate at RT for 20 minutes.
* Divide each plate into 8 sections. Mark each section with the correlating dilution. Spot 5 µl of each dilution of 1 time point taken on day 2 on one plate. Let the spots dry and incubate the plates at 37℃ for 2-3 hours.
* Determine the pfu/ml for each time point by counting the plaques on each plate.
  + E.g. at t=20 you see 8 single plaques at the spot with the 10^8 dilution sample, this means that the pfu at t=20 was 8/5µl \*10^8 pfu/µl = 1.6 \*10^8 pfu/µl = 16 \* 10^10 pfu/ml.

[1] Ellis, E. L., & Delbrück, M. (1938). Supplemental Information 2: NCBI genome database accession information (PDF file). The Journal of General Physiology. doi:10.7717/peerj.6447/supp-2

Phage plaque size over time

With this experiment we want to compare the plaque size and the appearance of the plaque. Virulent strains or lytic lead to more clear plaques, thus the virulence of different phages can be compared with this experiment. In addition we can compare the speed that virions diffuse as this is indicated by the plaque size.

#### Equipment

* 0.6% top agar
* LB agar plates
* 37℃ stove
* 37℃ shaker
* BL21(DE3)
* T7 wild type
* Phage buffer (1x PBS, 1mM MgCl2, 1mM MgSO4)

#### Protocols

Day 1:

* Inoculate BL21(DE3) in fresh medium for an overnight culture at 37℃ 150 rpm.

Day 2:

* Ten times serial dilute the T7 wildtype phage stock until 10^1, 10^2 and 10^3 pfu/ml.
* Aliquot 3 times 3 ml of liquid 0.6% top agar in 25 ml tubes, keep them in hot water to prevent the top agar from solidifying.
* Add 300µl of fresh BL21(DE3) overnight culture to the top agar (make sure the agar is not too hot). Immediately add 100µl of phage dilution and pour it over the LB agar plates.
* Incubate the plates at RT for 20 minutes.
* Incubate the plates at 37℃ for 2 hours
* Count and measure plaques size
* Measure plaque size.
  + After around 2 hours plaques will appear, from then onwards measure every 30 minutes and take a picture (Exact plaque formation time may vary for different phages).

## RNA

qPCR for RNA

1. Perform standard curve:
   * Prepare dilution with 2.75µM, 0.918µM, 0.306µM, 0.102µM, 0.034µM RNA (5 data points over 3 orders of magnitude, always below 100ng RNA)
     + To go to the wished pM range, these dilutions should be diluted 105 times. To do this with the least possible error, samples should be diluted first 2 times by 100. In order to perform these dilutions with the least possible error, two separated dilutions should be performed in which 3 µl of Starting dilution X will be added to 297µl of MQ water
2. Prepare following solutions

| Mix 1 (One mix per RNA sample) | Volumes for 1 reaction |
| --- | --- |
| Reverse primer | Variable |
| RNA template (from highest dilution) | Variable |
| RNase-free H2O | Up to 10 µl of the final reaction (see **Table 1**) |
| Final volume Mix 1 | Variable |
| Mix 2 (One common mix for all) | Volumes for 1 reaction |
| Forward primer | Variable |
| Power SYBR Green RT-PCR Mix (2X) | 5µl |
| RT-enzyme mix (125X) | 0.08 µl |
| Final volume Mix 2 | Variable |
| Final reaction volume | Final volume Mix 1 + Final volume Mix 2 = 10 µl |

Standard qPCR protocols demand to incubate the Reverse primer with the RNA at 65°C for 5 min inside a regular PCR machine in order to manage the binding between them. Mix 1 should always be prepared first and incubated in the mentioned conditions in PCR tubes. Right after, Mix 1 should be let at room temperature for 10 minutes. Preferably, Mix 2 should be prepared while Mix 1 is resting at room temperature.

![](https://static.igem.org/mediawiki/2020/b/be/T--TUDelft--Power-interest-grid.png)
3. Set thermal profile in PCR for standard curve:

| Stage | Step | Temp | Time |
| --- | --- | --- | --- |
| Holding | Reverse transcription | 48 °C | 30 min |
| Holding | Activation of AmpliTaq Gold DNA Polymerase, UP (Ultra Pure) | 95 °C | 10 min |
| Cycling (40 cycles) | Denature | 95 °C | 15 sec |
|  | Anneal/Extend | 60°C | 1 min |
| Melt curve (Optional) | Denature | 95°C | 15 sec |
|  | Anneal | 60°C | 15 sec |
|  | Denature | 95°C | 15 sec |

RNA electrophoresis

#### Preparation of the samples:

Work RNase-free! Treat your workspace with RNaseZAP prior to your experiment and work with gloves. Don't talk while handling your samples!

1. Prepare the electrophoresis samples in 0.2 mL PCR tubes; 2 µL of 2X RNA Loading Buffer per 2 µL of sample (check the appropriate preparation for each marker).
2. Put your samples at 90 °C for 5 minutes.
3. Leave your samples on ice for 2 minutes. Store your samples at 4 °C when not loaded directly on the gel

#### Preparation of the gel:

Work clean! Handle all material labelled as EtBr contaminated with gloves. Don't take it outside of the EtBr area and don't touch anything that is not labelled as EtBr contaminated with gloves.

1. Prepare TBE buffer: take the 10X concentrated TBE from the chemicals cabinet and dilute it 10 times with milli-Q.
2. Weigh agarose for a 2% gel. For 100 mL, 2 g of agarose is necessary.
3. Mix the TBE solution with the agarose and heat the solution (in a microwave) until it is completely dissolved.
4. Add 10,000 X SYBR Safe to the solution and mix well. Meaning that for a large gel of 100 mL you have to add 10 µL of SYBR Safe.
5. Pour the solution into the mould, making sure there are no bubbles. Add a comb to create wells for the samples. Let it solidify (~10 minutes).

#### RNA electrophoresis:

1. Transfer the gel to the electrophoresis cell minding the arrow that indicates the direction of DNA/RNA migration. Remove the combs and cover it with TBE-buffer.
2. Load the RNA ladder in the first well (check the appropriate volume for each marker) and load 4 µL of the samples in the other wells, according to the order in your lab journal.
3. Connect the cables following the colour code and run at 80 V for 60 min.

RNA isolation (RNeasy MiniElute)

1. Adjust the sample to a volume of 100 µL with nuclease free water.
2. Add 250 µL of absolute ethanol and mix well by pipetting.
3. Immediately(!) transfer the sample to an RNeasy MinElute spin column placed in a 2 mL collection tube. Centrifuge for 15 s at 8000 x g.
4. Place the RNeasy MinElute spin column in a new 2 mL collection tube and add 500 µL RPE buffer. Centrifuge for 15 s at 8000 x g.
5. Discard the flow-through and add 500 µL of 80% ethanol to the RNeasy MinElute spin column. Centrifuge for 2 minutes at 8000 x g.
6. Place the RNeasy MinElute spin column in a new 2 mL collection tube and centrifuge for 5 minutes at full speed.
7. Place the RNeasy MinElute spin column in a new 1.5 mL collection tube and add 14 µL nuclease free water. Centrifuge for 1 minute at full speed, your RNA is now in your tube.

RNA production (T7 RiboMax Promega)

Urea PAGE

#### Denatures secondary DNA and RNA

Preparation chamber

1. Assemble the gel according to manufacturer's description and fix the gel in the gel-casting chamber. Use 0.5-1.5 mm thick spacers.
2. Prepare the appropriate polyacrylamide solution. Use ultrapure urea and mix with the desired amount of acrylamide. Add TBE buffer to the gel mix to get a final concentration of 0.5-1 x TBE and fill up the volume with deionized, distilled water. Heat the solution for 20 seconds in the microwave and mix it gently.
3. Pour the gel immediately using a serological pipette and an automatic pipette aid between the two glass plates. Avoid introducing air bubbles.Insert the comb and let the gel polymerize for 30-60 minutes.
4. Dismount the gel from the casting chamber and assembly it to the gel apparatus
5. Fill the lower buffer chamber with running buffer (0.5-1 x TBE) that the glass plates will be submerged 2-3 cm with buffer. Fill the upper buffer chamber up to the top of the gel with running buffer.
6. Carefully remove the comb and rinse the wells with running buffer by using a pipette and gel loading tips.
7. Attach the lid of the gel system and plug in the cables to a high voltage power supply. Before you can load your samples you have to pre run the gel for at least 30 minutes to heat the gel up and to remove remaining urea from the gel. The optimal temperature should be between 45-55 °C.

Preparation samples

1. Add 2 x gel loading mix to your sample.
2. Denature the samples between 70-90 °C for a few minutes.

Run gel

1. Rinse pockets thoroughly
2. Transfer the samples into pockets.
3. Run gel at constant voltage

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# Team:TUDelft/Results

PHOCUS

[![](https://static.igem.org/mediawiki/2020/b/b3/T--TUDelft--Logo_TUDelft.png)](https://2020.igem.org/Team%3ATUDelft)

Team

[Team Members](https://2020.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2020.igem.org/Team%3ATUDelft/Collaborations)
[Team Bonding](https://2020.igem.org/Team%3ATUDelft/Team_Bonding)

Project

[Project Description](https://2020.igem.org/Team%3ATUDelft/Description)
[Contribution](https://2020.igem.org/Team%3ATUDelft/Contribution)
[Design](https://2020.igem.org/Team%3ATUDelft/Design)
[Engineering Success](https://2020.igem.org/Team%3ATUDelft/Engineering)
[Partnership](https://2020.igem.org/Team%3ATUDelft/Partnership)
[Sustainability](https://2020.igem.org/Team%3ATUDelft/Sustainable)

Lab

[Experimental Details](https://2020.igem.org/Team%3ATUDelft/Experiments)
[Notebook](https://2020.igem.org/Team%3ATUDelft/Notebook)
[Results](https://2020.igem.org/Team%3ATUDelft/Results)

Models

[Overview](https://2020.igem.org/Team%3ATUDelft/Model)
[Phage infection and toxin production](https://2020.igem.org/Team%3ATUDelft/Model/Toxin_Production)
[2D biofilm simulation](https://2020.igem.org/Team%3ATUDelft/Model/Biofilm_Simulation)

Parts

[Overview](https://2020.igem.org/Team%3ATUDelft/Part_Collection)
[Basic parts](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section1)
[Composite parts](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section2)
[Part collection](https://2020.igem.org/Team%3ATUDelft/Part_Collection#section3)

Human Practices

[Human Practices](https://2020.igem.org/Team%3ATUDelft/Human_Practices)
[Integrated Human Practices](https://2020.igem.org/Team%3ATUDelft/Human_Practices/iHP)
[Education & Engagement](https://2020.igem.org/Team%3ATUDelft/Education)
[Inclusion](https://2020.igem.org/Team%3ATUDelft/Inclusion)
[Proposed Implementation](https://2020.igem.org/Team%3ATUDelft/Implementation)
[Safety](https://2020.igem.org/Team%3ATUDelft/Safety)

Entrepreneurship

[Overview](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship)
[Customer](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Customer)
[Product](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Product)
[Company](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Company)
[Business Plan](https://2020.igem.org/Team%3ATUDelft/Entrepreneurship/Business_Plan)

Acknowledgements

[Attributions](https://2020.igem.org/Team%3ATUDelft/Attributions)
[Crowdfunding](https://2020.igem.org/Team%3ATUDelft/Crowdfunding)
[Sponsors](https://2020.igem.org/Team%3ATUDelft/Sponsors)

Achievements

[Judging Form](https://igem.org/2020_Judging_Form?id=3407)
[Achievements](https://2020.igem.org/Team%3ATUDelft/Achievements)
[Awards and Nominations](https://2020.igem.org/Team%3ATUDelft/Awards)

#

# Results

* [1. Bacteriophages as delivery vectors](#Section1)
* [2. Production and characterization of locust toxic molecules](#Section2)
* [3. RNA interference (RNAi)](#Section3)

## 1. Bacteriophages as delivery vectors

PHOCUS is a biopesticide consisting of a cocktail of bacteriophages that will be engineered to express molecules toxic to locusts after infection of their target locust gut bacteria. We have used the T7 bacteriophage as the phage model and  *Escherichia coli* (*E. coli*) BL21 (DE3) as the host model for phage engineering, as bacteria present in the locust gut belong to the same genus as *E. coli,* the  *Enterobacter genus*  [Dr. O. Lavy, personal interview]. We engineered the T7 bacteriophage replacing a phage non-essential gene with a GFP reporter gene, that would ultimately be substituted by our desired locust toxic molecule.

#### Phage engineering

##### Introduction

To engineer a phage to encode toxic molecules to locusts, we first investigated whether we could successfully engineer bacteriophages by replacing non-essential genes. We identified the three non-essential T7 genes as possible candidates: 0.6A (early gene), 1.1 (early gene) and 4.3 (middle gene)  [[1]](#reference). In order to determine which of these genes should be substituted with our recombinant gene, we performed  **proof of concept experiments using enhanced GFP del6 (229) (eGFP) as a reporter** , substituting each of the target T7 non-essential genes separately.

The non-essential genes were replaced by a double-stranded DNA (dsDNA) construct, coding for enhanced GFP, through Bacteriophage Recombineering of Electroporated DNA (BRED), designed by Marinelli et al. [[2]](#reference). This construct was designed with 100 nucleotides homologous to flanking regions on each side of these aforementioned non-essential genes. As the first product of BRED results in a mixture of wild-type and engineered phages [[2]](#reference), a CRISPR/Cas9 system was designed and expressed in  *E. coli*  BL21 (DE3) as described by Kiro et al [[3]](#reference) to speed up the selection process for the engineered phage. Cas9 is an endonuclease guided by single-guide RNA (sgRNA) that can be encoded on a plasmid [[4]](#reference). The plasmid encoding the sgRNA was designed to contain a protospacer of the wild type T7 genes 0.6A, 1.1 and 4.3. Guided by this sgRNA, Cas9 binds to the target sequence and makes a double stranded break at this certain position, resulting in the cleavage of the T7 wild-type phage DNA.

##### Results

BRED substrate
The dsDNA substrates encoding eGFP were constructed by PCR amplification of plasmid pUC57-OriLR-deGFP. The expected size of the amplified fragment was 878 bp; composed of 678bp from eGFP and 200bp from the homologous regions. **Figure 1** shows the amplified PCR fragments, all of them at the expected 878bp size.

![](https://static.igem.org/mediawiki/2020/b/b7/T--TUDelft--dsDNAsubstrate.png)

*Figure 1. 1% agarose gel with PCR product of the amplification of pUC57-OriLR-deGFP to obtain the dsDNA substrates for BRED. Each lane from left to right: L: DNA ladder used is the Smartladder (Eurogentec). Three lanes on the right show PCR product of eGFP with flanks homologous to those of genes 0.6A, 1.1 and 4.3 respectively. Three lanes contain a band of size ~900bp*

Confirmation of the gene replacement
The phage samples obtained after BRED were screened by PCR for the presence of the insert, using both internal and external primers (**Figure 2A**). The external PCR products of the BRED samples were of the same size as the product from the wildtype phage, indicating that the wild-type phages were still abundant in the phage samples after BRED . The internal PCR products did show amplification of DNA at each position, because the internal primer only anneals to GFP, therefore showing that  **there were engineered phages present in each sample after BRED.** A second band was shown at position 0.6A and 1.1 of unexpected size. Due to these results, it was decided to continue working with the sample containing T7 engineered at position 4.3. (**Figure 2B**). Additionally, the iGEM team from Austin advised us to continue experiments with T7 engineered at gene 4.3, as, in the T7 phage, early genes do not have a strong promoter and that upstream terminators can hinder expression of later genes downstream ([see Partnership](https://2020.igem.org/Team%3ATUDelft/Partnership)).

To select for the engineered phage, the BRED sample was plated on *E. coli* BL21 (DE3) with an induced CRISPR/Cas9 system targeting the wild-type gene 4.3 expecting a higher concentration of the engineered phage in the resulting plaques. This transformed host bacteria contained the pKDsgRNA\_4.3 plasmid containing the sgRNA targeting the T7 gene 4.3 [(BBa\_K3407025)](http://parts.igem.org/Part%3ABBa_K3407025), as well as the Cas9 plasmid pCas9-CR4 [[5]](#reference). Plaques were screened with primer binding inside the eGFP gene (**Figure 2A, subsection I**), resulting in one positive plaque (**Figure 2C, line 4**). The positive plaque was replated on *E. coli* BL21 (DE3) with an induced CRISPR/Cas9 system targeting the wild-type gene 4.3 and picked, as well as lysed for screening with primer binding inside the eGFP gene (**Figure 2A, subsection I**) (**Figure 2D**). Amplification of most plaques with those primers showed DNA bands, however, not at the expected size of 727 bp, but around 300 bp (**Figure 2D**). Some of the positive plaques obtained were screened for the presence of wild-type T7 phage by using both external and internal primers (**Figure 2E**). This amplification showed high concentrations of amplified DNA of approximately 600 bp, the expected size for the wild type phage. This indicates that the secondary plaques still contained high concentration of the wild type phage. We were not able to confirm the presence of the eGFP by sequencing.

![](https://static.igem.org/mediawiki/2020/c/c4/T--TUDelft--Results_Overview_phages.png)

*Figure 2. T7 bacteriophage engineering replacing gene 4.3 by eGFP using BRED. A) Representation of the amplification of the engineered region of T7 with external primers marked as E, and one internal and one external primer marked as I. B) PCR screening of BRED samples for replacement of non-essential genes 0.6A, 1.1 and 4.3 (lanes 2 through 7) using internal and external primers. Lane 8 (wt) shows amplification of the entire lysate of T7 wild-type obtained after electroporation without the addition of dsDNA substrate. C) PCR Screening of primary plaques obtained after plating the sample of BRED on  *E. coli*  expressing a Cas9 system targeting gene 4.3 using internal primers. D) PCR Screening of secondary plaques and secondary lysate (P) with internal primers. E) PCR Screening of secondary plaques with both internal and external primers. Each gel consisted of 1% agarose, the DNA ladder used is the Smartladder (Eurogentec).*

##### Conclusion

BRED was performed to replace non-essential genes 0.6A, 1.1 and 4.3 of the wild type T7 phage by eGFP,  **successfully obtaining positive engineered phages** in each of the positions after screening by PCR. T7 phages engineered in the 4.3 gene position were re-plated further in order to obtain isolated engineered phages, as the wild type phage was still present in the samples. Further PCR screening should be done to isolate the T7 phage engineered at position 4.3, using the constructed *E. coli BL21* (DE3) host strain containing the CRISPR/Cas9 system targeting the 4.3 gene of the wild-type T7 phage.

##### Future prospects

Future experiments will include the characterisation of GFP expression of an isolated T7 bacteriophage engineered containing eGFP in the 4.3 gene position. This would provide information about the expression levels of a gene replacing the non-essential native 4.3 gene. The ultimate goal would include engineering the T7 bacteriophage containing GFP, substituting the eGFP with our desired locust toxic molecules.

#### Validation of the Phage and Toxin Production model

In [Model 1- Phage and Toxin Production](https://2020.igem.org/Team%3ATUDelft/Model/Toxin_Production), the change of the number of bacteria, phages and toxin production over time were modeled. The objective of this model was to investigate if enough toxin could be produced by the infected gut bacteria to kill the locust in less than 7 days. To validate one part of the model, the biomass (OD600) of susceptible bacteria upon infection as well as the phage titer (P) were measured over time. After cells had grown to an OD of 0.3, measurements were taken every 5 minutes. This experiment was done in singlet. The obtained experimental profiles (**Figure 3**) show clear similarities to the predicted concentration profiles obtained with the model in terms of trend and value. This demonstrates that **our equations and parameters are a fair representation of reality**. An elaborate analysis of the data obtained from these experiments can be found [here](https://2020.igem.org/Team%3ATUDelft/Model/Toxin_Production#section2).

![](https://static.igem.org/mediawiki/2020/9/90/T--TUDelft--model1_wetlab_int.png)

*Figure 3. Experimental validation. a) Total cell concentration and b) bacteriophage concentration against time. The concentration is marked as a blue line for the model data and red dots for the experiments data.*

#### Partnership iGEM Vienna

iGEM team of BOKU-Vienna has also worked with T7 bacteriophage for their project. Their aim was also to engineer the T7 bacteriophage using BRED, but replacing another gene: the g10 gene. We decided to design experiments to compare the effect of the replacement of these different genes and the use of these different phages on the infectivity of the phage.

The experiments were performed using the wild-type phages of each team as no engineered phages were created and isolated in time. Both teams conducted experiments determining the **phage titer** needed to clear a liquid culture of susceptible bacteria ([infection dose assay](https://static.igem.org/mediawiki/2020/4/47/T--TUDelft--Partnership_Delft_Infectiondose.pdf)) to determine the infectivity of the phages. In addition, a [plaque assay](https://static.igem.org/mediawiki/2020/8/81/T--TUDelft--Partnership_Delft_Plaqueassay.pdf) was done wherein the **diameter of the plaque** size was measured over time to compare the diffusion speed and virulence of the phages. We obtained different results for both experiments and we concluded that the experiments should be repeated to find out what caused this difference. More elaborate results from both teams can be found in the [Partnership](https://2020.igem.org/Team%3ATUDelft/Partnership) page.

## 2. Production and characterization of locust toxic molecules

Our approach to killing locusts is based on a unique combination of the insecticidal activity of the crystal toxin Cry7Ca1 and the gene silencing effect of RNA interference (RNAi). Cry7Ca1 punctures the locust gut lining, harming the locust and allowing the RNAi precursors to reach the hemolymph, where they mature and silence the expression of vital locust genes.

#### Cry7Ca1

##### Introduction

The Cry7Ca1 protein is a crystal protein from *Bacillus thuringiensis* (Bt) strain BTH-13 that has been reported to have activity against the locust species *Locusta migratoria manilensis* [[6]](#reference). This protein turns into an activated toxin when cleaved by insect gut proteases. The Cry7Ca1 activated toxin is more efficient against *L. migratoria* than the protoxin [[6]](#reference). In our study, An **expression cassette was designed for the Cry7Ca1 activated toxin, using the viral promoter and terminator from T7**, the model phage used in phage engineering experiments. The goal of these experiments was to confirm the expression of our composite part [BBa\_K3407017,](http://parts.igem.org/Part%3ABBa_K3407017) that allows expression of the Cry7Ca1 under viral T7 promoter and terminator. The toxin was designed to be expressed with an N-terminal His-tag with a thrombin cutting site, giving a protein molecular weight of 68.5 kDa. This would allow the further removal of the His-tag for characterization experiments, as it would be inserted in the engineered phage without this tag.

##### Results

Optimal conditions for Cry7Ca1 toxin overexpression
In order to check the overexpression of the Cry7Ca1 toxin, we incubated the *E. coli* BL21 (DE3) (Negative control) and *E. coli* BL21 (DE3) transformed with the plasmid pTWIST\_Cry7Ca1 containing the Biobrick [BBa\_K3407017](http://parts.igem.org/Part%3ABBa_K3407017) at 37ºC until the OD600 reached approximately 0.6A. In order to find the best induction conditions, we added different IPTG concentrations (0.5 and 1 mM) and incubated the cultures for 4h and overnight. The total protein content of the cells and the soluble proteins obtained after cell lysis were analysed by SDS-PAGE electrophoresis (**Figure 4**).

![](https://static.igem.org/mediawiki/2020/8/85/T--TUDelft--Figure4_Cry_overexpression.png)

*Figure 4. SDS-PAGE to verifyCry7Ca1 overexpression at different IPTG conditions. A) Total protein content. B) Soluble proteins after lysis using FastBreak™ Cell Lysis Reagent (Promega). *E. coli* (DE3) is the negative control, induced with the higher IPTG concentration used in this experiment (1 mM). *E. coli* BL21 (DE3) Cry7Ca1 contains the plasmid pTWIS\_Cry7Ca1 ([BBa\_K3407017](http://parts.igem.org/Part%3ABBa_K3407017)), and it is induced with 0.5 mM IPTG and 1 mM IPTG. The non-induced sample is a negative control to check leaky expression. MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight). All the samples used corresponded to the same OD600.*

**Figure 4** shows **successful overexpression of Cry7Ca1 toxin** with all IPTG concentrations used, as a band corresponding to the molecular weight of the Cry7Ca1 toxin can be observed in samples from *E. coli*BL21 (DE3) Cry7Ca1 (**Figure 4A**). After lysis with FastBreak™ Cell Lysis Reagent (Promega), bands corresponding to the size of the Cry7Ca1 toxin are also observed (**Figure 4B**), although at lower intensity. These results indicated that a final IPTG concentration of 0.5 mM is enough for the overexpression of our BioBrick. They also suggest that our protein may not be completely soluble after lysis, as the fraction of soluble protein recovered seems lower than that of the total overexpressed amount. Moreover, we noticed a leaky expression of our protein, as a band was observed after an overnight culture of *E. coli* BL21 (DE3) pTWIST\_Cry7Ca1 without inducer. This may be due to the absence of a repressor in our constructed plasmid. In order to avoid leaky expression, another host could be used such as *E. coli* BL21 (DE3) pLysS. The [BBa\_K3407017](http://parts.igem.org/Part%3ABBa_K3407017) could also be modified to include a repressor such as the lacI gene.

Purification of Cry7Ca1 toxin
We purified the Cry7Ca1 using the identified IPTG concentration for overexpression. As the recombinantly expressed Cry7Ca1 toxin contains a His-tag at the N-terminal part, we purified it using affinity chromatography with the HisLink protein purification system (Promega). All samples obtained from the purification process were analysed by SDS-PAGE electrophoresis (**Figure 5**).

![](https://static.igem.org/mediawiki/2020/e/e5/T--TUDelft--Figure5_Cry_purified2.png)

*Figure 5. SDS-PAGE of Cry7Ca1 toxin purification by affinity chromatography.  *E. coli*  BL21 (DE3) is the negative control, and  *E. coli*  BL21 (DE3) Cry7Ca1 contains the pTWIS\_Cry7Ca1 ([BBa\_K3407017](http://parts.igem.org/Part%3ABBa_K3407017)). MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), ON (overnight), FT (flow-through), W (Washing), E (Elution). All the samples used corresponded to the same OD600.*

A clear band of the expected size was obtained in the elution sample (**Figure 5**), indicating that the **Cry7Ca1 toxin has successfully been purified**. The fact that the toxin protein is also present in the flow through sample suggests that further optimization is possible.

##### Conclusion

From these results, we conclude that the Cry7Ca1 toxin is overexpressed in *E. coli* cells after induction with 0.5 mM IPTG (although there is some leaky expression). In this expression conditions, the Cry7Ca1 toxin can be further **successfully purified** by affinity chromatography thanks to its N-terminal Histag.

##### Future prospects

The next step in our project would be to encode the Cry7Ca1 toxin in the T7 phage genome to see whether this protein can be expressed upon infection of the target bacteria. As the toxin can be successfully purified, it could also be used for in vitro assays to examine its toxicity against different insect gut cells, including gut bacteria of our target locust species, the desert locust (*Schistocerca gregaria*).

## 3. RNA interference (RNAi)

##### Introduction

RNA interference (RNAi) is a eukaryotic intracellular defence against viral infections and transposable elements triggered by double-stranded RNA (dsRNA) [[7]](#reference). RNAi will be used against locusts swarms by designing short hairpin RNA (shRNAs) containing a short sequence of **dsRNA targeting locust vital genes. Dicer** is the cytoplasmic enzyme in charge of producing siRNA from the shRNA, the first step of the siRNA-mediated gene silencing.

The components of a theoretical fusion protein ([see Design - Future perspectives](https://2020.igem.org/Team%3ATUDelft/Design#section7)) were designed to **deliver the shRNA produced in the locust gut into the hemolymph** where dsRNA causes a strong gene silencing. A component of this cargo is Fox-1 RNA binding domain (Fox-1 RBD), which was chosen to bind specifically our shRNAs by the loop of the hairpins. Theoretically provided with a fused transporter peptide (TMOF), Fox-1 RBD will deliver the shRNA across the locust gut’s epithelium into the hemolymph, triggering ubiquitous RNAi response. In principle, shRNAs would be transcribed in tandem (tshRNA) and **mini-3**, the dsRNA counterpart of classical dsDNA restriction enzymes, will cut its target sequence located at the base of each individual hairpin, detaching them from the transcript. This cleavage produces a GG overhang in the 3’ end.

We aim to prove our concepts by:

* Testing whether **in vitro transcription by T7 RNA polymerase can efficiently generate shRNAs** with strong secondary structures. A shRNA targeting GFP was used as a model ([BBa\_K3407022](http://parts.igem.org/Part%3ABBa_K3407022)), and the same shRNA with two point mutations in the loop (G6A and C3U) was used as a control of later experiments ([BBa\_K3407023](http://parts.igem.org/Part%3ABBa_K3407023)).
* Expressing and purifying **RNA binding domains (RBD) from Fox-1**: wild type ([BBa\_K3407020](http://parts.igem.org/Part%3ABBa_K3407020)) and mutated less active version ([BBa\_K3407024](http://parts.igem.org/Part%3ABBa_K3407024)). With this we can further test the binding activity of Fox-1 RBD when compared to a mutated inactive version.
* Testing whether the  **Fox-1 RBD domain ([BBa\_K3407020](http://parts.igem.org/Part%3ABBa_K3407020)) binds specifically to our shRNA**.
* Showing that  **our designed shRNAs ([BBa\_K3407022](http://parts.igem.org/Part%3ABBa_K3407022)) with GG 3’ overhang is a good substrate for Dicer**, therefore being able to perform the first enzymatic conversion from the RNAi pathway.

##### Results

Fox-1 RBD and RBD\* (mutated) overexpression and purification
In order to check the overexpression of Fox-1 RBD and its mutated version, we incubated the *E. coli* BL21 (DE3) (Negative control) and *E. coli* BL21 (DE3) transformed either with the pBbB7a\_Fox-1\_RBD ([BBa\_K3407020](http://parts.igem.org/Part%3ABBa_K3407020)) or pBbB7a\_Fox-1\_RBD\* ([BBa\_K3407024](http://parts.igem.org/Part%3ABBa_K3407024)) at 37ºC until the OD600 reached ~ 0.6. We induced the cultures with a final IPTG concentration of 1 mM and incubated them 4h at 37ºC and overnight at 30ºC. The total protein content of the cells was analysed by SDS-PAGE electrophoresis (**Figure 6A**). As the recombinantly expressed Fox-1 RBD and RBD\* (mutated) contain a His-tag at their N-terminal part, we purified them using affinity chromatography with the HisLink protein purification system (Promega). Samples obtained from the different purification steps were analysed by SDS-PAGE electrophoresis (**Figure 6B**).

![](https://static.igem.org/mediawiki/2020/8/8f/T--TUDelft--Figure6_FOX2.png)

*Figure 6. Purification of Fox-1 RBD and mutated Fox-1 RBD\*. A) SDS-PAGE gel of the overexpression samples before and after induction with IPTG. B) SDS-PAGE of samples from the different purification steps. *E. coli BL21* (DE3) is the negative control, *E. coli BL21* (DE3) Fox-1 RBD contains the part [BBa\_K3407020](http://parts.igem.org/Part%3ABBa_K3407020) and *E. coli BL21* BL21 (DE3) Fox-1 RBD contains the part [BBa\_K3407024](http://parts.igem.org/Part%3ABBa_K3407024). MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight), FT (flow-through), W1 and W2 (Washing), E (Elution). All the samples used corresponded to the same OD600.*

Additional protein bands corresponding to molecular weights of 13.6 kDa (Fox-1 RBD) and 13.4 kDa (Fox-1 RBD\*, mutated) were observed after induction (**Figure 6A**), indicating that **both proteins were successfully overexpressed**. Moreover, a clear band is obtained in the elution samples (E) (**Figure 6B**), demonstrating that both Fox-1 RBD and RBD\* were successfully purified. These two purified cargo proteins were subsequently used in Electrophoretic Mobility Shift Assay (EMSA) to verify binding with in vitro produced shRNA.

shRNA can be transcribed with T7 RNA polymerase
Hairpin structures can hamper transcription, causing premature termination. This is the case for the T7 viral RNA polymerase, whose commonly used terminator is a hairpin itself [[8]](#reference). To test whether T7 RNA polymerase is able to transcribe the designed shRNAs, two types of hairpins with GG overhangs were produced: one with the Fox-1 binding sequence in its loop, denoted as shRNA ([BBa\_K3407022](http://parts.igem.org/Part%3ABBa_K3407022)), and a second one, named shRNA\* ([BBa\_K3407023](http://parts.igem.org/Part%3ABBa_K3407023)) that contains two point mutations in the loop (G6A and C3U), which reduces its affinity with Fox-1 RBD [[9]](#reference).

For each short hairpin, a pair of complementary primers were annealed to form a DNA template for transcription (see [Design page](https://2020.igem.org/Team%3ATUDelft/Design)). The DNA template was designed to possess a T7 promoter followed by a 27nt inverted repeat sequence taken from the eGFP gene (nt 78 to 105), and linked together by the 9nt sequence containing the target of Fox-1 RBD. On the 3’ termini, two GG were added to produce the desired overhang.

![](https://static.igem.org/mediawiki/2020/6/69/T--TUDelft--shRNA_Resultsl.jpeg)

*Figure 7. Designed short-hairpin RNAs (shRNA). A) Scheme about the different parts of the designed shRNA. B) Urea-PAGE analysis of transcription products from T7 RiboMAX™ Express RNAi System. Primers were annealed as recommended in the kit, and transcription was performed at 42ºC for 2 hours. DNAse treatment was performed as indicated in the kit and the product was purified with MinElute RNA columns (Qiagen). shRNA ([BBa\_K3407022](http://parts.igem.org/Part%3ABBa_K3407022)), shRNA\* ([BBa\_K3407023](http://parts.igem.org/Part%3ABBa_K3407023))*.

The commercial kit T7 RiboMAX™ Express RNAi System (Promega) containing the T7 RNA polymerase was used to produce the shRNA hairpins (**Figure 7**). The expected sizes of the transcript are ~65bp for ssRNA when denatured, and ~32,5bp for dsRNA with its native hairpin structure if the loop is assumed to be part of the dsRNA (which represents an approximation). We would expect the dsRNA ladder to be denatured into ssRNA in the conditions of the gel. On the other hand, shRNA bands are located near 30nt, which indicates the gel denaturing conditions might not have been enough to undo the strong secondary structures of the hairpins, making them unable to move in the gel as their expected ~65 bp ssRNA but rather as ~32,5bp. This phenomenon is commonly seen when studying hairpins in Urea-PAGE assays [[10]](#reference). We conclude both **shRNA variants have successfully been transcribed in vitro** and they can be used for binding assays with the Fox-1 RBD protein and Dicer processing experiments.

Fox-1 RBD binds to shRNA

To study RNA-protein interaction, an **Electrophoretic mobility shift assay (EMSA)** was performed . When a protein binds to RNA, the complex migrates slower in the gel, shifting the RNA band to a higher position. shRNA ([BBa\_K3407022](http://parts.igem.org/Part%3ABBa_K3407022)) and Fox-1 RBD ([BBa\_K3407020](http://parts.igem.org/Part%3ABBa_K3407020)) were first incubated in 1:1 and 1:2 molar ratios shRNA:Fox in binding buffer. 400ng of RNA were loaded in each lane corresponding to 10 fmol, along with 10 fmol of Fox-1 RBD (1:1 ratio) or 20 fmol (1:2 ratio) in a total of 15 μL reaction. To demonstrate binding specificity, the same experiment was performed with Fox-1 RBD\* ([BBa\_K3407024](http://parts.igem.org/Part%3ABBa_K3407024)), a mutated version of Fox-1 with a reduced affinity for its binding sequence. In addition, the assays were also carried out with shRNA\* ([BBa\_K3407023](http://parts.igem.org/Part%3ABBa_K3407023)), a version of the shRNA with two mutations in the loop, which inhibits binding to Fox-1 RBD [[9]](#reference) (**Figure 8**). Samples were then loaded in a native polyacrylamide gel to avoid protein denaturation (**Figure 9**).

![](https://static.igem.org/mediawiki/2020/b/b6/T--TUDelft--EMSA_Expected.jpeg)

*Figure 8. Illustration of Fox-1 domains used for the assay and their expected interaction with the target sequence of the shRNA or shRNA\*. Mutations in either proteins or RNA are depicted as red dots. Mutated sequences of RNA or proteins are colored in orange, while wt protein domains Fox-1 RBD and wt binding sequence in the loop are colored in green.*

![](https://static.igem.org/mediawiki/2020/b/bd/T--TUDelft--EMSA_sucess.jpeg)

*Figure 9. EMSA assay of Fox-1 RBD binding to shRNA. Stars (\*) indicate mutated versions of the molecules, where sh\* is an shRNA with two mutations in the loop region recognised by Fox-1 RBD (G6A and C3U), and Fox\* is the RBD of Fox-1 with two mutations (F160A and F126A) rendering it unable to recognise the RNA target sequence. Control (-) is shRNA incubated in the same conditions without Fox. Incubation was performed in the binding buffer (10 mM Tris pH 7,5; 1 mM EDTA; 100 mM KCl; 0.1 mM DTT; 5% Glycerol; 0.01 mg/ml BSA) in different molar ratios at room temperature for one hour. Electrophoresis with 19:1 (Acrylamide:Bisacrylamide) 12% gel in TAE buffer. Electrophoresis was run at 120 V for about 3 hours in a TAE 1x buffer, stained with Sybr Safe staining solution (0.1% vol./vol. Sybr Safe in TAE buffer).*

The non-bound shRNA and shRNA\* appear on the gel as ~30bp dsRNA (negative control, **Figure 9**). Incubation of shRNA and ahRNA\* with wild-type Fox-1 RBD leads to a notable shift of the RNA band at both 1:1 and 1:2 molar ratios. This upper band corresponds to the shRNA-Fox-1 RBD complex, showing that both purified compounds are active. The lower fraction of free shRNA in the 1:2 molar ratio indicates that increasing the concentration of Fox-1 RBD yields a higher amount of complex formed.

Interestingly, incubation of shRNA with mutated Fox\* RBD does not lead to complex formation. This results corroborates literature data showing that **the introduced mutations strongly reduce the affinity of the protein to the target RNA sequence**. Surprisingly, Fox-1 RBD (denoted as Fox), but not Fox-1 RBD\* (denoted as Fox\*), is also able to bind to mutated shRNA\*, suggesting that under the studied conditions the mutations in the RNA loop sequence do not abolish complex formation (**Figure 10**). Different results were previously obtained using surface plasmon resonance [[9]](#reference), which may be explained by differences in the experimental conditions.

![](https://static.igem.org/mediawiki/2020/b/bf/T--TUDelft--EMSA_results.jpeg)

*Figure 10. Illustration of Fox-1 domains used for the assay and their obtained results from interaction assay with the target sequence of the shRNA or shRNA\*. Mutations in either proteins or RNA are depicted as red dots. Mutated sequences of RNA or proteins are colored in orange, while wt protein domains Fox-1 RBD and wt binding sequence in the loop are colored in green.*

Hairpins with GG 3’ overhangs are suitable Dicer substrates

Dicer processing is the first step of the RNAi-mediated gene silencing where small interfering RNAs (siRNAs) are produced. Dicer-2 isoform products are later used to cleave messenger RNAs (mRNAs) with a 100% complementarity with one of the siRNA’s strand [[11]](#reference). Not all dsRNA structures are processed with the same efficiency due to the preferences Dicer shows towards certain substrates. For instance, dsRNA sequences embedded in a longer RNA transcript or with capped ends have been reported to show less processability than those at the termini, which generally show an increased siRNA production rate when ending with 3’ overhangs in absence of ATP [[12]](#reference). In particular, GG overhangs have shown a medium level of processivity among all possible combinations in humans [[13]](#reference), but it has not been proven in insects yet. A scheme about how shRNAs would be processed by Dicer-2 is shown in **Figure 11**.

![](https://static.igem.org/mediawiki/2020/9/9d/T--TUDelft--DICER_enchilada.jpeg)

*Figure 11. Illustrated representation of Dicer-2 processing of shRNA into siRNA. dsRNA sequences are represented in red, GG overhang in purple, and the overhang made by Dicer at the cleaving site by light blue.*

With this in mind, we decided to study whether **GG protruding 3’ overhangs** emulating those left by mini-3 cleavage are suitable for the production of free, mature shRNA with RNAi triggering potential. shRNAs were designed with the aforementioned overhang and their processivity was tested with a Dicer cleaving assay using purified Dicer-2 from the insect species *Drosophila melanogaster* (**Figure 12**), kindly provided by TU Delft BN department.

![](https://static.igem.org/mediawiki/2020/1/1c/T--TUDelft--DICER_Time.jpeg)

*Figure 12. shRNA processed by Dicer at different incubation times. Two ladders were added to compare the mobility of dsRNA with conventional DNA ladder. 400 ng of shRNA were incubated at 25ºC in Dicer buffer (Tris pH 7.5 10 mM, MgCl2 1 mM, NaCl 37.5 mM, Glycerol 5%) with 0.2 µM Dicer. Each tube was snap-frozen at the desired time point with liquid nitrogen until the end of the assay (5 hours). Control (-) sample consists of shRNA incubated 5 hours under the same buffer and temperature conditions without Dicer. Urea-PAGE 15% of 19:1 (Acrylamide:Bisacrylamide) ratio. Electrophoresis ran for ~3 hours at 120 V.*

The unprocessed shRNA gives a clear band on the gel, slightly above the 30nt size marker of the dsRNA ladder (**Figure 12**). Incubation with Dicer yields two lower bands, likely corresponding to the two cleavage products: the siRNA and the loop. Increasing the reaction duration from 1 hour to longer times is accompanied by a higher fraction of both enzymatic products. These results demonstrate that the **in vitro transcribed shRNA containing GG 3’ overhangs can be processed by Dicer from the insect Drosophila melanogaster**, adding evidence that our shRNAs have potential to silence any targeted gene from the locusts by means of RNAi.

##### Conclusion

**T7 viral RNA polymerase is a suitable candidate to transcribe our designed shRNAs shRNA** ([BBa\_K3407022](http://parts.igem.org/Part%3ABBa_K3407022) and [BBa\_K3407023](http://parts.igem.org/Part%3ABBa_K3407023)).successfully. The combination of the selected promoter and inverted repeats linked with a loop leads to the transcription of the desired shRNAs. As in vitro production has already been proven, the designed template DNA can further be brought to the next level and engineer the phage to contain it.

Although mobility in Urea-PAGE is unpredictable, results show **Dicer is able to process the shRNA to form siRNA**, constituting the first step for a successful RNAi-mediated gene silencing. The designed overhangs seem to yield good processivity with Dicer-2 from the insect D. melanogaster, setting the first evidence that mini-3 is an acceptable candidate for cleaving the shRNAs from the tandem transcript, producing free and mature shRNA with RNAi triggering potential in insects.

From the hypothetical cargo that would deliver shRNAs into the hemolymph, at least **Fox-1 RBD can bind most of the shRNA in 1:2 molar ratio**. This finding suggests the domain can be used as a part of a fusion protein, and further research should be done to elucidate whether the other domains of the cargo are also suitable for our purpose. Although the binding takes place, it does not show the expected specificity towards its target sequence under our conditions. This result makes evident the necessity of a deeper study of the interaction between the loop structure and Fox-1 RBD, where the dsRNA part or the secondary structure might influence the specificity.

The theoretical delivery molecule requires further research to prove its functionality, such as the influence of the hairpin structure in the specific binding to Fox-1 RBD, or ultimately testing the ability to cross the gut barrier. Phages seem to be a realistic candidate to produce shRNAs in gut bacteria to target insects such as *Schistocerca gregaria*.

##### Future prospects

In order to produce multiple shRNAs and maximise the chances of silencing vital functions of the locusts, the shRNAs can be produced as a **single tandem transcript RNA (tshRNA)**. Each shRNA could be generated after cleavage by an enzyme such as Mini-3 from Bacillus subtilis. **Mini-3** shows a high sequence associated cleavage, cutting dsRNA containing “ACCU” with little off-targets [[14]](#reference), leaving 3’ protruding overhangs, as depicted in **Figure 13**.

![](https://static.igem.org/mediawiki/2020/1/16/T--TUDelft--tshRNA_mini3.jpeg)

*Figure 13. Illustration of mini-3 role in shRNA production through processing of tshRNA transcript. mini-3 is represented as a dimer molecule coloured in garnet attached to its target sequence of dsRNA colored in purple.*

As the designed overhangs in our shRNA ([BBa\_K3407022](http://parts.igem.org/Part%3ABBa_K3407022)) seem to yield good processivity with Dicer-2 from the insect *D. melanogaster,* mini-3 would represent an acceptable candidate for cleaving the shRNAs from the tshRNA, producing free and mature shRNA with RNAi triggering potential in insects.

In order to protect shRNAs from degradation by RNAses, the protein **YmdB** could be co-expressed. This enzyme has shown to inhibit the RNAse III from *E. coli* [[15]](#reference), which we expected to help in production of shRNA in vivo. In this project, both YmdB and mini-3 have been cloned and overexpressed in compatible plasmid backbones for their characterization (see [Design for more information](https://2020.igem.org/Team%3ATUDelft/Design)). Preliminary experiments have been conducted in this direction and are described below.

Cloning of Mini-3 and YmdB from bacterial genome

We obtained the DNA sequences encoding for the enzyme Mini-3 and the protein YmdB by genome amplification from *Bacillus subtilis* and *E. coli* BL21 (DE3) respectively (**Figure 14**).

![](https://static.igem.org/mediawiki/2020/3/39/T--TUDelft--YmdB_mini3.jpeg)

*Figure 14. Genome amplification of mini III and YmdB. Agarose gel 1% containing the bands corresponding to Mini-3 and YmdB with flanking regions to be later cloned by Gibson Assembly in the respective receptor vectors.*

As it can be seen in **Figure 14**, both **genes were successfully amplified by PCR** as bands corresponding to their molecular weights can be observed. The amplified sequences were purified from the gel and cloned by Gibson Isothermal Assembly in the correspondent amplified backbone vector. Mini-3 was cloned into the vector pBbE8c, which contains an araBAD promoter and a T7Te terminator and is induced by anhydrotetracycline (aTc), thus obtaining a new composite part ([BBa\_K3407018](http://parts.igem.org/Part%3ABBa_K3407018)). YmdB was cloned into the vector pBbA2k, which contains a tetR/tetA promoter and a T7Te terminator and can be induced by L-arabinose, thus obtaining a new composite part ([BBa\_K3407019](http://parts.igem.org/Part%3ABBa_K3407019)). The assembled new plasmids were transformed into *E. coli* DH5-α. Plasmids purified from the transformed colonies were sequenced and transformed in *E. coli* BL21 (DE3) for protein expression.

Overexpression of Mini-3 and YmdB.

To overexpress Mini-3 and YmdB, we incubated at 37ºC *E. coli* BL21 (DE3) (negative control) and *E. coli* BL21 (DE3) transformed with the plasmids pBbE8c\_mini3 or pBbA2k\_YmdB. When the cultures reached OD600 ≈ 0.6, they were induced with 20 mM L-arabinose and 400 mM anhydrotetracycline respectively, at 37ºC for 4 hours and overnight at 20ºC. The total protein content of the cells was analysed by SDS-PAGE electrophoresis (**Figure 15**).

![](https://static.igem.org/mediawiki/2020/4/4f/T--TUDelft--Figure15_mini3_YmdB2.png)

*Figure 15. Overexpression of mMini-3 and YmdB. *E. coli* BL21 (DE3) is the negative control. A)  *E. coli*  BL21 (DE3) pBbE8c\_mini3 contains the part [BBa\_K3407018](http://parts.igem.org/Part%3ABBa_K3407018). B) *E. coli* BL21 (DE3) pBbA2k\_YmdB contains the part [BBa\_K3407019](http://parts.igem.org/Part%3ABBa_K3407019). MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight). All the samples used corresponded to the same OD600.*

As seen in the SDS-PAGE of the total protein content (**Figure 15**), both **Mini-3 and YmdB proteins were successfully overexpressed** after induction with 20 mM L-arabinose and 400 mM anhydrotetracycline, respectively. No additional bands can be observed in *E. coli* BL21 (DE3) pBbE8c\_mini3 or pBbA2k\_YmdB without induction, indicating that there is no leaky expression.

Future experiments will include **purification** of the overexpressed Mini-3 to use it for in vitro activity assays. If the tshRNA is produced, then the purified Mini-3 could be used for its cleavage. Furthermore, **co-expression of tshRNA, Mini-3 and YmdB** experiments could also be performed, therefore enabling the production of multiple shRNAs *in vivo*.

References

- Bacteriophages: Biology and Applications - Google Books. (n.d.). Retrieved October 24, 2020, from https://books.google.nl/books?hl=nl&lr=&id=6sTBd2EJpmYC&oi=fnd&pg=PA29&dq=phages+infect+bacteria&ots=j0fY0fm6fs&sig=tIckCDuOjW0sZwV13HHBBTZNtI8&redir\_esc=y#v=onepage&q=phages%20infect%20bacteria&f=false
- Marinelli, L. J., Piuri, M., Swigoňová, Z., Balachandran, A., Oldfield, L. M., van Kessel, J. C., & Hatfull, G. F. (2008). BRED: A Simple and Powerful Tool for Constructing Mutant and Recombinant Bacteriophage Genomes. PLoS ONE, 3(12), e3957. https://doi.org/10.1371/journal.pone.0003957
- Kiro, R., Shitrit, D., & Qimron, U. (2014). Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system. RNA Biology. https://doi.org/10.4161/rna.27766
- Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. https://doi.org/10.1126/science.1225829
- Reisch, C. R., & Prather, K. L. J. (2015). The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli. Scientific Reports. https://doi.org/10.1038/srep15096
- Wu, Y., Lei, C. F., Yi, D., Liu, P. M., & Gao, M. Y. (2011). Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis. Applied and Environmental Microbiology. https://doi.org/10.1128/AEM.02462-10
- Obbard, D. J., Gordon, K. H. J., Buck, A. H., & Jiggins, F. M. (2009). The evolution of RNAi as a defence against viruses and transposable elements. Philosophical Transactions of the Royal Society B: Biological Sciences. https://doi.org/10.1098/rstb.2008.0168
- Schwartz, A., Rahmouni, A. R., & Boudvillain, M. (2003). The functional anatomy of an intrinsic transcription terminator. EMBO Journal. https://doi.org/10.1093/emboj/cdg310
- Auweter, S. D., Fasan, R., Reymond, L., Underwood, J. G., Black, D. L., Pitsch, S., & Allain, F. H. T. (2006). Molecular basis of RNA recognition by the human alternative splicing factor Fox-1. EMBO Journal. https://doi.org/10.1038/sj.emboj.7600918
- Soler Canton, A. (2019) Synthetic biology meets liposome-based drug delivery. Casimir PhD series, Delft-Leiden 2019-35. https://doi.org/10.4233/uuid:b46cc724-c6cf-4282-b3ba-3817e8bebf87
- Lam, J. K. W., Chow, M. Y. T., Zhang, Y., & Leung, S. W. S. (2015). siRNA versus miRNA as therapeutics for gene silencing. Molecular Therapy - Nucleic Acids. https://doi.org/10.1038/mtna.2015.23
- Sinha, N. K., Trettin, K. D., Aruscavage, P. J., & Bass, B. L. (2015). Drosophila dicer-2 cleavage is mediated by helicase- and dsrna termini-dependent states that are modulated by loquacious-PD. Molecular Cell. https://doi.org/10.1016/j.molcel.2015.03.012
- Vermeulen, A., Behlen, L., Reynolds, A., Wolfson, A., Marshall, W. S., Karpilow, J., & Khvorova, A. (2005). The contributions of dsRNA structure to Dicer specificity and efficiency. RNA. https://doi.org/10.1261/rna.7272305
- Głów, D., Pianka, D., Sulej, A. A., Kozłowski, Ł. P., Czarnecka, J., Chojnowski, G., … Bujnicki, J. M. (2015). Sequence-specific cleavage of dsRNA by Mini-III RNase. Nucleic Acids Research. https://doi.org/10.1093/nar/gkv009
- Głów, D., Pianka, D., Sulej, A. A., Kozłowski, Ł. P., Czarnecka, J., Chojnowski, G., … Bujnicki, J. M. (2015). Sequence-specific cleavage of dsRNA by Mini-III RNase. Nucleic Acids Research. https://doi.org/10.1093/nar/gkv009