iGEM 2022 — Team thessaloniki-meta

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{% block title %}Engineering Success{% endblock %}
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## Introduction

---

Our journey began aiming to apply the engineering principles to every aspect of our research project. The
engineering steps of Design, Build, Test and Learn helped us to evaluate, solve and finally optimize every arising
problem we faced. These principles helped us to find new ways to approach troubleshooting for the occurring
issues. Generally, we understood that the engineering process can be applied to many aspects of a
multidisciplinary research project.

## Cycle 1

---

#### Cloning GG to 3A for final cr-cas plasmid

It is well established that in vivo crispR/cas systems provide immunity against viruses and plasmids in bacteria
and archaea ([{{context.references.Rath\_2015.label}}](#Rath_2015)).
The silencing of foreign nucleic acids is achieved through a ribonucleoprotein
complex of Cas molecule preloaded with crRNA molecules that act as guides for targeting the degradation of the
invading nucleic acid ([{{context.references.Rath\_2015.label}}](#Rath_2015)).
Once this duplex is necessary for the system to be active, we decided to
compose a Cas13a-crRNA plasmid for each crRNA molecule in order to produce efficiently and promptly the
ribonucleoprotein complex. It is worth noting that RNA molecules in vitro easily misfold and are usually unstable
tending to be degraded in presence of RNases or get trapped in inactive conformations if not preserved in the
proper way (Flores and Ataide, 2018). Thus, ribonucleoprotein duplexes protect and stabilize the RNA molecules
reducing the risk of degradation. Considering the advantages of the ribonucleoprotein complex we decided to
synthesize the cloned Cas13a-crRNA plasmid.

#### Design stage

In order to synthesize the final Cas13a-crRNA plasmid some factors had to be considered. More specifically, after
the sequences of each crRNA as well as Cas13a were amplified and their amplification was successfully confirmed,
the most efficient method had to be chosen to integrate the inserts into the pSB1C3 backbone vector. According to
bibliography one of the most commonly used assembly methods is Golden Gate assembly. This method is based on an
all-in-one mix reaction consisting of a type IIS restriction enzyme and T4 DNA ligase for efficient assembling of
the sequences, reducing handling time and increasing cloning efficiency. The DNA sequences get digested via the
restriction enzyme and are ligated by the DNA ligase. This process is repeated in cycles, and the elements are
forming the desired products since the properly assembled parts lack the restriction sites that exist at the
unincorporated DNA parts. Taking into account the advantages of this method, we chose Golden Gate assembly not
only for the composition of this specific plasmid but generally as a cloning method for our project in order to
achieve the least possible waste of time and the maximum cloning efficiency.

#### Build stage

We followed a modified protocol of Golden gate called Seva Brick based Golden Gate assembly developed by Damalas
Stamatios et. al. 2020 as described thoroughly in the [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
More significantly, the reaction consists of the SevaBrick Assembly mix (BsaI, T4 ligase, T4 ligase.
buffer, DpnI, BSA) and the entry parts as well as the vector backbone. The DNA amounts of the final reaction were
calculated via an excel file, introduced by Damalas Stamatios et. al. 2020. The final mix underwent the following
thermocycling conditions: 37 °C, 20 min; (16 °C, 4 min; 37 °C, 3 min) x 30; 50 °C, 10 min; 80 °C, 10 min.

After the end of the reaction, the samples were transformed into chemically competent E. coli DH5a cells using
heat shock transformation. Afterwards, the cells were cultured in LB antibiotic plates at 37 °C overnight.

To confirm if the cloning was successful, samples from the colonies underwent colony PCR and the results were
analyzed through an agarose gel.

[![]({{  context.figures.fig1.thumb  }})](%7B%7B%20%20context.figures.fig1.image%20%20%7D%7D)

Golden Gate assembly, with two inserts, BsaI as a restriction enzyme and T4 DNA ligase.

#### Test stage

Several tests were performed to achieve the cloning of the two entry parts with the backbone vector. However, its
synthesis was inefficient. In order to understand the reasons our protocol was not effective, we tried to improve
several factors. More specifically, we firstly checked the initial design of the plasmid in the SnapGene software.
Consequently, and as long as we confirmed that the design was correct, we resynthesized each part of the reaction
to confirm that the proper sequences were used. Then, we checked the reaction conditions and the enzymes'
functionality to confirm that they are working normally. However, the synthesis of the cloned Cas13a-crRNA plasmid
was not achieved (Figure 2).

[![]({{  context.figures.fig2.thumb  }})](%7B%7B%20%20context.figures.fig2.image%20%20%7D%7D)

Golden Gate assembly for the crRNA, Cas13a and the pSB1C3 backbone vector. The synthesis of the final plasmid
was unsuccessful

#### Learning stage

Since Golden Gate assembly was inefficient, we sought in the literature to determine the possible reasons why the
final plasmid was formed incorrectly and to find solutions. Based on the results, we observed that the high size
difference of the parts was probably the main cause of the experimental failure. While searching for a more
functional cloning method for this particular combination of inserts, we found that iGEM also suggests 3A assembly
as a cloning method, which has been extensively used to ligate molecules with a difference in sequence size and
was firstly described by [{{context.references.Shetty\_2011.label}}](#Shetty_2011).
More significantly, 3A assembly stands for 3 Antibiotic assembly and
is based on the integration of two parts in a backbone vector. The selection of the correct assembly products is
achieved through antibiotics. 3A assembly exploits the negative and positive selection in order to minimize the
number of possible incorrect assemblies. The inserts of the reaction are perceived in a) upstream part plasmid, b)
downstream part plasmid and c) the destination vector. Each plasmid has a prefix and a suffix region where the
restriction sites are. The upstream part is digested with EcoRI and SpeI, the downstream part is digested with
XbaI and PstI and the vector with EcoRI and PstI. The plasmids are then mixed to synthesize the final plasmid
(Figure 3). The final sample is then transformed into competent cells. The cells are cultured in LB plates in 37
°C overnight. According to [{{context.references.Shetty\_2011.label}}](#Shetty_2011)
this method can be used to assemble parts ranging in length from 12bp to 3-4kb. Thus, it could be a more effective
method for the synthesis of cloned Cas13a-crRNA.

Verily, we applied 3A assembly method to create cloned Cas13a-crRNA plasmid and we observed that our experiment
was crowned with success (Figure 4). In our experiment the upstream part was Cas13a (5422bp) and the downstream
part was each crRNA molecule (102-107bp). The destination vector was the pSB1C3 backbone vector (2029bp).
According to the 3A assembly the vector must have a different antibiotic resistance than the two input plasmids.
However, in our case, the vector had the same antibiotic as the input parts but the result was still successful.
Thus, we can safely support that 3A assembly is an effective method for the integration of inserts with high
difference in size.

[![]({{  context.figures.fig3.thumb  }})](%7B%7B%20%20context.figures.fig3.image%20%20%7D%7D)

3A assembly

[![]({{  context.figures.fig4.thumb  }})](%7B%7B%20%20context.figures.fig4.image%20%20%7D%7D)

The successful cloning of Cas13a-CrRNA plasmid after ligation with 3A assembly method. The band depicted down of
the desired band in 5pm3 colony is a byproduct caused by the undesired hybridization of the primers with another
part of the bacterial DNA.

## Cycle 2

---

#### CRISPR/Cas13a detection assay

After successive engineering cycles and troubleshooting processes we finally managed to produce the system's basic biological
components in quantities sufficient for the final DIAS detection assay. The LbuCas13a was expressed from the cloned final
T7-SUMO-LbuCas13 ([BBa\_K4170022](%7B%7B%20context.parts.BBa_K4170022.url%20%7D%7D)) plasmid and purified utilizing a standardized
methodology developed by our lab. In addition, the crRNA DNA sequences were successfully cloned into pSB1C3 plasmid
([BBa\_K4170023](%7B%7B%20context.parts.BBa_K4170023.url%20%7D%7D) - [BBa\_K4170023](%7B%7B%20context.parts.BBa_K4170023.url%20%7D%7D))
employing the methodology described at our custom [crPrep crRNA preparation kit](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)
and produced in satisfactory quantities via in vitro transcription. The next important step towards the
implementation of our project was to verify the functionality of the LbuCas13a/crRNA duplex in recognizing the
target miRNA and subsequently activating the trans-cleavage activity of the enzyme providing a green fluorescent
signal.

#### Design stage

To develop a detection system with increased sensitivity and broad clinical applicability a lot of factors
affecting the CRISPR/Cas13a enzyme reaction need to be investigated. Thus multiple experiments were conducted to
estimate the suitable LbuCas13a, crRNA and reporter concentrations to achieve the optimal conditions for the
detection assay and better understand the kinetics of the enzyme reactions. The system's detection method is based
on a detection standard curve; a bioanalytical method which represents a linear relationship between concentration
of an analyte (independent variable-miRNA added) and response (dependent variable-fluorescence intensity).
Finally, this relationship is used to predict the unknown concentration of the analyte in a complex matrix such as
the blood or serum. To achieve the detection system's maximum effectiveness we had to investigate factors
affecting the enzyme reaction such as the appropriate reporter concentration and the specific time point since
reaction initiation that the fluorescence signal should be obtained.

#### Build stage

For the investigation of factors that affect the kinetics of the reaction such as the optimal reporter
concentration we designed a simplified detection protocol. The basic steps of the protocol are the preparation of
the CC reaction mixture along with the different RS-P and RS-N reaction mixtures as described in detail below
(figure 5).

##### RS reaction mixtures preparation

The RS-P positive reaction mixtures contained a predefined concentration of target miRNA (1nM) in 1X reaction
buffer. However, different concentrations of Reporter RNA (1μM, 0.75 μΜ, 0.5μΜ, 0.25μΜ, 01μM) were added in each
reaction mixture to investigate the optimal reporter concentration for the enzyme reaction. In addition, the RS-N
negative reaction mixtures contained the same RNA reporter concentration as for the RS positive reaction mixtures
without the addition of the target miRNA.

* After the addition of the necessary reagents the RS-P and RS-N reaction mixtures remained in ice until use.

##### CC reaction mixture preparation

The CC final reaction mixture contains 20nM LbuCas13a, 20nM crRNA in 1X reaction buffer.

* The CC reaction mixture after its preparation, remained on ice until further use.
* Before its addition in the microplate, the mixture was left at 37 °C for 10 min without agitation to enable the
  efficient association of Cas13a enzymes with the crRNAs in the reaction mixture.

##### Microplate preparation for fluorescence experiments

The CC final reaction mixture contains 20nM LbuCas13a, 20nM crRNA in 1X reaction buffer. The CC reaction mix
contains the reagents that are listed in table X on the experiments page in a final volume of 20μl.

* After CC reaction mixture incubation at 37 °C and RS reaction mixtures preparation, a specific volume of the CC
  mixture (20μl) was added to each RS mixture generating the final detection master mixtures which were gently
  flicked. Afterwards, 50μl of the final detection master mixtures were added to each corresponding well of the
  microplate.
* The microplate was incubated for 60 min in a fluorescence plate reader with fluorescence measurements (FAM
  channel, \( \lambda\_{ex} \) 494 nm, \( \lambda\_{em} \) 518 nm) taken every 1 min.

[![]({{  context.figures.C2pre.thumb  }})](%7B%7B%20%20context.figures.C2pre.image%20%20%7D%7D)

The initial experimental workflow followed to study the kinetics of the enzyme reaction and
investigate the factors which affect the assay performance

#### Test stage

From the real time kinetic measurement of Cas13a reactions incubated with different reporter concentrations and a
predefined miRNA concentration (1nM), we observed a conservative pattern on how the fluorescence intensity changed
over time. During the first minutes of the reaction the emitted fluorescence from the RNA reporter increases
linearly until the reaction reaches the plateau phase when the fluorescence intensity starts to decrease. On the
contrary, the negative control samples exhibit constant background fluorescence at all time points. But, are these
the only conclusions that can be drawn from the fluorescence curves?

To construct the following standard curves which plot the miRNA concentration (standards) versus time, the
fluorescence capture point should be in the linear part of the curve before the plateau phase. Inspecting
thoroughly the graphs (Figure 6), we can easily understand that the samples loaded first at the microplate (1μM
and 0.75μΜ) show a limited linear section in the curve which corresponds to the first 1-5 minutes since reaction
initiation. On the other hand, the samples loaded last at the microplate (0.25μΜ and 0.1 μΜ) show a wider linear
section on the curve that corresponds to the first 1-12 minutes since reaction initiation. This limited linear
section of the curve may hinder the practical implementation of the detection system, due to variations in the
detected fluorescence caused by the fast enzyme reaction speed and fluctuations in researcher's handling time
required for reaction preparation.

**1.00μM**

[![]({{  context.figures.C21000.thumb  }})](%7B%7B%20%20context.figures.C21000.image%20%20%7D%7D)

**0.75μM**

[![]({{  context.figures.C20750.thumb  }})](%7B%7B%20%20context.figures.C20750.image%20%20%7D%7D)

**0.50μM**

[![]({{  context.figures.C20500.thumb  }})](%7B%7B%20%20context.figures.C20500.image%20%20%7D%7D)

**0.25μM**

[![]({{  context.figures.C20250.thumb  }})](%7B%7B%20%20context.figures.C20250.image%20%20%7D%7D)

**0.10μM**

[![]({{  context.figures.C20100.thumb  }})](%7B%7B%20%20context.figures.C20100.image%20%20%7D%7D)

Fluorescence intensity changes over time

#### Learning stage

Taking into account the above, we decided to investigate solutions to limit the effect of researcher's handling
time on assay results. To achieve this, we conducted all the following enzyme reactions on ice, keeping the
microplate's temperature at 4°C until its insertion on the fluorescence plate reader. The low temperature prevents
reaction initiation until the microplate is inserted in the plate reader which keeps a 37 °C constant temperature.
In addition, we removed the 10 min pre-incubation step of the CC reaction mixture at 37 °C. We concluded that the
LbuCas13a enzyme can be efficiently associated with the crRNA directly inside the microplate well without the
pre-incubation step.

In summary on the following experiments we made the following experimental modifications:

* All reagents, the reaction tubes and the microplate should be kept on ice until the fluorescence measurement.
* The pre-incubation of LbuCas13a enzyme with the crRNA was skipped, to limit the effect of handling time on assay results.

[![]({{  context.figures.C2post.thumb  }})](%7B%7B%20%20context.figures.C2post.image%20%20%7D%7D)

The optimized experimental workflow which emerged from the engineering cycle. This protocol was then
followed to study the assay performance at subsequent experiments. The pre-incubation step of LbuCas13a enzyme
with crRNA is skipped and all the experimental procedures are conducted on low temperature keeping the
microplate on ice before insertion at the fluorescence plate reader.

## Cycle 3

---

#### Microfluidic device manufacturing

One of the directions of our engineering cycles was the process of manufacturing our microfluidic device. This
aspect of the project is very crucial since utilizing the technology of microfluidics, we can achieve droplet
production resulting into increase of LOD (Level of Detection),
([{{context.references.Kaminski\_2021.label}}](#Kaminski_2021)).

#### Design stage

The Design stage of this engineering cycle involves the general design of the microfluidic device and the process
of device manufacturing. Since we aim to generate droplets, the microfluidic device should exhibit droplet
generation behavior. The chosen design was a flow-focusing T-junction device because it is reported to generate
droplet with high mono-dispersity and the diameter of the droplets are a function of the flow conditions and
channel dimensions ([{{context.references.Ibrahim\_2021.label}}](#Ibrahim_2021)).
The channel geometry and the general device are demonstrated in Figure 8 and 9 respectively. The design is
selected to have 3 inlets and 1 outlet with 2 oil inlets and 1 water inlet since the produced droplets must Water
in Oil emulsions. The channel depth is designed to be 500μm and the width to be 400,500 and 700 for the oil, water
inlets channels and outlet channels respectively. The selected geometry was based on Computational Fluid Dynamics
since it exhibited appropriate segmented flow.

[![]({{  context.figures.fig8.thumb  }})](%7B%7B%20%20context.figures.fig8.image%20%20%7D%7D)

Channel geometry of the microfluidic channels

[![]({{  context.figures.fig9.thumb  }})](%7B%7B%20%20context.figures.fig9.image%20%20%7D%7D)

Microfluidic Device.

The manufacturing process chosen was the Fused Deposition Modelling since it is reported to be able to produce
functional microfluidic devices ([{{context.references.Nelson\_2019.label}}](#Nelson_2019)).
Fused Deposition Modelling is a 3D printing
technique based on the deposition of melted thermoplastic material layer after layer until the 3D device is
finished. The material used in the FDM process is a filament of polymeric material that is softened and melted
with the help of heat and then extruded. The polymer chosen for this type of device was a regular Polylactic Acid
(PLA) with a black color filament and a transparent Polylactic Acid filament. The regular PLA exhibits more suitable
rheological behavior for FDM technique in comparison with the transparent one. Some of the advantages of the FDM process
are the low cost of the materials, the wide range of slicing software and the simple machinery design that makes it
suitable for beginners ([{{context.references.Kristiawan\_2021.label}}](#Kristiawan_2021)).

[![]({{  context.figures.photoC3.thumb  }})](%7B%7B%20%20context.figures.photoC3.image%20%20%7D%7D)

#### Build stage

After the general design of the microfluidic device and the selection of the 3D printing technique, the next step
is to select the appropriate parameters for the manufacturing of the device. The geometry of the design was
selected to be suitable for printing. One of the main disadvantages of the FDM technique is the accuracy
limitation.

Having that in mind, we decided to use 2 types of nozzles for the material extrusion. The first was 0.4 mm and the
second 0.2 mm. The smaller the nozzle diameter, the higher the accuracy and also higher the total time of printing
will be. A general rule for selecting the appropriate nozzle is to understand that the nozzle diameter is
affecting the XY resolution, and the layer height is affecting the resolution on the Z axis. Since the smaller
channel width was 400 μm, hypothetically the 0.4 mm = 400μm nozzle would be fine.

The next step was the selection of the Slicing software for the device and the parameters of the printing. The
slicing software used was the Ultimaker Cura. The parameters we used were chosen based on the literature
([{{context.references.Nelson\_2019.label}}](#Nelson_2019) and
[{{context.references.Zhang\_2019.label}}](#Zhang_2019)).

FDM printing Parameters

| FDM parameter | Value |
| --- | --- |
| Nozzle Temperature | 210 /( ^oC /) |
| Bed Temperature | 60 /( ^oC /) |
| Layer Height | 0.1 /( mm /) |
| Printing Speed | 50 /(mm s^{-1}/) |
| Infill Density | 100% |

[![]({{  context.figures.fig10.thumb  }})](%7B%7B%20%20context.figures.fig10.image%20%20%7D%7D)

Ultimaker Cura slicing software for the microfluidic device.

#### Test stage

The final stage of the process is the manufacturing of the device and the test of the microfluidic behavior. The
manufacturing stage was a challenging aspect. After printing several devices with slightly different parameters to
achieve the desired result, the overall outcome was the inability of general liquid flow. The printing process can
overall divide into 3 main parts. The first part includes the printing of all the layers beneath the channels that
were printed with accuracy. The next part was the layers of the channel geometry. The channel geometry, during the
printing with the 0.4 mm nozzle, resulted with the unwanted material inside since the extruder did not retract the
material in time. The outcome was that not even one device could perform fluidic behavior. The final part of the
manufacturing was all the layers above the channels. The problem in this stage is that if the extruded material is
not solidified right after extrusion, unwanted material can again result in the channels. This problem could not
be solved by just changing the nozzle.

A second approach to the problem was to split the model and print the first and the second part together and the
third alone. In this way, we could evade the unwanted material inside the channels during the printing process.
After printing the two components, we tried to assemble them together to test the whole device. The method
involved heating the first layer of the upper piece and then putting it on the first piece. Unfortunately, upon
testing it with a pumping system for the droplet generation, it showed major leakage issues resulting in failure.

[![]({{  context.figures.fig11.thumb  }})](%7B%7B%20%20context.figures.fig11.image%20%20%7D%7D)

Microfluidic device printed with FDM 3D printing technique.

#### Learning Stage

Facing all those problems with the FDM technique, we tried different approaches to manufacturing a functional
device that will exhibit appropriate properties for our project. We understood that even changing the printing
parameters and the initial 3D models, we had to face the FDM limitations such as surface roughness and leakage
issues ([{{context.references.Zhang\_2019.label}}](#Zhang_2019)).

So, we decided to move in another direction. The direction we followed was to change the printing technique to
Digital Light Projection (DLP). The process is based on the conversion of liquid to solid, using computer
spatially controlled photopolymerization to create solid objects from a vat of liquid resins under light
irradiation. DLP offers lower printing times and far better surface roughness, resulting in more accurate
manufacturing of the channels ([{{context.references.Quan\_2020.label}}](#Quan_2020)).
The materials used for this printing technique are called photopolymers or resins and the materials that we used
were a standard silver resin and a transparent yellow resin.

The process of manufacturing the device was like the FDM approach. We printed 2 different parts of the
microfluidic device, first to check that the channels would be open and second for better control of the unwanted
material inside in the channel geometry.

[![]({{  context.figures.fig12.thumb  }})](%7B%7B%20%20context.figures.fig12.image%20%20%7D%7D)

The 2 different parts of the microfluidic device for FDM and DLP techniques

The surface of the printing device with the DLP technique is far greater. For the FDM device, the channels are
rough and can affect the droplet generation. The DLP channels, on the other hand, exhibit a smoother surface and
the channels are appropriate for liquid flow. Having a lot of issues trying to assemble them correctly to perform
the ideal behavior we tried to print the whole model directly to save time and effort. Unfortunately, not all the
channels were printed clear, without undesired resin, meaning that the post-processing was a major drawback.

[![]({{  context.figures.fig13.thumb  }})](%7B%7B%20%20context.figures.fig13.image%20%20%7D%7D)

Whole microfluidic device with 3 inlets and 1 outlet printed with DLP technique.

Finally, after printing the same device under different conditions with undesired results, we observed that nearly
all the channels were cleared of the resin, but having 3 inlets and 1 outlet, always one of them would be blocked.
Having that in mind, we tried to modify the model of the microfluidic device to involve only 2 inlets, 1 water and
1 oil inlet. Having only 2 inlets made the post-processing successful, and we achieved droplet generation
behavior. The selected flow conditions based on the model were 40 μL/min for oil (continuous phase) and 8 μL/min
for water (dispersed phase).

[![]({{  context.figures.fig14.thumb  }})](%7B%7B%20%20context.figures.fig14.image%20%20%7D%7D)

Experimental setup with 1 oil and 1 water inlet and 1 outlet.

{% include 'references.html' %}
{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

## Protocols

---

#### Bacterial Cultures

### LB Medium preparation

##### Introduction

---

LB Medium is a nutrient-rich microbial growth medium used for the
cultivation of *E. coli*. We used pre-mixed powder according to
Lennox from PanReac.

[Details](%7B%7Bcontext.docs.pdf.lb_med%7D%7D)

##### Reagents and equipment

---

* Pre-mixed LB-Medium powder according to Lennox.
* dH2O
* Flask
* Sterile Bottle
* Autoclave
* Weighing Machine

##### Composition of pre-mixed LB-Medium powder

* NaCl 5 g/L
* Tryptone 10 g/L
* Yeast extract 5 g/L

##### Procedure

---

1. For the preparation of 1 L of LB-agar weigh 20g of the pre-mixed
   powder.
2. Dissolve it in 1 L of dH2O. For larger or smaller volumes of the
   mixture scale up or down respectively.
3. Autoclave the mixture.
4. If the addition of antibiotics is needed, it can be added after the
   mixture has cooled, since it is sensitive to high temperatures. This is
   approximately the temperature that you can hold the flask that contains
   the LB-Medium only with lab gloves.

### LB Agar preparation

##### Introduction

---

LB Agar is a nutrient-rich microbial growth medium used for the preparation
of LB Agar plates for cultivation of *E. coli*. We used pre-mixed
powder according to Lennox from PanReac.

[Details](%7B%7Bcontext.docs.pdf.lb_agar%7D%7D)

##### Reagents and equipment

---

* Pre-mixed LB-agar powder according to Lennox.
* dH2O
* Flask
* Sterile Bottle
* Autoclave
* Weighing Machine

##### Composition of pre-mixed LB-Medium powder

* Agar 15 g/L
* NaCl 5g/L
* Tryptone 10 g/L
* Yeast extract 5 g/L

##### Procedure

---

1. For the preparation of 1 L of LB-agar weigh 35 g of the pre-mixed
   powder.
2. Dissolve it in 1 L of dH2O. For larger or smaller volumes of the
   mixture scale up or down respectively.
3. Autoclave the mixture.
4. Let the mixture cool down approximately at 60o C before you add the
   appropriate quantity of antibiotic if it is needed.
   If you are using a 1000x stock solution of antibiotic, calculate the volume
   of the stock solution that you should add to the mixture by dividing the
   volume of the LB-agar you are preparing with 1000.
5. Add the antibiotic with a pipette directly into the mixture and swirl
   the bottle in order to ensure that the antibiotic is distributed
   evenly.

### LB agar plates preparation

##### Introduction

---

Preparation of LB-agar plates with different antibiotics is a crucial
procedure in a microbiology lab as they are utilized for culturing bacteria
in the lab. This protocol is based on Addgene's " Pouring LB Agar Plates"
protocol. For the preparation of LB-agar we used premixed powder according
to LENOX.

[Details](https://www.addgene.org/protocols/pouring-lb-agar-plates/)

##### Reagents and equipment

---

* Pre-mixed LB-agar powder according to Lennox.
* dH2O
* Flask
* Sterile Bottle
* Autoclave
* Weighing Machine
* Sterile pipettes
* Ice bucket
* Sterile plates of your desired size
* Antibiotic at 1000 x concentration dissolved in the appropriate liquid
  solvent

##### Procedure

---

1. For the preparation of 1 L of LB-agar weigh 35 g of the pre-mixed
   powder.
2. Dissolve it in 1 L of dH2O. For larger or smaller volumes of the
   mixture scale up or down respectively.
3. Autoclave the mixture.
4. Light the flame at the plate pouring station and dilute your antibiotic
   into your 50-60oC molten gel mix using sterile technique.
5. Swirl the agar bottle to ensure even distribution of the antibiotic
   throughout the agar
6. Open one plate at a time next to the flame and begin pouring.
7. Leave your plates out on the bench to solidify.

### Glycerol stock preparation

##### Introduction

---

Bacterial glycerol stocks are essential for long-term storage of plasmids.
A glycerol stock of bacteria can be stored stably at -80oC for
many years. This protocol is based on Addgene's protocol "Creating
Bacterial Glycerol Stocks for Long-term Storage of Plasmids".

[Details](https://www.addgene.org/protocols/create-glycerol-stock/)

##### Reagents and equipment

---

* Liquid bacterial cultures
* 50% glycerol
* Pipette & tips
* Tubes

##### Procedure

---

###### Preparation of the liquid bacterial culture

1. For the preparation of the liquid bacterial culture follow the protocol:
   "Inoculation of liquid bacterial cultures”.

###### Preparation of glycerol stock

2. Into a tube, add 500 ul of the overnight liquid culture and 500ul of 50%
   glycerol
3. Mix gently by inverting the tube
4. Freeze and store the glycerol stock tube at -80oC

###### Recovery of bacterial cells

5. Use a sterile loop to scrape some of the bacteria off on the top of the
   glycerol stock and streak a plate with the appropriate antibiotic. Do not
   unthaw the glycerol stock.
6. Grow overnight the bacteria at the appropriate temperature.

### Streaking and isolating bacteria on a LB agar plate

##### Introduction

---

This protocol describes two crucial procedures that are followed for
isolating a single clonal population of bacteria from a glycerol stock or a
stab culture. The protocol is based on Addgene's protocol l "Streaking and
Isolating Bacteria on an LB Agar Plate".

[Details](https://www.addgene.org/protocols/streak-plate/)

##### Reagents and equipment

---

* Sterile toothpicks or wire loop
* Bunsen burner
* Incubator
* Marker
* LB Agar plate with appropriate antibiotic
* Bacterial stab

##### Procedure

---

###### Preparation

1. Get an LB agar plate with the appropriate antibiotic
2. Label the bottom of the plate with information like: the plasmid name,
   the date and antibiotic resistance.
3. Sterilize your lab bench using 70% ethanol and working near a flame or
   Bunsen burner in order to maintain sterility.
4. Get the desirable glycerol stock or bacterial stab.

###### Streaking

- Using a sterile loop, touch the bacteria growing within the punctured
  area of the stab culture or the top of the glycerol stock.
- Gently spread the bacteria over a section of the plate, as shown in the
  diagram, to create streak #1
- Using another sterile toothpick, or freshly sterilized loop, drag
  through streak #1 and spread the bacteria over a second section of the
  plate, to create streak #2.
- Using a third sterilized loop, drag through streak #2 and spread the
  bacteria over the last section of the plate, to create streak #3.

![]({{  context.figures.streak.thumb  }})

###### Isolation

5. Incubate the plate with newly plated bacteria overnight (12-18 hours)
   at 37oC
6. After the incubation, single colonies should be visible. A single
   colony should look like a white dot growing on the solid medium. If the
   bacterial growth is too dense and you do not see single colonies,
   re-streak onto a new agar plate to obtain single colonies.
7. Once there are single colonies, they can be picked and then proceed to
   recovering plasmid DNA or use the individual colonies for other
   experiments.

### Preparation of Chemically competent cells

##### Introduction

---

We used this protocol to prepare competent cells, able to be transformed by
our parts.

##### Reagents and equipment

---

* 350 ml 0,1 M CaCl2
* 100% Glycerol
* Erlenmeier sterile flasks
* Centrifuge bottles
* Incubator
* Cooling centrifuge
* Ice bucket filled with ice

##### Procedure

---

1. Plate the cells on a plate. If the cells have any drug resistance
   integrated in them, use LB-Agar plates containing this resistance;
   otherwise use plain LB-Agar plates.
2. Pick one colony and grow overnight in 25 ml LB at 37oC
   (containing the drug if resistance present)
3. Transfer the cells to 500 ml LB final volume
4. Incubate at 37oC for ~2,5h (until the OD reaches ~0.5)
5. Centrifuge cells at 4oC for 15min at 5000RPM (Sorval)
6. Discard the supernatant and re-suspend the pellet in 250ml 0,1M CaCl 2
7. Incubate on ice for 20 min
8. Centrifuge again as before
9. Discard supernatant and re-suspend the pellet in 43ml 0,1M CaCl 2 (8.6ml per 50ml Falcon)
10. Incubate for 2-12h (usually 4h)
11. Add 7ml 100% glycerol and mix well (1.4ml per 50ml Falcon)
12. Distribute in 1,5ml tubes
13. Quick freeze in liquid nitrogen
14. Store at -80oC

### Inoculating a Liquid Bacterial Culture

##### Introduction

---

A liquid bacteria culture is used to grow up enough bacteria, crucial to
isolate enough plasmid DNA for experimental use. The protocol is based on
Addgene's protocol “Inoculating a Liquid Bacterial Culture".

[Details](https://www.addgene.org/protocols/inoculate-bacterial-culture/)

##### Reagents and equipment

---

* Liquid LB Medium
* LB agar plate with bacterial cultures
* Bunsen burner
* Incubator
* Falcon tubes
* Pipette and tips

##### Procedure

---

1. When ready to grow your culture, add liquid LB medium to a tube or
   flask and add the appropriate antibiotic to the correct concentration.

| Antibiotics | Ampicillin | Chloramphenicol | Kanamycin | Gentamycin | Tetracycline |
| --- | --- | --- | --- | --- | --- |
| Recommended concentration | 100 μg/ml | 25 μg/ml | 50 μg/ml | 10 μg/ml | 10 μg/ml |

2. Select a single colony from the LB agar plate, using a sterile pipette
   tip or toothpick.
3. Drop the tip or toothpick into the liquid LB with antibiotic and swirl.
4. Loosely cover the culture with sterile aluminum foil.
5. Incubate bacterial culture at 37°C for 12-18 hours in a shaking
   incubator.
6. After incubation, check for growth, which is characterized by a cloudy
   haze in the media.

#### Cloning experiments

### Primer preparation

##### Introduction

---

For the reconstitution of the lyophilized primers we followed the provided
by the Integrated DNA Technologies (IDT).

[Details](https://www.idtdna.com/pages/education/decoded/article/tips-for-resuspending-and-diluting-your-oligonucleotides)

##### Reagents and equipment

---

* Lyophilized primers
* ddH2O
* Pipette & tips
* Microfuge tubes

##### Procedure

---

###### Before reconstitution

1. Before opening, spin down the pellet for 30 sec at full speed, to
   collect all the powder at the bottom of the vial.
2. Open the vial carefully, without touching the inside of the lid.

###### Reconstitution of the primer (100μM storage solution)

3. Find the oligo yield information (in nmol) on the tube label or
   specification sheet
4. Multiply this number by 10
5. The resulting product is the amount of ddH2O needed, in µL,
   to prepare a 100 µM solution.
6. Mix gently by inverting the tube. Avoid mixing by pipetting, shaking or
   vortex.

###### Preparation of a working stock (10μM)

7. Dilute one part of the primer stock by adding 9 parts water, in order to
   create a 10 pmol/ul working stock concentration (1:10)

### PCR Amplification

##### Introduction

---

Polymerase chain reaction (PCR) is a simple technique for the amplification
of targeted DNA fragments in vitro. We followed the PCR protocol for “Q5
High-Fidelity DNA Polymerase”(New England Biolabs) and the biphasic PCR
protocol introduced by Stamatios G.Damalas. We used the biphasic protocol
in cases where our primers were long in base pairs and with high
complexity.

[Details](https://international.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491)
[More...](https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/1751-7915.13609)

##### Reagents and equipment

---

* 5x Q5 Reaction Buffer
* 10 mM dNTPs
* 10 μM Forward primer
* 10 μM Reverse primer
* Template DNA
* Q5 High-Fidelity DNA Polymerase
* Nuclease free water
* Ice bucket filled with ice
* Pipette & tips
* Thermocycler

##### Procedure

---

1. Add the following components in a PCR tube and gently mix (for a 25 μl
   reaction).

|  |  |
| --- | --- |
| 5X Q5 Reaction Buffer | 5 μl |
| 10 mM dNTPs | 0.5 μl |
| 10 μM Forward primer | 1.25 μl |
| 10 μM Reverse primer | 1.25 μl |
| Template DNA | variable |
| Q5 High-Fidelity DNA Polymerase | 0.25 μl |
| Nuclease-free water | to 25 μl |

2. Collect all liquid to the bottom of the tube by a quick spin if
   necessary.
3. Introduce the PCR tubes in the thermocycler and use the following PCR
   protocol

###### Standard PCR protocol

|  |  |  |
| --- | --- | --- |
| **Step** | **Temperature** | **Time** |
| Initial Denaturation | 98oC | 30 sec |
| 25-35 cycles | 98 oC (Denaturation) | 5-10 sec |
|  | 50-72 oC (Annealing) | 10-30 sec |
|  | 72 oC (Extension) | 20-30 sec/kb |
| Final Extension | 72 oC | 2 min |
| Hold | 4-10 oC | ∞ |

###### Biphasic PCR protocol

| Step | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98oC | 2 min |
| **Phase 1 X** 10 cycles | 98 oC | 20 sec |
|  | 50oC | 20 sec |
|  | 72 oC | Part dependent |
|  | 98 oC | 20 sec |
| **Phase 2 X** 25 cycles | 76oC | 20 sec |
|  | 72oC | Part dependent |
|  | 72 oC | 2 min |
| Hold | 4-10 oC | ∞ |

### Site-directed mutagenesis PCR

##### Introduction

---

PCR Mutagenesis is a simple method for generating site-directed
mutagenesis. We followed the PCR protocol for “Q5 High-Fidelity DNA
Polymerase” (New England Biolabs).

[Details](https://international.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491)

##### Reagents and equipment

---

* 5x Q5 Reaction Buffer
* 10 mM dNTPs
* 10 μM Forward mutagenic primer
* 10 μM Reverse primer
* Template DNA
* Q5 High-Fidelity DNA Polymerase
* Nuclease free water
* Ice bucket filled with ice
* Pipette & tips
* Thermocycler

##### Procedure

---

1. Add the following components in a PCR tube and gently mix (for a 25 μl
   reaction).

|  |  |
| --- | --- |
| 5X Q5 Reaction Buffer | 5 μl |
| 10 mM dNTPs | 0.5 μl |
| 10 μM Forward mutagenic primer | 1.25 μl |
| 10 μM Reverse primer | 1.25 μl |
| Template DNA | variable |
| Q5 High-Fidelity DNA Polymerase | 0.25 μl |
| Nuclease-free water | to 25 μl |

2. Collect all liquid to the bottom of the tube by a quick spin if
   necessary.
3. Introduce the PCR tubes in the thermocycler and use the following PCR
   protocol

| Step | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98oC | 30 sec |
| 25-35 cycles | 98 oC (Denaturation) | 5-10 sec |
|  | 50-72 oC (Annealing) | 10-30 sec |
|  | 72 oC (Extension) | 20-30 sec/kb |
| Final Extension | 72 oC | 2 min |
| Hold | 4-10 oC | ∞ |

### Colony PCR

##### Introduction

---

Colony PCR is a high-throughput method for determining the presence or
absence of insert DNA in plasmid constructs. This method validates the
correct assembly of our genetic parts with the plasmid backbone. We
followed the PCR protocol for “Taq DNA Polymerase with Standard Taq Buffer”
(New England Biolabs).

[Details](https://international.neb.com/protocols/0001/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273)

##### Reagents and equipment

---

* 10x Standard Taq Reaction Buffer
* 10 mM dNTPs
* 10 μM Forward primer
* 10 μM Reverse primer
* Template DNA
* Taq DNA Polymerase
* Nuclease free water
* Ice bucket filled with ice
* Pipette & tips
* Thermocycler

##### Procedure

---

1. Add the following components in a PCR tube and gently mix (for a 25 μl
   reaction).

|  |  |
| --- | --- |
| 10X Standard Taq Reaction Buffer | 2.5 μl |
| 10 mM dNTPs | 0.5 μl |
| 10 μM Forward primer | 0.5 μl |
| 10 μM Reverse primer | 0.5 μl |
| Template DNA | variable |
| Taq DNA Polymerase | 0.125 μl |
| Nuclease-free water | to 25 μl |

2. Collect all liquid to the bottom of the tube by a quick spin if
   necessary.
3. Introduce the PCR tubes in the thermocycler and use the following PCR
   protocol

| Step | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 95oC | 30 sec |
| 25-35 cycles | 95 oC (Denaturation) | 15-30 sec |
|  | 45-68 oC (Annealing) | 15-60 sec |
|  | 68 oC (Extension) | 1 min/kb |
| Final Extension | 68 oC | 5 min |
| Hold | 4-10 oC | ∞ |

### Agarose gel electrophoresis

##### Introduction

---

Agarose gel electrophoresis is a standard lab procedure for separating DNA
or RNA by size and allows the visualization and purification of the
amplified PCR products. We followed the Addgene's protocol "Agarose Gel
Electrophoresis"

[Details](https://www.addgene.org/protocols/gel-electrophoresis/)

##### Reagents and equipment

---

* Agarose
* TAE Buffer Solution (10x)
* TAE Buffer Solution (1x)
* Ethidium Bromide (EtBr)
* dH2O
* Casting tray
* Well combs
* Conical Flask
* Pipette & tips
* Weighing machine
* Microwave
* Voltage source
* Gel box
* UV light source
* Electrophoresis device

##### Procedure

---

###### Gel preparation 1% agarose

1. Weigh 0.7 gr of agarose and place it in a flask.
2. Mix agarose powder with 70ml 1x TAE .
3. Microwave for 2-3 min until the agarose is completely dissolved.
4. Let agarose solution cool down with cold water (for about 2 minutes
   under the tab).
5. Add 4 μl ethidium bromide (EtBr).
6. Place the well comb into a gel tray and pour the agarose into it.
7. Let the gel at room temperature for 25 min, until it has completely
   solidified.

###### Samples preparation and running the agarose gel

8. Add 4 μl loading buffer 6X to each DNA sample (20 μl).
9. Once the agarose gel is solidified, place it into the electrophoresis
   device.
10. Fill the gel box with 1x TAE, until the agarose gel is covered.
11. Load the DNA samples and 1 μl of a molecular weight ladder.
12. Run the gel at 80-150 V for about 1 hour, depending on the gel
    concentration and voltage.
13. Turn Off power and carefully remove the gel from the gel box.
14. Visualize the DNA fragments, utilizing any UV light source.

### Golden Gate Assembly

##### Introduction

---

Golden Gate Assembly is a molecular cloning method that allows the
simultaneous and directional assembling of multiple DNA fragments into a
plasmid backbobe. We followed the “Golden Gate-based **SevaBrick Assembly**"
method introduced by Stamatios G. Damalas.

[Details](https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/1751-7915.13609)

##### Reagents and equipment

---

* BsaI HF v2
* T4 ligase
* T4 ligase Buffer
* DpnI
* BSA 20 mg/ml
* Biobrick parts
* Nuclease free water
* Ice bucket filled with ice
* Pipette & tips
* Sterile PCR tubes
* Thermocycler

#####

##### Procedure

---

1. Calculate DNA amounts and dilutions using an Excel file that is provided
   to the SEVA 3.1 paper. (See the link above)
2. Prepare the Golden Gate Master Mix by adding the following components in
   a PCR tube and gently mix. For a 8 μl reaction, use 2 μl of Golden Gate
   Master Mix.

|  |  |
| --- | --- |
| BsaI HF v2 | 12 μl |
| T4 ligase | 10 μl |
| T4 ligase Buffer | 18 μl |
| DpnI | 1 μl |
| BSA 20 mg/ml | 1 μl |

3. Add the appropriate amounts of Golden Gate Master Mix and the DNA parts
   in a PCR tube and gently mix.
4. Collect all liquid to the bottom of the tube by a quick spin if
   necessary.
5. Introduce the PCR tubes in the thermocycler and use the following PCR
   protocol

| Cycles | Temperature | Time |
| --- | --- | --- |
|  | 37oC | 20 min |
| **X** 30 | 16 oC | 4 min |
|  | 37 oC | 3 min |
|  | 50 oC | 10 min |
|  | 80oC | 10 min |
| Hold | 4-10 oC | ∞ |

### 3A Assembly ligation

##### Introduction

---

3A Assembly ligation is a method for assembling two part samples and
selecting for correct assemblies through antibiotics. We followed the 3A
Assembly protocol introduced by iGEM Registry.

[Details](https://parts.igem.org/Help%3AProtocols/3A_Assembly)

##### Reagents and equipment

---

* EcoRI-HF
* SpeI
* PstI
* XbaI
* DNA templates of the 2 parts and the plasmid backbone
* NEB Buffer 2
* dH2O
* BSA
* T4 DNA ligase Buffer
* T4 DNA ligase
* Ice bucket filled with ice
* Pipette & tips
* PCR tubes
* Heating block machine

##### Procedure

---

###### Digestion

1. Restriction digestion of the two parts that will be assembled and the
   plasmid backbone. Add 4 μl of the linearized plasmid, Part A, Part B and 4
   μl of the corresponding enzyme Master Mix.

| Enzyme M.M for Plasmid backbone | Enzyme M.M for Part A | Enzyme M.M for Part B |
| --- | --- | --- |
| 5 μl NEB Buffer 2 | 5 μl NEB Buffer 2 | 5 μl NEB Buffer 2 |
| 0.5 μl BSA | 0.5 μl BSA | 0.5 μl BSA |
| 0.5 μl EcoRI-HF | 0.5 μl EcoRI-HF | 0.5 μl XbaI |
| 0.5 μl PstI | 0.5 μl SpeI | 0.5 μl PstI |
| 0.5 μl DpnI |  |  |
| 18 μl dH2O | 18.5 μl dH2O | 18.5 μl dH2O |

2. Digest the 3 reactions at 37oC for 30 min
3. Heat kill the restriction enzymes at 80oC for 20 min

###### Ligation

4. Add in a PCR tube all the components below.

|  |  |
| --- | --- |
| Plasmid backbone | 2 μl |
| Part A | <3 μl equimolar to Part B |
| Part B | <3 μl equimolar to Part A |
| T4 DNA ligase buffer | 1 μl |
| T4 DNA ligase | 0.5 μl |
| dH2O | to 10 μl |

5. Place the tube at 16 oC for 30 min and then at 80 oC for 20 min.
6. Transformation with 1-2 μl of ligation product into appropriate
   bacterial competent cells.

### Transformation for DH5a/BL21 competent cells

##### Introduction

---

Transformation is the process by which foreign DNA is introduced into a
bacterial cell. In order to transform our E.coli competent cells with the
desired plasmids we followed the “High Efficiency Transformation Protocol”
from New England Biolabs.

[Details](https://international.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987)

##### Reagents and equipment

---

* LB agar plate (with appropriate antibiotic)
* LB or SOC media
* Competent cells
* DNA you would like to transform
* Shaking incubator at 37 °C
* Stationary incubator at 37 °C
* Ice bucket filled with ice
* Microcentrifuge tubes
* Sterile spreading device
* Heating-block

##### Procedure

---

1. Thaw a tube of competent E. coli cells on ice until the last ice crystal
   disappears.
2. Mix gently and pipette 50 μl of cells into a transformation tube on ice.
3. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture.
   Carefully flick the tube 4-5 times to mix cells and DNA. **Do not vortex.**
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
6. Place on ice for 5 minutes. Do not mix.
7. Pipette 950 µl of room temperature SOC into the mixture.
8. Place at 37°C for 60 minutes. Shake vigorously 250 rpm.
9. Warm selection plates to 37°C.
10. Mix the cells thoroughly by flicking the tube and inverting, then
    perform several 10-fold serial dilutions in SOC.
11. Spread 50-100 µl of each dilution onto a selection plate and incubate
    overnight at 37°C.

### Restriction enzyme digestion

##### Introduction

---

For the design of all the different single and double digestion that we
performed, we used the NEB restriction digestion tools: “Heat Inactivation”
and “NEBcloner”.

[Details](https://international.neb.com/tools-and-resources/usage-guidelines/heat-inactivation)
[More...](https://nebcloner.neb.com/#!/redigest)

##### Reagents and equipment

---

* 10X rCutSmart Buffer
* Appropriate restriction enzymes
* Nuclease-free Water
* DNA that will be digested
* Ice bucket filled with ice
* Pipette & tips
* PCR tubes
* Heating block machine

##### Procedure

---

###### Single digestion by SapI

1. Set up reaction as follows:

| Component | 50 μl Reaction |
| --- | --- |
| DNA | 1 μg |
| 10X rCutSmart Buffer | 5 μl (1X) |
| SapI | 1.0 μl (10 units) |
| Nuclease - free Water |To 50 μl

2. Incubate at 37 oC for 5-15 minutes.
3. Incubate at 65 oC for 20 minutes for enzyme inactivation.

###### Double digestion

1. Use NEBcloner to find the best conditions for the reaction according to
   the restriction enzymes.
2. Add the reaction components in a sterile tube.
3. Incubate at the best conditions according to NEBcloner.
4. Incubate for enzyme inactivation according to the Heat Inactivation
   table.

#### DNA Isolation and Purification for cloning

### Plasmid-DNA isolation (Miniprep)

##### Introduction

---

In order to isolate the plasmid DNA from liquid bacterial cultures, we used
the NucleoSpin Plasmid kit for plasmid DNA isolation (Macherey-Nagel). We
followed the protocol supplied with the kit by the manufacturer for
isolation of high-copy plasmid DNA (pSB1C3) from E.coli.

[Details](%7B%7Bcontext.docs.pdf.plasmid%7D%7D)

##### Reagents and equipment

---

* Resuspension Buffer A1
* Lysis Buffer A2
* Neutralization Buffer A3
* Wash Buffer AW
* Wash Buffer
* Elution Buffer AE
* RNase A
* Nucleospin® Plasmid Columns
* Collection tubes
* 96-100% ethanol
* Pipette & tips
* 1,5 ml microcentrifuge tubes
* Manual pipettes
* Microcentrifuge
* Vortex mixer
* Heating-block

##### Procedure

---

###### Cultivate and harvest bacterial cells

1. Use 1-5 mL of a saturated E.coli LB culture, pellet cells in a
   microcentrifuge for 30 s at 11,000 x g.
2. Discard the supernatant and remove as much of the liquid as possible.

###### Cell lysis

3. Add 250 μL Buffer A1. Resuspend the cell pellet completely by
   vortexing. Make sure no cell clumps remain before addition of Buffer
   A2!
4. Add 250 μL Buffer A2. Mix gently by inverting the tube 6–8 times.
   Incubate at room temperature for up to 5 min.
5. Add 300 μL Buffer A3. Mix thoroughly by inverting the tube 6–8 times
   until blue samples turn colorless completely! Do not vortex to avoid
   shearing of genomic DNA!

###### Clarification of lysate

6. Centrifuge for 5 min at 11,000 x g at room temperature. Repeat this
   step in case the supernatant is not clear!

###### Bind DNA

7. Place a NucleoSpin® Plasmid/Plasmid (NoLid) Column in a Collection Tube
   (2 mL) and pipette a maximum of 700 μL of the supernatant from step 6
   onto the column.
8. Centrifuge for 1 min at 11,000 x g.
9. Discard flowthrough and place the NucleoSpin® Plasmid/Plasmid (NoLid)
   Column back into the collection tube. Repeat this step to load the
   remaining lysate.

###### Wash silica membrane

10. Add 600 μL Buffer A4.
11. Centrifuge for 1 min at 11,000 x g.
12. Discard flowthrough and place the NucleoSpin® Plasmid / Plasmid (NoLid)
    Column back into the empty collection tube.

###### Dry silica membrane

13. Centrifuge for 2 min at 11,000 x g and discard the collection tube.

###### Elute DNA

14. Place the NucleoSpin® Plasmid / Plasmid (NoLid) Column in a 1.5 mL
    microcentrifuge tube and add 50 μL Buffer AE.
15. Incubate for 1 min at room temperature.
16. Centrifuge for 1 min at 11,000 x g.

### PCR clean-up Gel extraction

##### Introduction

---

For the PCR clean-up of the PCR samples and the extraction of the DNA
samples from agarose gel we used the NucleoSpin Gel and PCR Clean-up XS kit
(Macherey-Nagel). We followed the protocol supplied with the kit by the
manufacturer.

[Details](%7B%7Bcontext.docs.pdf.pcr_clean%7D%7D)

##### Reagents and equipment

---

* Binding Buffer NTI
* Wash Buffer NT3
* Elution Buffer NE
* Nucleospin® Gel and PCR Clean-up XS columns
* Collection tubes
* 96-100% ethanol
* Pipette & tips
* 1,5 ml microcentrifuge tubes
* Manual pipettes
* Centrifuge for microcentrifuge tubes
* Vortex mixer
* Heating-block
* Weighing machine
* Scalpel to cut agarose gels

##### Procedure

---

###### Before starting the preparation:

* Ensure that ethanol was added to Wash Buffer NT3.
* For gel extraction procedure, excise DNA band from agarose gel and
  determine the weight of the gel slice.
* Preheat a heating block to 50 oC.

---

###### Adjust DNA binding condition

1. Mix 1 volume of sample with 2 volumes of Buffer NTI.
2. Vortex
3. Incubate mixture at 50°C with constant shaking until the gel is
   completely dissolved. **(Only for agarose gel extraction)**

###### Bind DNA

4. Place a NucleoSpin® Gel and PCR Clean-up XS Column into a Collection
   Tube (2 mL) and load up to 500 μL sample
5. Centrifuge for 30 sec at 11,000 x g.
6. Discard flowthrough and place the column back into the collection tube.
7. If necessary, load the remaining sample and repeat steps 5,6.

###### Wash silica

8. Add 500 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up XS
   Column.
9. Centrifuge for 30 sec at 11,000 x g.
10. Discard flowthrough and place the column into a 1.5 mL microcentrifuge
    tube.

###### Wash and dry silica

11. Add 300 µL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up XS
    column.
12. Centrifuge for 1 min at 11,000 x g.
13. Discard flowthrough and place the column into a 1.5 mL microcentrifuge
    tube.

###### Elution

14. Add 6-12 µL Buffer NE and incubate at room temperature (18-25°C) for 1
    min.
15. Centrifuge for 1 min at 11,000 x g.
16. Reload the eluates onto the column and repeat steps 14, 15.

#### In Vitro Transcription

### In vitro transcription

##### Introduction

---

We used the HiScribe T7 High Yield RNA Synthesis kit for the in vitro
transcription of crRNAs using T7 RNA Polymerase (New England Biolabs). We
followed the protocol supplied with the kit by the manufacturer.

[Details](https://international.neb.com/protocols/0001/01/01/dna-template-preparation-e2040)
[More...](https://international.neb.com/protocols/0001/01/01/standard-rna-synthesis-e2040)

##### Reagents and equipment

---

* CTP
* UTP
* ATP
* Template DNA
* T7 RNA Polymerase Mix
* GTP
* 10X T7 Reaction Buffer
* Nuclease - free microfuge tubes
* Pipette & tips
* Incubator
* Centrifuge for microcentrifuge tubes
* Ice bucket filled with ice

##### Procedure

---

###### DNA template preparation - Plasmid template

1. Linearization of the plasmid template, using the appropriate restriction
   enzymes
2. Extract DNA with an equal volume of 1:1 phenol/chloroform mixture,
   repeat if necessary.
3. Extract twice with an equal volume of chloroform to remove residual
   phenol
4. Precipitate the DNA by adding 1/10th volume of 3 M sodium acetate, pH
   5.2, and two volumes of ethanol. Incubate at -20°C for at least 30 minutes.
5. Pellet the DNA in a microcentrifuge for 15 minutes at top speed.
   Carefully remove the supernatant.
6. Rinse the pellet by adding 500 μl of 70% ethanol and centrifuging for 15
   minutes at top speed. Carefully remove the supernatant.
7. Air dry the pellet and resuspend it in nuclease-free water at a
   concentration of 0.5-1 μg/μl.

###### RNA synthesis

8. Thaw the necessary kit components, mix and pulse-spin in microfuge to
   collect solutions to the bottom of tubes. Keep on ice.
9. If you are planning to run many reactions, it is convenient to prepare a
   master mix by combining equal volumes of the 10X reaction buffer and four
   ribonucleotide (NTP) solutions. Use 10 μl per reaction.
10. Assemble the reaction at room temperature in the following order:

|  |  |  |
| --- | --- | --- |
| Nuclease-free water | X μl |  |
| 10X Reaction Buffer | 2 μl |  |
| ATP (100 mM) | 2 μl | 10 mM final |
| GTP (100 mM) | 2 μl | 10 mM final |
| UTP (100 mM) | 2 μl | 10 mM final |
| CTP (100 mM) | 2 μl | 10 mM final |
| Template DNA | X μl | 1 μg |
| T7 RNA Polymerase Mix | 2 μl |  |
| Total reaction volume | 20 μl |  |

11. Mix thoroughly, pulse-spin in microfuge.
12. Incubate at 37°C for 2 hours. The yield will not be compromised if the
    incubation temperature is within the range of 35–40°C.
13. *Optional:* DNase treatment to remove DNA template.
14. Purification of synthesized RNA or analysis of transcription products
    by gel electrophoresis.

### Purification of RNA

##### Introduction

---

For the purification of the RNA sample from in vitro transcription (IVT) we
used the Monarch® RNA Cleanup Kit (New England BioLabs). We followed the
protocol supplied with the kit by the manufacturer.

[Details](https://international.neb.com/protocols/2018/06/28/monarch-rna-cleanup-kit-protocol)

##### Reagents and equipment

---

* Monarch® RNA Cleanup Columns (500 μg)
* Monarch® Collection Tubes II
* Monarch® RNA Cleanup Binding Buffer
* Monarch® RNA Cleanup Wash Buffer
* Nuclease-free Water
* 95-100% ethanol
* Pipette & tips
* 1,5 ml microcentrifuge tubes
* Manual pipettes
* Centrifuge for microcentrifuge tubes
* Vortex mixer
* Heating-block

##### Procedure

---

1. Add 100 μl RNA Cleanup Binding Buffer to the 50 μl sample.
A starting sample volume of 50 μl is recommended. For smaller samples,
nuclease-free water can be used to adjust the volume. For samples larger
than 50 μl, scale buffer volumes accordingly. Samples with a starting
volume > 150 μl will require reloading of the column during Step 3.
2. Add 150 μl of ethanol (≥ 95%) to your sample and mix by pipetting or
   flicking the tube. Do not vortex.
3. Insert column into collection tube and load the sample onto column and
   close the cap.
4. Centrifuge for 1 minute at 16,000 x g.
5. Discard flow-through.
6. Re-insert column into collection tube.
7. Add 500 μl RNA Cleanup Wash Buffer
8. Spin for 1 minute at 16,000 x g.
9. Discard the flow-through.
10. Repeat wash steps (Steps 6-9).
11. Transfer column to an RNase-free 1.5 ml microfuge tube
12. Elute in nuclease-free water 6-20 μl and spin for 1 min at 16,000x g **.**
13. The eluted RNA can be used immediately or stored at -70oC

#### SUMO-LbuCas13a Expression and Purification

### Liquid bacterial pre-culture (15ml)

##### Reagents and equipment

---

* LB-Medium
* E.coli BL21 (DE3) Bacteria transformed with the final-T7-LbuCas13a
  plasmid
* Chloramphenicol
* Falcon tube 50ml
* Pipette and tips
* Shaking incubator
* Waste filled with chlorine

##### Procedure

---

1. Under sterile conditions, pour 15 ml of LB-Medium in the Falcon tube
2. Thaw and add 7,5 μl Chloramphenicol
3. Under sterile conditions pick with a tip enough bacteria
4. Throw the tip into the falcon that contains the LB-Medium
5. Place the falcon in a shaking incubator
6. Grow the liquid culture overnight at 37 °C

### Liquid bacterial culture (1000 mL)

##### Reagents and equipment

---

* Sterile Flask 1000 ml with LB-Medium
* Liquid bacterial pre-culture (15ml)
* Chloramphenicol
* IPTG
* Incubator
* Photometer

##### Procedure

---

1. Under sterile conditions, pour 5 ml or more of the liquid bacterial
   pre-culture into the sterile Flask with 1000 ml LB-Medium, until the
   Optical Density (OD) is 0,1 units
2. Add chloramphenicol and place the culture in the incubator at 37 oC
3. Every 30 min measure the culture OD, until it reaches 0,5-0,6 units
4. Add IPTG 1mM
5. Place the culture again in the incubator at 37oC for the
   required hours depending on the experimental design.

### Collection of bacterial pellets

##### Reagents and equipment

---

* Binding buffer
* Glycerol 100%
* 5 Falcons (50 ml)
* Refrigerated centrifugation

##### Procedure

---

1. Divide the culture into 5 falcons
2. Centrifuge for 13 min, 4000 rpm , at 4oC
3. Discard the supernatant
4. Repeat steps 1-3 until the 1000 ml culture finishes
5. Add 23 ml Binding Buffer to one of the five falcons
6. Vortex until the pellet is completely dissolved
7. Pour the solution into the second falcon and repeat steps 6-7 until the
   solution is collected in a final falcon
8. Add to the final falcon glycerol 100% up to 25 ml
9. Vortex

### Freeze-thaw cycles

##### Reagents and equipment

---

* Falcon with the bacterial pellets
* dH2O
* Freezer approximately -60oC , -80 oC
* Thermometer

##### Procedure

---

1. Place the falcon in the freezer (-60oC, -80 oC)
   for 20 min
2. Place a beaker with water on a heat source
3. Place the falcon in it when the temperature reaches 42oC
   until the solution is thawed
4. Repeat steps 1-3
5. Store the falcon in the freezer ( -70oC )

### Lysis of Inclusion Bodies (IBs)

##### Reagents and equipment

---

* Falcon with the bacterial pellets after the freeze-thaw cycles.
* Lysozyme 100 mg/ml
* Triton X-100 280 μl
* DNase 1 mg/ml
* MgCl2 8 mM 2,4 ml
* Protease Inhibitor (PI) 1x 260 μl
* Refrigerator
* Ultrasound sonication
* Refrigerated centrifugation
* Rotator machine

##### Procedure

---

1. Thaw the falcon from the previous step
2. Add 1 ml lysozyme
3. Place the falcon in the refrigerator for 30 min
4. Add 280 μl triton x-100
5. Place it on ice
6. Sonication 30 sec, ice 1 min
7. Repeat step 6, 13 times
8. Add 560 μl DNase, 2,4 ml MgCl2 , 260 μl PI
9. Incubate the sample in a cold room for 2 hours using rotator machine
10. Centrifuge for 20 min, 14000 rpm , at 4oC
11. Discard the supernatant
12. Store the pellets at -20oC

### Processing of the Inclusion Bodies

##### Reagents and equipment

---

* Falcon that contains the lysed Inclusion Bodies
* Buffers A, B, C
* Protease Inhibitor (PI) 1x
* L-Arg
* Refrigerator
* Refrigerated centrifugation
* Rotator machine
* Weighing Machine

##### Procedure

---

1. Take out of the freezer the falcon of the previous step
2. Centrifuge for 5 min , 10000 rcf at 4oC
3. Discard the supernatant, if exists
4. Weigh the falcon and calculate the quantity of Buffer A and PI that is
   needed
5. Add Buffer A and PI and dissolve completely
6. Rotation in the refrigerator for 30 min
7. Centrifuge for 20 min , 15000 rcf at 4oC
8. Discard the supernatant
9. Weigh the falcon and calculate the quantity of Buffer B and PI that is
   needed
10. Add Buffer B and PI and dissolve completely
11. Rotation in the refrigerator for 15 min
12. Centrifuge for 15 min , 15000 rcf at 4oC
13. Discard the supernatant
14. Weigh the falcon and calculate the quantity of Buffer C and PI that is
    needed
15. Rotation in the refrigerator for 5 min
16. Centrifuge for 5 min , 6000 rcf at 4oC
17. Discard the supernatant
18. Steps 1-17 take place on ice
19. Add 800 μl L-Arg and dissolve
20. Centrifuge for 5 min , 4000 rcf at room temperature
21. Collect the supernatant on another falcon and store (1st
    spin)
22. Repeat steps 19-20
23. Collect the supernatant on another falcon and store (2nd
    spin)
24. Under sterile conditions, filter the 1st and 2nd
    spin with 45 mm filter
25. Store the falcons at room temperature

### Purification of SUMO-LbuCas13a

##### Reagents and equipment

---

* Cytiva His-trap column kit
* Cytiva His-trap buffer kit
* SUMO-LbuCas13a protein
* Centrifuge machine

##### Procedure

---

*Note* All centrifugations were performed at 2500g for 1min at Room
Temperature

1. Removal of column's storage buffer by centrifugation (discard the
   flowthrough).
2. Equilibration of the His-trap column by adding 600μl binding buffer of
   20mM imidazole and centrifuge (discard the flowthrough).
3. Place 600μl of the sample to the column (protein binding to the matrix)
   and centrifuge (discard the flowthrough).
4. Repeat step 3 until the column's capacity is reached.
5. Perform 3 washing steps using binding buffers of 40 mM imidazole, 80mM
   imidazole and 100mM imidazole (centrifuge and discard the flowthrough).
6. Addition of 200mM elution buffer containing 500mM of imidazole
   (centrifuge and store the flowthrough)
7. Repeat step 6.

### Cleavage of SUMO tag

##### Reagents and equipment

---

* 100 μl template (protein of interest)
* 20 μl SUMO protease buffer
* 10 μl ULP1 protease
* Up to 200 μl DEPC H2O
* Pipette & tips
* Sterile tubes
* Heat block machine

##### Procedure

---

1. Add all the reagents in a tube
2. Incubate for 2 hours at 37 oC

### SDS page electrophoresis

##### Reagents and equipment

---

* Bis-acrylamide 30%
* Tris-HCl 1M PH 8,80
* Tris-HCl 1M PH 6,8
* APS 10%
* TEMED
* Loading Dye 5X
* Running Buffer
* 2-Propanol
* SDS 10%
* dH2O
* Protein marker
* Coomassie blue staining
* Electrophoresis apparatus
* Glass cast
* Cast stand
* Thermoblock

##### Procedure

---

###### Gel preparation

1. Glass cast assembly
2. Build the separating phase and fill in the glass cast compartment
3. Add 2-propanol on top 1ml
4. Wait for 1 hour and 30 min for the gel to polymerize
5. Wash out the 2-propanol using dH2O
6. Dry the compartment between the glasses
7. Create the stacking phase and add it to the cast
8. Wait about 1 hour and 30 min for the gel to solidify

###### Sample preparation

9. For every 30 μl of sample add 7,5 μl of loading dye 5X
10. Boil the sample for 10 min at 95oC using a Thermoblock

###### Running of the polyacrylamide gel

11. Assembly the electrophoresis apparatus
12. Add the running buffer
13. Load the sample (30 μl max) and the protein marker (4 μl)
14. Start the electrophoresis at 80 V for 30 min and then raise the value
    at 140 V for 1h
15. Remove the gel from the cast and put it in staining or use it for
    Western Blot

### Western Blotting

##### Reagents and equipment

---

* Transfer Buffer (cold)
* PBST Buffer
* Methanol
* Blocking Buffer
* Primary antibody Anti- His Santa Cruz 1:2000 ( 4 ml PBST, 3% milk, 2 μl
  antibody)
* Alkaline phosphatase buffer
* NBT solution
* BCIP solution
* Western blot apparatus
* PVDF membrane
* Whatman filter papers
* Gel from SDS electrophoresis
* Stirring machine
* Rocking machine

##### Procedure

---

1. Soak the cassette pads in the transfer buffer.
2. Wet the membrane with methanol for 1 min to activate it and place it in
   the transfer buffer.
3. Cut 4 whatman filter papers and place them in the transfer buffer.
4. Remove gel from cast and leave it in the transfer buffer.
5. Assembly of cassette : place a pad, two whatman papers and on top of
   them the gel. Put the membrane on top of the gel plus two more whatman
   papers and another pad (leave out all air bubbles). Close the cassette and
   place it in the apparatus and fill up with transfer buffer. Add a magnet
   for stirring and ice if desired.
6. Transfer in the cold room (4oC) for 2 h at 100V, 0,3A.
7. Remove membrane and place it in blocking buffer for 1h and 30 min on the
   rocker machine.
8. Remove the blocking buffer and place the membrane in the primary
   antibody solution and leave it at 4 oC overnight.
9. The next day remove the primary antibody and perform four PBST washes of
   5 min each (5ml per membrane, rocker machine).
10. Add the secondary antibody solution and place it on the rocker machine
    for 1h and 20 min.
11. Remove the secondary antibody and perform again four PBST washes of 5
    min each (5 ml per membrane, rocker machine).
12. Start a new set of four washes using AP buffer (5 ml per membrane,
    rocker machine).
13. Add 4 ml AP buffer to the membrane plus 13,3 μl BCIP and 17,6 μl NBT
    (rocker machine).
14. Wait for the bands to appear.

#### Cell culturing and RNA isolation

### Cell culturing and sub-culting

##### Reagents and equipment

---

* Cell culture vessel
* DMEM High Glucose (4.5 g/l), with L-Glutamine, without Sodium Pyruvate
* 0.25% (w/v) Trypsin- 0.53 mM EDTA solution
* Fetal bovine serum (FBS)
* Penicillin-Streptomycin (10.000 U/mL)
* PBS 1X
* 75% ethanol
* Serological pipettes
* Pipette & tips
* Microcentrifuge
* Fume hood
* Tissue cell culture dishes

##### Procedure

---

###### Initiating frozen cultures

1. Prepare a culture vessel so that it contains the recommended volume of
   the appropriate culture medium as listed on the Product Sheet,
   equilibrated for temperature and pH (CO2).
2. Thaw the vial by gentle agitation in a water bath at 37°C or the normal
   growth temperature for that cell line. Thawing should be rapid,
   approximately 2 minutes or until ice crystals have melted.
3. Remove the vial from the water bath and decontaminate it by dipping in
   or spraying with 70% ethanol. Follow strict aseptic conditions in a
   laminar flow tissue culture hood for all further manipulations.
4. Unscrew the top of the vial and transfer the contents to a sterile
   centrifuge tube containing 9 mL of the recommended medium. Remove the
   cryoprotectant agent (DMSO) by gentle centrifugation (10 minutes at 125
   × g). Discard the supernatant, and resuspend the cells in 1 or 2 mL of
   complete growth medium. Transfer the cell suspension into the culture
   vessel containing the complete growth medium and mix thoroughly by
   gentle rocking.
5. Examine the cell cultures after 24 hours and subculture as needed.

###### Subculturing procedure

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA
   solution to remove all traces of serum which contains trypsin
   inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells
   under an inverted microscope until the cell layer is dispersed (usually
   within 5 to 15 minutes).

   Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by
   gently pipetting.
4. Add appropriate aliquots of the cell suspension to new culture vessels.
5. Incubate cultures at 37°C.

We followed a subcultivation ratio of 1:2 to 1:5 and the medium was changed
every 2 days.

### Total RNA extraction and purification from A549, MRC5 cell lines

##### Introduction

---

In order to extract and purify the total RNA from the A549 and MRC5 cell
lines we used the RNeasy Micro Kit (Qiagen Inc.) We followed the protocol
supplied with the kit by the manufacturer.

[Details](%7B%7Bcontext.docs.pdf.rna_extract%7D%7D)

##### Reagents and equipment

---

* Cell culture vessel
* DMEM High Glucose (4.5 g/l), with L-Glutamine, without Sodium Pyruvate
* 0.25% (w/v) Trypsin- 0.53 mM EDTA solution
* Fetal bovine serum (FBS)
* Penicillin-Streptomycin (10.000 U/mL)
* PBS 1X
* 75% ethanol
* Serological pipettes
* Pipette & tips
* Microcentrifuge
* Fume hood
* Tissue cell culture dishes
* RNeasy Micro Kit (Qiagen Inc.)
* RNase free microcentrifuge tubes
* Pipette & tips
* Fume hood

##### Procedure

---

###### Cell seeding and detaching

1. The cells were seeded in 6-well plate at a density of 0.3 x 10 6 in high glucose Dulbecco’s
   modified Eagle Medium (DMEM),
   supplemented with 10% FBS and 1% PS and incubate at 37oC, 5%
   CO2
2. When the cells reached at about 80% confluency, were detached with
   Trypsin-EDTA solution, washed with PBS 1X and the total number of cells
   was estimated using a Neubauer chamber.
3. 5X105/per cell line were used as the starting material for the RNA
   isolation procedure utilizing the RNeasy Micro Kit (Qiagen Inc.)

###### RNA isolation

4. Add 10 μl β-mercaptorthanol (β-ME) to 1 ml Buffer RLT before use. Store
   at room temperature.
5. The cells suspension was centrifuged at 1200xg for 5 minutes to collect
   the cell pelet
6. Add 350 μl Buffer RLT and homogenize.
7. Add 1 volume of 70% ethanol to the lysate and mix well by pipetting. Do
   not centrifuge! Proceed immediately to the next step!
8. Transfer the sample to a RNeasy MinElute spin column in a 2 ml
   collection tube. Close the lid and centrifuge for 15 sec at ≥8000 x g.
   Discard the flow-through.
9. Add 350 µl Buffer RW1 to the RNeasy MinElute spin column. Close the
   lid. Centrifuge for 15 s at ≥8000 x g. Discard the flow-through.
10. Add 10 µl DNase I stock solution to 70 µl Buffer RDD. Mix by inverting
    the tube. Add the DNase I incubation mix (80 µl) directly to the RNeasy
    MinElute spin column membrane. Place on the benchtop (20–30°C) for 15
    min.
11. Add 350 µl Buffer RW1 to the RNeasy MinElute spin column. Close the
    lid, and centrifuge for 15 sec at ≥8000 x g. Discard the collection
    tube.
12. Place the RNeasy MinElute spin column in a new 2 ml collection tube.
13. Add 500 µl Buffer RPE to the spin column. Close the lid, and centrifuge
    for 15 sec at ≥8000 x g. Discard the flow-through
14. Add 500 µl of 80% ethanol to the RNeasy MinElute spin column. Close the
    lid, and centrifuge for 2 min at ≥8000 x g. Discard the collection
    tube.
15. Place the RNeasy MinElute spin column in a new 2 ml collection tube.
    Open the lid of the spin column, and centrifuge at full speed for 5 min
    to dry the membrane. Discard the flow-through and collection tube.
16. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube.
    Add 14 µl RNase-free water directly to the center of the spin column
    membrane. Close the lid gently, and centrifuge for 1 min at full speed
    to elute the RNA.

#### Microfluidics

### 3D-printed microfluidic device manufacturing with DLP technology

##### Reagents and equipment

---

* DLP 3D printer
* Resin
* Isopropyl alcohol
* Syringe pumps

##### Procedure

---

1. Design the 3D model of the appropriate microfluidic device through a
   Computer-Aided Design.
2. Export the STL file of the device
3. Use the STL file as an input to the according slicing software of the
   DLP 3D printer.
4. Slice the model with layer height of 0.05 mm and Time exposure 40
   seconds for every layer.
5. Fill the resin vat with the resin of your choice.
6. Start the printing process.
7. Remove the device from the platform.
8. Clear the microfluidic channels with isopropyl alcohol.
9. Ready for testing!

![]({{  context.figures.microfluidics.thumb  }})

#### CRISPR/Cas13a bulk fluorescence experiments

### Quantification of the target miRNA in total RNA extracts isolated from cell cultures

##### Reagents and equipment

---

* PBS 1X
* 75% ethanol
* Serological pipettes
* Pipette & tips
* Fume hood
* RNase free microcentrifuge tubes
* Pipette, tips & falcon tubes
* Microcentrifuge
* 96-well black microplate (Greiner Bio-one)
* Fluorescence microplate reader

##### Procedure

---

###### Step 1 (RS mixture preparation)

The **RS reaction mix** contains the reagents that are shown
in the following table in a final volume of 30μl. The RS mixture is
prepared by adding the same volume of RNA reporter, a specified amount of
total RNA sample extracted by each cell line according to the RNeasy Micro
Kit (Qiagen Inc.), reaction buffer 5X and water. For estimating the miRNA
levels in RNA samples isolated from 2 different cell lines, 2 RS mixtures
should be prepared each having the corresponding RNA sample.

| RS mixture (1 for each cell line) | | |
| --- | --- | --- |

---

| Reagent | Quantity μl (for 20μl mix) | Multiply by the number of technical replicates (e.g. 4) |
| Reporter 5μM | 10 | 40 |
| RNA sample (83.3ng) | 10 | 40 |
| Reaction Buffer 5X | 6 | 24 |
| Water | 4 | 16 |

###### Step 2 (CC reaction mixture preparation)

The **CC reaction mix** contains the reagents that are shown
in the following table in a final volume of 20μl.

| CC mixture | | |
| --- | --- | --- |
| Reagent | Quantity μl (for 20μl mix) | X number of total samples (e.g. 2 cell lines X 4 replicates/ cell line =8) |
| LbuCas13a 2pmol/μl | 0.5 | 4 |
| crRNAx 100nM | 10 | 80 |
| Reaction Buffer 5X | 4 | 32 |
| Water | 5.5 | 44 |

###### Step 3 (microplate preparation)

20μl of the RS mix corresponding to the same cell line is added on the
specified wells of the microplate, followed by the addition of 30μl of the
CC reaction mixture. The mixture is pipetted up and down several times and
the enzyme reaction starts.

The above procedure is repeated for all the different RS mixtures which
correspond to the RNA extracts isolated from different cell lines.

###### Step 4 (fluorescence measurement)

The plate is inserted into the appropriate fluorescence plate reader and
the fluorescence detection should be conducted within the first 5 minutes
after reaction initiation for best results.

| Reaction Buffer 5X for Cas13a/crRNA assays | |
| --- | --- |
| Component | Concentration |
| Tris HCl | 10mM |
| KCl | 50mM |
| MgCl2 | 1.5mM |
| pH 8.3 | |

## Solutions Preparation

---

### Preparation of Glycerol 50%

##### Reagents and equipment

---

* 99% glycerol
* dH2O
* Duran bottle
* Serological pipette

##### Procedure

---

1. Add 1 part 99% glycerol in a sterile bottle
2. Add 1 part of sterile H2O and mix until the glycerol is completely dissolved
3. Autoclave the solution

### Preparation of Tris-Acetate-EDTA (TAE) Buffer 10x

##### Reagents and equipment

---

* 48.5 g Tris Base
* 11.4 mL glacial acetic acid
* 20 mL 0.5M EDTA (pH 8.0)
* Duran bottle
* Weighing machine

##### Procedure

---

1. Dissolve Tris in about 800 mL of deionized water
2. Add acetic acid and EDTA
3. Add deionized water to a final volume of 1L
4. Sterilize by autoclaving
5. Store at room temperature

### Chloroform: Isoamyl alcohol 24:1

##### Reagents and equipment

---

* 14,4 ml chloroform
* 600 μl isoamyl alcohol
* Duran bottle
* Fume hood

##### Procedure

---

1. Mix the two solutions
2. Store at 4oC

### Phosphate Buffered Saline 10X (PBS)

##### Reagents and equipment

---

* 80 gr NaCl
* 2 gr KCl
* 14.4 gr Na2HPO4
* Up to 1 L dH2O
* Duran bottle
* Weighing machine
* Autoclave
* pH meter

##### Procedure

---

1. Weigh the appropriate quantities of solid ingredients
2. Dissolve the reagents in dH2O
3. Adjust pH to 7.4
4. Sterilize by autoclaving
5. Store at Room Temperature

### Radioimmunoprecipitation Assay Buffer (RIPA)

##### Reagents and equipment

---

* 1.5 ml NaCl 5M
* 0.5 ml SDS 10%
* 5 ml Nonidet P-40 10%
* 0,5 g Deoxycholic acid sodium salt
* 0.5 ml Sodium phosphate pH 7.2
* Duran bottle
* Weighing machine
* Autoclave

##### Procedure

---

###### For 1L solution

1. Add all the solutions in a Duran bottle
2. Weigh 0.5 Deoxycholic acid sodium salt and add it to the bottle
3. Mix until complete dissolve
4. Sterilize by autoclaving
5. Store at 4oC

### 30% Bis-Acrylamide

##### Reagents and equipment

---

* 116,8 g Acrylamide
* 3.2 g Bis
* Up to 400 ml dH2O
* Sterile Duran bottle
* Weighing machine
* Protective mask

##### Procedure

---

###### For 400 ml solution

1. Weigh the appropriate amounts of solid ingredients
2. Dissolve them in about 400 mL of dH2O in a Duran bottle
3. Heat the solution for complete dissolve if it is needed
4. Store at 4oC

### Alkaline phosphatase buffer 10% (APS)

##### Reagents and equipment

---

* 0.1 g APS
* Up to 1 ml dH2O
* Sterile falcon
* Weighing machine
* Protective mask

##### Procedure

---

###### For 1 ml solution

1. Weigh the 0.1 g of APS
2. Dissolve it in 1 mL of dH2O
3. Store at 4oC

### Phosphate Buffered Saline with Tween 20 (PBST solution)

##### Reagents and equipment

---

* 2 ml Tween 10% solution
* 198 ml PBS 1x solution
* Sterile Duran bottle

##### Procedure

---

###### For 200 ml solution

1. Add the solutions in a Duran bottle and mix
2. Store at Room temperature

### Destaining solution

##### Reagents and equipment

---

* 300 ml Methanol
* 100 ml Acetic acid
* Up to 1000 ml dH2O
* Sterile Duran bottle

##### Procedure

---

###### For 1L solution

1. Add the solutions in a Duran bottle and mix
2. Add up to 1000 ml dH2O and dissolve
3. Store at Room temperature

### L-Arginine solution pH 7

##### Reagents and equipment

---

* 2,613 g L-arginine
* 1,2 ml Tris-HCl 1M pH: 7,6
* Up to 30 ml dH2O
* Sterile Duran bottle
* Weighing machine
* pH meter

##### Procedure

---

###### For 30 ml solution 0.5M

1. Weigh L-Arginine and dissolve it to Tris-HCl
2. Add up to 30 ml dH2O and dissolve
3. Adjust the pH to 7.0
4. Store at Room temperature

### DDT (Dithiothreitol)

##### Reagents and equipment

---

* 0,463 g DDT
* Up to 1 ml dH2O
* Sterile tube
* Weighing machine

##### Procedure

---

###### For 1 ml solution 3M

1. In a tube weigh 0,463 g DTT
2. Add up to 1 ml dH2O and dissolve
3. Store at - 20oC

### Isopropyl-beta-D-Thiogalactopyranoside (IPTG)

##### Reagents and equipment

---

* 2,383 g IPTG
* Up to 10 ml dH2O
* Sterile falcon
* Filter 0,22 μm
* Weighing machine

##### Procedure

---

###### For 10 ml solution 1M

1. In a falcon weigh 2,383 g IPTG
2. Dissolve IPTG in 8 ml dH2O
3. Add up to 10 ml dH2O
4. Filter the solution using a 0,22 μm filter
5. Store at - 20oC

### Lysozyme

##### Reagents and equipment

---

* 10 mg Lysozyme
* Up to 1 ml Glycerol
* Sterile tubes
* Filter 0,22 μm
* Weighing machine

##### Procedure

---

###### For 10 ml solution 1M

1. In a tube weigh 10 mg Ly-ozy me
2. Add up to 8 ml dH2O and dis-olv e
3. Filter the solution using a 0,22 μm -ilt er
4. Store at - 20oC

### Nitro Blue Tetrazolium (NBT)

##### Reagents and equipment

---

* 75 mg NBT
* 0,7 ml 70% v/v DMF
* Sterile tubes
* Weighing machine

##### Procedure

---

###### For 0,7 ml solution

1. In a tube weigh 75 mg NBT
2. Add 0,7 ml DMF and dissolve
3. Store at - 20oC

### 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP)

##### Reagents and equipment

---

* 50 mg BCIP
* 1 ml 100% DMF
* Sterile tubes
* Weighing machine

##### Procedure

---

###### For 1 ml solution

1. In a tube weigh 50 mg BCIP
2. Add 1 ml DMF and dissolve
3. Store at - 20oC

### Alkaline Phosphatase (AP) Buffer

##### Reagents and equipment

---

* 60 ml Tis-HCl 1M
* 12 ml NaCl 5M
* 30 ml MgCl2
* Up to 600 ml dH2O
* Sterile Duran bottle
* pH meter

##### Procedure

---

###### For 600 ml solution

1. Add the reagents in a Duran bottle and mix
2. Adjust pH to 9,5
3. Store at Room Temperature

### EDTA

##### Reagents and equipment

---

* 186.1 g disodium EDTA
* Up to 1000 ml dH2O
* Duran bottle
* pH meter
* Weighing machine
* Autoclave

##### Procedure

---

###### For 1L solution 0,5M (pH 8.0)

1. In a Duran bottle weigh 186.1 g disodium EDTA
2. Add dH2O up to 1000 ml and adjust the pH to 8
3. Stir vigorously on a magnetic stirrer
4. Sterilize by autoclaving
5. Store at Room Temperature

### Solutions for purification of bacterial inclusion bodies (IBs)

##### Reagents and equipment

---

| Buffer A | Buffer B | Buffer C |
| --- | --- | --- |
| 50 mM Tris-HCl pH 8.0 | 50 mM Tris-HCl pH 8.0 | 50 mM Tris-HCl pH 8.0 |
| 100 mM NaCl | 100 mM NaCl | 100 mM NaCl |
| 1 mM EDTA | 1 mM EDTA | 1 mM EDTA |
| 0,1% sodium deoxycholate DOC | 1% Nonidet P-40 | - |
| Up to 200 ml dH2O | Up to 100 ml dH2O | Up to 100 ml dH2O |
| Duran bottle | Duran bottle | Duran bottle |
| Weighing machine | Weighing machine | Weighing machine |

##### Procedure

---

| 1. | Add all the solutions in a Duran bottle and mix |
| 2. | Add the appropriate amount of dH2O |
| 3. | Store at Room Temperature |

### Loading Dye

##### Reagents and equipment

---

* 1.25 ml Tris-HCl 1M pH 6.8
* 0.5 g SDS 10%
* 0.0063 g Bromophenol Blue
* 2.5 ml glycerol 10%
* Up to 5 ml dH2O
* Sterile falcon
* Weighing machine

##### Procedure

---

1. Weigh the solid ingredients
2. Add all the reagents in a sterile falcon
3. Add dH2O up to 1000 ml and dissolve
4. Store at 4oC

### Transfer Buffer 1X

##### Reagents and equipment

---

* 100 ml Transfer buffer 10X (Glycine & Tris-Base)
* 200 ml Methanol
* Up to 1000 ml dH2O
* Sterile Duran bottle

##### Procedure

---

###### For 1L solution

1. Add all the reagents in a Duran bottle
2. Add dH2O up to 1000 ml and mix
3. Store at 4oC

### Staining solution

##### Reagents and equipment

---

* 180 ml Methanol
* 40 ml Acetic acid
* 400 mg Coomassie BB-R
* Up to 400 ml dH2O
* Sterile Duran bottle
* Weighing machine

##### Procedure

---

###### For 400 ml solution

1. Weigh the Coomassie
2. Add and mix all the reagents in a sterile Duran bottle
3. Add dH2O up to 400 ml and dissolve
4. Store at Room temperature

### Running Buffer 1X

##### Reagents and equipment

---

* 14.4 g Glycine
* 3 g Tris-Base
* 1 g SDS
* Up to 1000 ml dH2O
* Sterile Duran bottle
* Weighing machine

##### Procedure

---

###### For 1L solution

1. Weigh the solid ingredients
2. Dissolve glycine and Tris-Base in 800 ml dH2O
3. Add SDS and dissolve
4. Add dH2O up to 1000 ml and dissolve
5. Store at Room temperature

## Primer Sequences

---

### Primers for LbuCas13a

| Primer Name | Primer Sequence |
| --- | --- |
| cas13a P1 FWD | aGGTCTCGGAATTCGCGGCCGCTTCTAGatgggcagcagccatc |
| cas13a P1 RVS | aGGTCTCagctcCtctttgtccaggtagtacttg |
| cas13a P2 FWD | aGGTCTCagagcttaatgacaagaacatcaaatatg |
| cas13a P2 RVS | aGGTCTCttcagttggcgaaagattttcag |
| cas13a P3 FWD | aGGTCTCactgaaCtctgcgaatgtcttccgttatc |
| cas13a P3 RVS | aGGTCTCaatgcgcagcaacagaccttgcagcagatttaactcgttAaattcaaccttgtttttgagatg |
| cas13a P4 FWD | aGGTCTCcgcattcttcaccgtttagttggttatacgagcatttgggaaCGCgacctgcgttttcgtctg |
| cas13a P4 RVS | aGGTCTCGACTGCAGCGGCCGCTACTAGTAttattaattctcgcttttcttctcttcc |
| SUMOLESS FWD | aGGTCTCcatgaaagtcaccaaagtgggtg |
| SUMOLESS RVS | aGGTCTCttcatgccgctgctgtgatg |
| cas13a T7 P0 FWD | aGGTCTCGGAATTCGCGGCCGCTTCTAGAGttattactgcccgctttccag |
| cas13a T7 P0 RVS | aGGTCTCctagTggggaattgttatccgctc |
| cas13a T7 P1 FWD | aGGTCTCtactagaaataattttgtttaactttaagaaggag |
| cas13a P1b FWD | aGGTCTCaTACCatgggcagcagccatc |
| Pv | aGGTCTCacagtccggcaaaaaaggg |
| Ev | ccgGGTCTCaattccagaaatcatccttagcgaaagc |
| cas13a RBS FWD | aGGTCTCtactagagaaagaggagaaatactagatgggcagcagccatc |
| VF 2 | TGCCACCTGACGTCTAAGAA |
| VR | ATTACCGCCTTTGAGTGAGC |
| Ep | agctttcgctaaggatgatttctGGTCTCggaattcgcg |
| Pp | accttgcccttttttgccGGTCTCgactgcagcg |
| Sp | cccttttttgccggactgcagcggccgctaGGTCTCcTAGTa |
| Xp | ggatgatttctggaattcgcggccGGTCTCtACTAga |
| OP1 | cgctaaggatgatttctggaattcgcaggccgcttggtctcaactagagaaagaggagaaatactagatg |
| Prham.Brk-F | aggtctctgaattcgcggccgcttctagagttattaatctttctgcgaattgag |
| Prham.Brk-R | aggtctcgactgcagcggccgctactagtattcattacgaccagtctaaaaag |

### Primers for crRNAs

| Primer Name | Primer Sequence |
| --- | --- |
| Loop RVS standard | aGGTCTCaTTAGTCCCCTTCATTTTTGGGGTGGTCCTATAGTGAGTCGTATTACTCTAGAAGCGGCCGCGAATTC |
| 3ps spacer FWD interchangeable | aGGTCTCACTAAAACTACAAGTGCCTTCACTGCAGAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
| 3pm spacer FWD interchangeable | aGGTCTCACTAAAACTACAAGTGCCTTCACAGCAGAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
| 3pe spacer FWD interchangeable | aGGTCTCACTAAAACTACAAGTGCCTTCACTGCAGAGTGAAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
| 5ps spacer FWD interchangeable | aGGTCTCACTAAAACCCTGCACTGTAAGCACTTTGAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
| 5pm spacer FWD interchangeable | aGGTCTCACTAAAACCCTGCACTGTAAGCAGTTTGAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
| 5pe spacer FWD interchangeable | aGGTCTCACTAAAACCCTGCACTGTAAGCACTTTGGTGCTAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
| Loop FWD standard | aGGTCTCaGAAGATCCTTTGATCTTTTCTACGGGGTCTG |

## Cloning Strategy

---

#### A Golden Gate-based cloning pipeline for the construction of our final constructs.

##### Introduction

A fundamental goal of our project design is to construct functional genetic elements in accordance with the
principles of standardization, interchangeability and modularity. Synthetic biology engineering approaches are
based on a combination of multiple genetic elements that can be easily interchanged between biological systems to
provide new capabilities and applications. In this context, we designed and constructed all our genetic elements
in accordance with the BioBrick RFC[10] standard. Our genetic parts are flanked by the prefix and suffix sequence
at the beginning and the end of the part respectively enabling the easy interchangeability of the genetic
sequences utilizing standard cloning methods. The restriction sites that also appear at the prefix and suffix and
could interfere with the assembly methods have been successfully removed from the inserted sequences with
mutagenesis PCR. Considering that the iGEM registry uses pSB1C3 high copy number plasmid as its shipping standard,
we cloned all our parts in pSB1C3 before admission to the registry.

##### Golden Gate-based "SevaBrick Assembly" method.

Given its advantages and as we were attempting to clone multiple inserts for protein fusion constructs in the
pSB1C3 plasmid backbone, the Golden Gate-based "SevaBrick Assembly" method introduced by Stamatios G. Damalas and
colleagues[seva 3.1] as the primary cloning method for our project (Damalas et al., 2020). This method consists of
standardized primers and protocols and facilitates the straightforward one-step assembly of multiple genetic
elements into the SEVA 3.1 or the pSB#X# (# is determined by the identity of the replication origin and the letter
X is determined by the antibiotic resistance marker) backbones in a fast and reliable process. The SevaBrick
Assembly is a method where all the parts and backbones to be assembled are PCR amplified from any SEVA 3.1 or
BioBrick vector, using a core set of standard long primers. All SevaBrick primers anneal on standard sequences of
the SEVA 3.1 or BioBrick vectors, introducing BsaI recognition sites for directional multipart assembly via Golden
Gate (Figure 1).

[![]({{  context.figures.Figure_1.thumb  }})](%7B%7B%20%20context.figures.Figure_1.image%20%20%7D%7D)

Graphical overview of the SevaBrick Assembly. Inserts and backbones are amplified using a core set of
standard primers which introduce compatible Bsal sites, allowing the construction of expression vectors with
varying degrees of complexity via Golden Gate (Figure modified from Damalas et al., 2020).

The majority of our parts were large in kb and thus the Gibson Assembly was inappropriate for our cloning
strategy. In contrast to Gibson assembly which requires three different enzymes and can be challenging, the Golden
Gate-based cloning method that we followed requires only a type II S restriction enzyme and a DNA ligase for the
efficient assembling of genetic sequences. However, the tremendous advantage of this method is that it is
typically performed as an all-in-one-pot reaction minimizing handling time and maximizing cloning efficiency.
Specifically, the properly designed DNA parts, the type II S restriction enzyme and a DNA ligase are mixed in a
PCR tube and placed in a thermocycler. The DNA elements get digested due to restriction enzyme functionality and
are ligated by the DNA ligase. This process is repeated over and over again and the elements are forming the
desired products since the properly assembled parts lack the restriction sites that exist at the unincorporated
DNA parts.

Utilizing the Golden Gate-based "SevaBrick Assembly" method in combination with additional proprietary primers and
method modifications, we have successfully assembled all our basic parts such as protein-coding sequences, into
the pSB1C3 plasmid backbone. Using this methodology we succeed in the proper assembly of 5 different parts at the
desired vector on the first try and with the least possible waste of valuable time. All our parts are compatible
with the BioBrick RFC[10] standard facilitating their incorporation into complicated genetic devices and composite
parts. The pSB1C3 plasmids with integrated coding sequences of our basic parts are called Repository (Repos)
plasmids for convenience. Specific details regarding the description and the individual cloning process that we
followed for the assembling of our final constructs are described below.

[Detailed Description](%7B%7Bcontext.docs.details%7D%7D)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

## Overview

---

This year our team decided to deal with a project which combines innovative synthetic biology-based methodologies
and engineering tools with state-of-the art technologies for 3D-printed microfluidics manufacturing. Knowledge
from different scientific fields including molecular biology, biochemistry and nanotechnology was employed. To
construct the final DIAS detection platform for accurate target miRNA detection, multiple basic “components” had
to be developed from scratch and subsequently efficiently assembled as a functional detection system with high
reliability. After conceptualizing the idea behind DIAS system, considerable in silico work had to be done to
efficiently design the desire genetic parts for our system. And then comes the lab work. Several PCR methodologies
along with multiple cloning procedures were employed to construct the final RFC [10] compatible plasmids bearing
the desired sequences for the LbuCas13a expression and the crRNAs in vitro transcription. Afterwards, several
experiments were conducted to efficiently express and purify the LbuCas13a protein for subsequent experiments in
the detection assay. Regarding the microfluidic chips manufacturing, several trial-and-error methodologies were
employed to construct the chip in its final form. And last but not least, the system's "components" produced in
the previous experimental steps had to be tested as a "whole functional machine" in several detection assays.
Below we provide an extensive description of our experimental results which could be divided into 4 main
categories:

* [PCR amplification and cloning experiments.](#section_2)
* [LbuCas13a protein expression and purification.](#section_3)
* [Droplet microfluidic chip manufacturing.](#section_4)
* [Feasibility of LbuCas13a/crRNA system for miRNA detection in bulk solution.](#section_5)

This section provides all the necessary experimental results regarding the PCR amplification steps and cloning
experiments that we followed to construct the final pSB1C3 plasmid. Further information regarding the cloning
strategy that employed for the construction of each plasmid is provided on the
[experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

## Cloning experiments

---

#### Results from cloning experiments

##### Construction of Cloned SUMO-LbuCas13a Repos ([BBa\_K4170014](%7B%7B%20context.parts.BBa_K4170014.url%20%7D%7D)) plasmid

This plasmid contains the coding sequence (CDS) of codon-optimized LbuCas13a protein in fusion with small
ubiquitin-like modifier (SUMO) protein fixed to pSB1C3 backbone utilizing two standard techniques: a) mutagenesis
PCR amplification; b) Golden Gate Assembly.

Mutagenesis PCR was performed for Cas13a sequence by combining 4 pair of primers, fragmenting LbuCas13a into 4 parts
(Cas13a P1, Cas13a P2, Cas13a P3, Cas13a P4), as described in the
[experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.
After the completion of the mutagenesis PCR amplifications, 1% agarose gel electrophoresis was conducted. As shown in
Figure 1 all the PCR amplifications DNA ladder 100bp succeeded. Cas13a P1 part (1240 bp), Cas13a P2 part (734 bp),
Cas13a P3 part (1338 bp), Cas13a P4 part (646 bp) and pSB1C3 backbone linearized (2052 bp) are depicted in Figure 1.

[![]({{  context.figures.Figure_1.thumb  }})](%7B%7B%20%20context.figures.Figure_1.image%20%20%7D%7D)

1 % agarose gel electrophoresis of the PCR amplified SUMO-Cas13a parts and pSB1C3 backbone. DNA ladder:
100bp DNA Marker NIPPON Genetics EUROPE. Cas13a P1 part: Cas13a P1 part amplified from
pGJK\_His-SUMO-LbuCas13a with Cas13a P1 FWD and Cas13a P1 RVS primers (1240 bp). Cas13a P2 part: Cas13a P2
part amplified from pGJK\_His-SUMO-LbuCas13a with Cas13a P2 FWD and Cas13a P2 RVS primers (734 bp). Cas13a P3
part: Cas13a P3 part amplified from pGJK\_His-SUMO-LbuCas13a with Cas13a P2 FWD and Cas13a P3 RVS primers
(1338 bp). Cas13a P4 part: Cas13a P4 part amplified from pGJK\_His-SUMO-LbuCas13a with Cas13a P4 FWD and
Cas13a P4 RVS primers (646 bp). DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE. pSB1C3 backbone
linearized: pSB1C3 backbone linearized amplified from Bba\_J36400 (2022 DNA distribution Kit) with Ev
and Pv primers (2052 bp). DNA ladder: DNA ladder 100bp DNA Marker NIPPON Genetics EUROPE.

| Name of amplified part | Primer 1 | Primer 2 | Template DNA | Sequence Length (bp) |
| --- | --- | --- | --- | --- |
| Cas13a P1 Part ([BBa\_K4170002](%7B%7B%20context.parts.BBa_K4170002.url%20%7D%7D)) | Cas13a P1 FWD | Cas13a P1 RVS | pGJK\_His-SUMO-LbuCas13a | 1240 |
| Cas13a P2 Part ([BBa\_K4170003](%7B%7B%20context.parts.BBa_K4170003.url%20%7D%7D)) | Cas13a P2 FWD | Cas13a P2 RVS | pGJK\_His-SUMO-LbuCas13a | 734 |
| Cas13a P3 Part ([BBa\_K4170004](%7B%7B%20context.parts.BBa_K4170004.url%20%7D%7D)) | Cas13a P3 FWD | Cas13a P3 RVS | pGJK\_His-SUMO-LbuCas13a | 1338 |
| Cas13a P4 Part ([BBa\_K4170005](%7B%7B%20context.parts.BBa_K4170005.url%20%7D%7D)) | Cas13a P4 FWD | Cas13a P4 RVS | pGJK\_His-SUMO-LbuCas13a | 646 |
| pSB1C3 backbone linearized | Ev | Pv | BBa\_J36400 (2022 DNA distribution Kit) | 2052 |

Afterwards, Golden Gate assembly of the PCR amplified Cas13a P1, P2, P3, P4 parts with the linearized pSB1C3
vector was performed for the efficient construction of the SUMO-LbuCas13a coding sequence into PSB1C3 plasmid. The
products of the Golden Gate assembly underwent transformation into E.coli DH5a competent cells. To verify the
successful Golden Gate assembly, colony PCR was performed using the primers VR and VF2. Then the samples were
loaded and run in 1 % agarose gel electrophoresis. As depicted on Figure 2 from all colonies the desired
SUMO-LbuCas13a (5908 bp) part has been amplified. For the colony PCR procedure, from the agar plate half amount of
each colony was picked and diluted on 10 μl of dH20 performing the template DNA of the colony PCR. The other half
amount was picked for the overnight liquid culture.

[![]({{  context.figures.Figure_2.thumb  }})](%7B%7B%20%20context.figures.Figure_2.image%20%20%7D%7D)

Colony PCR of E-coli DH5a transformants using VR and Cas13a-p4-RVS primers, after Golden Gate assembly. 1 %
agarose gel electrophoresis. Lanes 1,2,3,5,6,7,9,10,11: SUMO-LbuCas13a Repos (5908 bp) from different
colonies, using VR and Cas13a-p4-RVS primers. DNA ladder: DNA ladder 1kb plus NEB. The bands shown down of
the desired band are byproducts because some the primers VR-VF2 enhanced another part of the bacteria.

| Name of amplified part | Primer 1 | Primer 2 | Sequence Length (bp) |
| --- | --- | --- | --- |
| UMO-LbuCas13a part | VR | VF2 | 4152 |

##### Construction of Cloned final T7-SUMO-LbuCas13a ([BBa\_K4170016](%7B%7B%20context.parts.BBa_K4170016.url%20%7D%7D)) plasmid

This plasmid contains all the basic genetic parts required for the bacterial expression of codon-optimized
LbuCas13a protein in fusion with small ubiquitin-like modifier (SUMO) under the transcription control of T7
promoter fixed to pSB1C3 backbone. For the construction of this part two standard techniques were utilized: a) PCR
amplification; b) Golden Gate assembly.

PCR amplifications were performed utilizing 3 pairs of primers and then 1 % agarose gel electrophoresis was
conducted. As shown in Figures 3 and 4 all the PCR amplifications succeeded. In Figure 3 T7-Cas13a P1 part (1261
bp) and T7- Cas13a P0 part (1560 bp) are depicted. In Figure 4 T7-Cas13a P2-P4 part (2678 bp) is depicted. The
linearized pSB1C3 backbone was not amplified again, as the amount of its first amplification was enough for all
the experiments that followed (Figure 1).

[![]({{  context.figures.Figure_3.thumb  }})](%7B%7B%20%20context.figures.Figure_3.image%20%20%7D%7D)

1 % agarose gel electrophoresis of the PCR amplified T7-Cas13a P1 and P0 parts. T7-Cas13a part P1:
T7-Cas13a part P1 amplified from pGJK\_His-SUMO-LbuCas13a, with T7 P0 FWD and T7 P0 RVS primers (1261
bp). DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE. T7-Cas13a part P0: T7-Cas13a part P0 amplified
from pGJK\_His-SUMO-LbuCas13a, with T7 P1 FWD and T7 P1 RVS primers (1560 bp).

[![]({{  context.figures.Figure_4.thumb  }})](%7B%7B%20%20context.figures.Figure_4.image%20%20%7D%7D)

1 % agarose gel electrophoresis of the PCR amplified codon-optimized SUMO-Cas13a part P2-P4. T7-Cas13a
part P2-P4: T7-Cas13a part P2-P4 amplified from Cas13a repos plasmid with P2 FWD and P4 RVS primers
(2678 bp). DNA ladder: 1kb plus DNA ladder NEB.

| Name of amplified part | Primer 1 | Primer 2 | Template DNA | Sequence Length (bp) |
| --- | --- | --- | --- | --- |
| T7-Cas13a P0 Part | T7 P0 FWD | T7 P0 RVS | pGJK\_His-SUMO-LbuCas13a | 1560 |
| T7-Cas13a P1 Part | T7 P1 FWD | T7 P1 RVS | pGJK\_His-SUMO-LbuCas13a | 1261 |
| T7-Cas13a P2-P4 Part | P2 FWD | P4 RVS | SUMO-LbuCas13a Repos plasmid ([BBa\_K4170014](%7B%7B%20context.parts.BBa_K4170014.url%20%7D%7D)) | 2678 |

Afterwards, Golden Gate assembly of the PCR amplified T7-Cas13a P0, P1, P2-P4 parts with the linearized pSB1C3
vector was performed for the efficient construction of the fina T7-SUMO-LbuCas13a plasmid. The products of the
Golden Gate assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was performed,
using two pairs of primers: VR / VF2 and T7-Cas13a-P0-FWD / Cas13a-P4-RVS. For the colony PCR procedure, from the
agar plate half amount of each colony was picked and diluted on 10 μl of dH20. The other half amount was picked
for liquid overnight culture. The samples were loaded and run 1 % agarose gel electrophoresis we concluded that
the Golden Gate assembly was successful. As depicted in Figure 5 the outcomes of the colony PCR (VR/VF2) five out
of six colonies have received the desirable insert (5713 bp). The next five samples are the outcomes of the colony
PCR (T7-Cas13a-P0-FWD / Cas13a-P4-RVS) from 5 different colonies. All the colonies have received the desired
insert (5446 bp).

[![]({{  context.figures.Figure_5.thumb  }})](%7B%7B%20%20context.figures.Figure_5.image%20%20%7D%7D)

Colony PCR of E-coli DH5a transformants using two pairs of primers, after Golden Gate assembly. 1 % agarose
gel electrophoresis. Lanes 1-6 : Final T7-SUMO-LbuCas13a using VR and Cas13a-p4-RVS primers (5713 bp) from
different colonies. Lanes 7-11: Final T7-SUMO-LbuCas13a using T7-Cas13a-P0-FWD and Cas13a-P4-RVS primers
(5446 bp) from different colonies. DNA ladder: 1kb plus DNA ladder NEB.

| Name of amplified part | Primer 1 | Primer 2 | Sequence Length (bp) |
| --- | --- | --- | --- |
| T7-SUMO-LbuCas13a | VR | VF2 | 5713 |
| T7-SUMO-LbuCas13a | T7-Cas13a-P0-FWD | Cas13a-P4-RVS | 5446 |

##### Construction of Cloned T7-LbuCas13a-SUMOLESS [BBa\_K4170017](%7B%7B%20context.parts.BBa_K4170017.url%20%7D%7D)

This plasmid contains all the basic genetic parts required for the bacterial expression of codon-optimized
LbuCas13a protein without the small ubiquitin-like modifier (SUMO) protein **fixed to pSB1C3 backbone**
utilizing **two standard techniques: a) PCR amplification; b) Golden Gate Assembly**.

PCR amplification was performed utilizing two pairs of primers and then the samples were loaded and run in 1 %
agarose gel electrophoresis. As shown in Figure 6 both PCR amplifications succeeded. On the left side the
SUMOLESS-Cas13a part (3521 bp) is depicted and on the right side SUMOLESS-T7 part (1640 bp). The linearized pSB1C3
backbone was not amplified again, as the amount of its first amplification was enough for all the experiments that
followed (Figure 1).

[![]({{  context.figures.Figure_6.thumb  }})](%7B%7B%20%20context.figures.Figure_6.image%20%20%7D%7D)

1 % agarose gel electrophoresis of the PCR amplified SUMOLESS-Cas13a parts. DNA ladder: 1kb plus DNA ladder
NEB. SUMOLESS-Cas13a part: SUMOLESS-Cas13a part amplified from final T7-SUMO LbuCas13a with SUMOLES FWD and
Cas13a P4 RVS primers (3521 bp). DNA ladder: 1kb plus DNA ladder NEB. SUMOLESS-T7: SUMOLESS-T7 amplified
from final T7-SUMO-LbuCas13a with T7 P0 FWD and SUMOLSS RVS primers (1640 bp).

| Name of amplified part | Primer 1 | Primer 2 | Template | Sequence Length (bp) |
| --- | --- | --- | --- | --- |
| SUMOLESS-Cas13a | SUMOLESS FWD | Cas13a P4 RVS | final T7-SUMO-LbuCas13a [BBa\_K4170016](%7B%7B%20context.parts.BBa_K4170016.url%20%7D%7D) | 3521 |
| SUMOLESS-T7 | T7 P0 FWD | SUMOLESS RVS | final T7-SUMO-LbuCas13a [BBa\_K4170016](%7B%7B%20context.parts.BBa_K4170016.url%20%7D%7D) | 1640 |

Afterwards, Golden Gate assembly of the PCR amplified SUMOLESS-Cas13a and SUMOLESS-T7 parts with the linearized
pSB1C3 vector was performed for the efficient construction of the T7-LbuCas13a-SUMOLESS plasmid. The products of
the Golden Gate assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was
performed, using VR and VF2 primers. For the colony PCR procedure, from the agar plate half amount of each colony
was picked and diluted on 20 μl of dH20. The other half amount was picked for liquid culture. The samples were
loaded and run in 1% agarose gel electrophoresis and then we concluded that the Golden Gate assembly was
successful. As depicted in Figure 7 from all colonies the SUMOLESS Cas13a (5395 bp) has been amplified.

[![]({{  context.figures.Figure_7.thumb  }})](%7B%7B%20%20context.figures.Figure_7.image%20%20%7D%7D)

Colony PCR of E-coli DH5a transformants using VR and VF2 primers, after Golden Gate assembly. 1 % agarose
gel electrophoresis. DNA ladder: 1kb plus DNA ladder NEB. Lines 1-3: SUMOLESS Cas13a using VR and VF2
primers (5395 bp) from different colonies.

| Name of amplified part | Primer 1 | Primer 2 | Sequence Length (bp) |
| --- | --- | --- | --- |
| SUMOLESS Cas13a | VR | VF2 | 5395 |

##### Construction of Cloned Rhamnose Repos [BBa\_K4170054](%7B%7B%20context.parts.BBa_K4170054.url%20%7D%7D)

This plasmid contains the rhaBAD operon and rhaR-rhaS activators of E.coli flanked by BioBrick Preffix and Suffix
regions into the PSBIC3 backbone. For the composition of this part two standard techniques were utilized: a)
colony PCR from DH5a competent E.coli cells; b)Golden Gate Assembly.

After the completion of the colony PCR amplifications, 1 % agarose gel electrophoresis was conducted. As shown in
Figure 8 Rha (2091 bp) from colonies 1 and 3 were slightly amplified while amplification from colony 2 was
unsuccessful. For our next experiments we used RhaBAD-rhaR-rhaS colony part amplified from colony 4 which was the
most successfully amplified colony.

[![]({{  context.figures.Figure_8.thumb  }})](%7B%7B%20%20context.figures.Figure_8.image%20%20%7D%7D)

Colony PCR of E-coli DH5a using Prham.Brk-F and Prham.Brk-R primers from 4 different colonies in order to
amplify the Rha sequence. 1 % agarose gel electrophoresis. Lanes 1-4: Rha (2091 bp) using Prham.Brk-F and
Prham.Brk-R primers from different colonies. DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE.

| Name of amplified part | Primer 1 | Primer 2 | Template | Sequence Length (bp) |
| --- | --- | --- | --- | --- |
| rhaBAD-operon | Prham.Brk-F | Prham.Brk-R | E.coli DH5a bcteria | 2091 |

[![]({{  context.figures.Figure_9.thumb  }})](%7B%7B%20%20context.figures.Figure_9.image%20%20%7D%7D)

Colony PCR of E-coli DH5a transformants using VR and VF2 primers, after Golden Gate assembly. 1 % agarose
gel electrophoresis. Lines 1-4: Rhamnose repos using VR and VF2 primers (2345 bp) from different colonies.
DNA ladder: 1kb plus DNA ladder NEB

Afterwards, Golden Gate assembly of the colony PCR amplified rhaBAD-operon part with the linearized pSB1C3 vector
was performed for the efficient construction of the Rhamnose Repos plasmid. The products of the Golden Gate
assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, **using VR and
VF2 primers**. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted
on 10 μl of dH20. The other half amount was picked for liquid overnight culture. Picking samples from different
colonies and then evaluating the results on a 1% agarose gel electrophoresis, we concluded that the Golden Gate
assembly was successful on **colonies 3 and 4**. As depicted on Figure 9 from 3 and 4 colonies the Rhamnose-Repos
(2345 bp) has been amplified, while from **1 and 2** colonies has not.

| Name of amplified part | Primer 1 | Primer 2 | Sequence Length (bp) |
| --- | --- | --- | --- |
| RhaBAD-operon | VR | VF2 | 2345 |

##### Construction of Cloned Rhamnose-SUMO-LbuCas13a BBa\_K4170055

This plasmid contains all the basic genetic parts required for the bacterial expression of codon-optimized
LbuCas13a protein with the small ubiquitin-like modifier (SUMO) protein under the transcriptional control of
rhaBAD promoter regulated by rhaS and rhaR activators into PSBIC3 backbone. For the composition of this part two
standard techniques were utilized: a)PCR amplification; b) Golden Gate assembly.

After the completion of the PCR amplifications, 1 % agarose gel electrophoresis was conducted. As shown in Figure
10 both PCR amplifications succeeded. On the left side the rhaR-rhaS part (2119 bp) is depicted and on the right
side Cas13a-RBS-P4 RVS part (3903 bp).

[![]({{  context.figures.Figure_10.thumb  }})](%7B%7B%20%20context.figures.Figure_10.image%20%20%7D%7D)

1 % agarose gel electrophoresis of the PCR amplified Rhamnose-SUMO-LbuCas13a parts. rhaR-rhaS part:
rhaR-rhaS part amplified from Rhamnose Repos, with Ep and Sp primers (2119 bp). Cas13a-RBS-P4 RVS part:
Cas13a-RBS-P4 RVS part amplified from SUMO-Cas13a Repos, with Cas13a RBS and P4 RVS primers (3903 bp). DNA
ladder: 100bp DNA Marker NIPPON Genetics EUROPE.

| Name of amplified part | Primer 1 | Primer 2 | Template | Sequence Length (bp) |
| --- | --- | --- | --- | --- |
| rhaBAD | Ep | Sp | Rhamnose Repos ([BBa\_K4170054](%7B%7B%20context.parts.BBa_K4170054.url%20%7D%7D)) | 2119 |
| Cas13a-RBS-P4 RVS part | Cas13a RBS FWD | P4 RVS | SUMO-Cas13a Repos ([BBa\_K4170014](%7B%7B%20context.parts.BBa_K4170014.url%20%7D%7D)) | 3903 |

##### Final T7-SUMO-LbuCas13a diagnostic digestions

After the cloning of plasmid final T7-SUMO-LbuCas13a, that contains all the basic genetic parts required for the
bacterial expression of codon-optimized LbuCas13a protein in fusion with small ubiquitin-like modifier (SUMO)
protein, diagnostic digestions followed, in order to verify with a different method than colony PCR that the
cloning was successful. We performed a double digestion using the restriction enzymes EcoRI and SpeI and a single
digestion using the restriction enzyme EcoRI. Then a 1% agarose gel electrophoresis was conducted. From the double
digestion two bands should arise (5422 bp and 2047 bp), while from the simple digestion with EcoRI only a 7469bp
band should arise. From this experiment final T7-SUMO-LbuCas13a plasmids that have been isolated from 6 different
colonies were used. As depicted in Figure 11 all the digestions were successful.

| DNA template | Restricted enzymes | Sequence length (bp) |
| --- | --- | --- |
| Final T7-SUMO-LbuCas13a | EcoRI, SpeI | 5422 and 2047 |
| Final T7-SUMO-LbuCas13a | EcoRI | 7469 |

[![]({{  context.figures.Figure_11.thumb  }})](%7B%7B%20%20context.figures.Figure_11.image%20%20%7D%7D)

1 % agarose gel electrophoresis after diagnostic digestion of final T7-SUMO-Cas13a. Lanes 1-6: Double
digestion with EcoRI and SpeI restriction enzymes (5422 bp and 2047 bp). DNA ladder: 1kb plus DNA ladder
NEB. Lanes 7-12: Single digestion with EcoRI restriction enzyme (7469 bp). DNA ladder: 1kb plus DNA ladder
NEB

##### Construction of Cloned crRNAs

For the purposes of this project, we conducted the cloning of six crRNAs:
crRNA-3ps, [BBa\_K4170019](%7B%7B%20context.parts.BBa_K4170019.url%20%7D%7D),
crRNA-3pm, [BBa\_K4170021](%7B%7B%20context.parts.BBa_K4170021.url%20%7D%7D),
crRNA-3pe, [BBa\_K4170022](%7B%7B%20context.parts.BBa_K4170022.url%20%7D%7D),
crRNA-5ps, [BBa\_K4170042](%7B%7B%20context.parts.BBa_K4170042.url%20%7D%7D),
crRNA-5pm, [BBa\_K4170043](%7B%7B%20context.parts.BBa_K4170043.url%20%7D%7D),
crRNA-5pe.[BBa\_K4170044](%7B%7B%20context.parts.BBa_K4170044.url%20%7D%7D).
crRNA is a fundamental part of CRISPR/Cas13a system and its structure consists of a stem-loop supporting the
Cas13a/crRNA duplex and a spacer performing the guidance of the system.

* **crRNA-3ps** targets the miRNA-17-3p since it contains the nucleotide sequence for the perfect hybridization
  with the mature miRNA-17-3p.
* **crRNA-3pm** consists of the loop sequence for the construction of Cas13a/crRNA duplex and a spacer sequence
  with a mismatch \( (T \to A) \) in position 14 generating no perfect hybridization of the spacer with the mature
  miRNA-17-3p.
* **crRNA-3pe** consists of the loop sequence for the construction of Cas13a/crRNA duplex and a spacer sequence
  containing an extra hairpin loop in the 3' end.
* **crRNA-5ps** targets the miRNA-17-5p since it contains the nucleotide sequence for the perfect hybridization
  with the mature miRNA-17-5p.
* **crRNA-5pm** consists of the loop sequence for the construction of Cas13a/crRNA duplex and a spacer sequence
  with a mismatch \( (C \to G) \) in position 14 generating no perfect hybridization of the spacer with the mature
  miRNA-17-5p.
* **crRNA-5pe** consists of the loop sequence for the construction of Cas13a/crRNA duplex and a spacer sequence
  containing an extra hairpin loop in the 5' end.

Each plasmid was constructed with direct PCR amplification of PSB1C3 plasmid utilizing 2 pairs of primers followed
by a Golden Gate-based "SevaBrick Assembly" method. The first pair consists of a primer (**loop RVS standard**)
binding to the Biobrick prefix sequence containing an overhang with the stem-loop sequence of the crRNA and a
primer (**loop FWD standard**) binding to the backbone of the plasmid. The second pair of primers consists of a primer
(**crRNA spacer FWD interchangeable**) binding to the Biobrick suffix sequence containing an overhang with the spacer
sequence of the crRNA and a primer (**spacer RVS standard**) binding to the backbone of the plasmid. This strategy
enables the handy and fast alternation of the overhang sequence of the interchangeable primer for the construction
of any crRNA flanked in PSB1C3 plasmid keeping the other primers as customary components. Each plasmid contains
all the basic genetic parts required for the in vitro transcription of the different crRNAs regulated by T7
promoter.

| Name of amplified part | Primer 1 | Primer 2 | Template | Sequence Length (bp) |
| --- | --- | --- | --- | --- |
| Loop part | Loop RVS standard | Loop FWD standard | pSB1C3 | 1209 |
| 3ps-spacer part | 3ps spacer FWD interchangeable | spacer RVS standard | pSB1C3 | 867 |
| 3pm-spacer part | 3pm spacer FWD interchangeable | spacer RVS standard | pSB1C3 | 867 |
| 3pe-spacer part | 3pe spacer FWD interchangeable | spacer RVS standard | pSB1C3 | 867 |
| 5ps-spacer part | 5ps spacer FWD interchangeable | spacer RVS standard | pSB1C3 | 867 |
| 5pm-spacer part | 5pm spacer FWD interchangeable | spacer RVS standard | pSB1C3 | 867 |
| 5pe-spacer part | 5pe spacer FWD interchangeable | spacer RVS standard | pSB1C3 | 867 |

[![]({{  context.figures.Figure_12.thumb  }})](%7B%7B%20%20context.figures.Figure_12.image%20%20%7D%7D)

1 % agarose gel electrophoresis of the PCR amplified crRNA loop part and crRNA-spacer parts. DNA ladder:
100bp DNA Marker NIPPON Genetics EUROPE. Loop part: loop part amplified from pSB1C3 with Loop RVS standard
and Loop FWD standard primers (1209 bp). 3ps spacer part: 3ps spacer part amplified from pSB1C3 with 3ps
spacer FWD interchangeable and spacer RVS standard primers (867 bp). 3pm spacer part: 3pm spacer part
amplified from pSB1C3 with 3pm spacer FWD interchangeable and spacer RVS standard primers (867 bp). 5ps
spacer part: 5ps spacer part amplified from pSB1C3 with 5ps spacer FWD interchangeable and spacer RVS
standard primers (867 bp). DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE. 5pm spacer part: 5pm spacer
part amplified from pSB1C3 with 5pm spacer FWD interchangeable and spacer RVS standard primers (867 bp). 5pe
spacer part: 5pe spacer part amplified from pSB1C3 with 5pe spacer FWD interchangeable and spacer RVS
standard primers (867 bp). The display of DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE. The bands
depicted down of the amplified parts is the primer-dimers (PD) byproducts which were formed because of
strings of complementary bases in primers.

After the completion of the PCR amplifications, 1% agarose gel electrophoresis was conducted. The samples also
contain DNA templates complementary to the crRNA molecules. According to Figure 12 all the PCR amplifications
succeeded, using the appropriate primers as shown on the above table. Loop part (1209 bp) and 3ps, 3pm, 3pe, 5ps,
5pm, 5pe spacer parts are depicted.

Then, Golden Gate assembly of the PCR amplified loop part and each crRNA-spacer part was conducted, for the
efficient construction of the crRNA coding sequence under the transcriptional control of the T7 promoter. The
Golden Gate assembly products underwent transformation into E.coli DH5a competent cells and then colony PCR was
performed, using the primers VR and VF2. Picking sample from different colonies and then evaluating the results on
a 1 % agarose gel electrophoresis we concluded that the Golden Gate assembly was successful. As depicted on Figure
13 from all colonies has been amplified the desired inserts. For the colony PCR procedure, from the agar plate
half amount of each colony was picked and diluted on 10 μl of dH20. The other half amount was picked for liquid
overnight culture.

[![]({{  context.figures.Figure_13.thumb  }})](%7B%7B%20%20context.figures.Figure_13.image%20%20%7D%7D)

Colony PCR of E-coli DH5a transformants using VR and VF primers, after Golden Gate assembly. 1 % agarose gel
electrophoresis. DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE.

| Name of amplified part | Primer 1 | Primer 2 | Sequence Length (bp) |
| --- | --- | --- | --- |
| Cloned crRNA-3ps [BBa\_K4170019](%7B%7B%20context.parts.BBa_K4170019.url%20%7D%7D) | VR | VF2 | 390 |
| Cloned crRNA-3pm [BBa\_K4170021](%7B%7B%20context.parts.BBa_K4170021.url%20%7D%7D) | VR | VF2 | 390 |
| Cloned crRNA-3pe [BBa\_K4170022](%7B%7B%20context.parts.BBa_K4170022.url%20%7D%7D) | VR | VF2 | 395 |
| Cloned crRNA-5ps [BBa\_K4170042](%7B%7B%20context.parts.BBa_K4170042.url%20%7D%7D) | VR | VF2 | 390 |
| Cloned crRNA-5pm [BBa\_K4170043](%7B%7B%20context.parts.BBa_K4170043.url%20%7D%7D) | VR | VF2 | 390 |
| Cloned crRNA-5pe [BBa\_K4170044](%7B%7B%20context.parts.BBa_K4170044.url%20%7D%7D) | VR | VF2 | 395 |

##### Digestions of the cloned crRNA plasmids

For further assurance that the DNA templates complementary to the crRNAs have been inserted to the pSB1C3 plasmid,
diagnostic digestions followed. As depicted in Figure 14 a double enzymatic digestion by XbaI and PstI restriction
enzymes indicatively was performed for the cloned crRNA 3pm (102 bp) and cloned crRNA 5pm (102 bp).

[![]({{  context.figures.Figure_14.thumb  }})](%7B%7B%20%20context.figures.Figure_14.image%20%20%7D%7D)

Diagnostic double enzymatic digestion by XbaI and PstI restriction enzymes of cloned crRNA 5pm and 3pm. 1 %
agarose gel electrophoresis. 5pm: Cloned crRNA 5pm digested (102 bp). DNA ladder: 100bp DNA Marker NIPPON
Genetics EUROPE. 3pm: Cloned crRNA 3pm digested (102 bp).

##### In vitro transcription of crRNAs

For the in vitro transcription of crRNAs the plasmids were linearized utilizing the enzyme SapI which recognized
the restriction site we had insert in the plasmid downstream of the crRNA sequence.

[![]({{  context.figures.Figure_15.thumb  }})](%7B%7B%20%20context.figures.Figure_15.image%20%20%7D%7D)

Enzymatic digestion by SapI restriction enzyme of cloned crRNA 5pe, 5pm and 5ps. 1% agarose gel
electrophoresis. DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE. Psb1C3: undigested pSB1C3 circular
plasmid (2070bp). 5pe: Cloned crRNA 5pe digested (2151bp). DNA ladder:100bp DNA Marker NIPPON Genetics
EUROPE. 5pm: Cloned crRNA 5pm digested (2146bp). 5pm: Cloned crRNA 5pm digested (2146bp).

The linearized DNA template bands were purified by Nuclospin Gel and PCR Clean-up kit (Macherey-Nagel, Duren,
Germany) and a second purification step with phenol/chloroform was performed. The next step was the
template-directed synthesis of the crRNAs through in vitro transcription using T7 Polymerase (HiScribe T7 High
Yield RNA Synthesis Kit, NEB) and the purification of the products, using Monarch RNA Cleanup Kit (NEB). In Figure
16 the crRNA transcripts are depicted on a 3% agarose gel electrophoresis after in vitro transcription, while in
Figure 17, crRNA transcripts are depicted after the clean-up procedure. In both Figures, the samples were loaded
with the following order: crRNA-3p standard design (59bp), crRNA-3p mismatch design (59bp), crRNA-3p extra-loop
(64bp), crRNA-5p standard design (59bp), crRNA-5p mismatch design (59bp), crRNA-5p extra-loop design (64bp)

[![]({{  context.figures.Figure_16.thumb  }})](%7B%7B%20%20context.figures.Figure_16.image%20%20%7D%7D)

Results of the In Vitro transcription of the crRNAs in 3% agarose gel. 3ps: crRNA-3p standard (59bp). 3pm:
crRNA -3p mismatch design (59bp). 3pe: crRNA-3p extra-loop (64bp). 5ps: crRNA-5p standard design (59bp).
5pm: crRNA-5p mismatch design (59bp). 5pe: crRNA-5p extra-loop design (64bp).

[![]({{  context.figures.Figure_17.thumb  }})](%7B%7B%20%20context.figures.Figure_17.image%20%20%7D%7D)

Purified products of In Vitro transcription of crRNAs. 3% agarose gel. 3ps: crRNA-3p standard (59bp). 3pm:
crRNA-3p mismatch design (59bp). 3pe: crRNA-3p extra-loop (64bp). 5ps: crRNA-5p standard design (59bp). 5pm:
crRNA-5p mismatch design (59bp). 5pe: crRNA-5p extra-loop design (64bp).

##### Construction of Cloned LbuCas13a-SUMO with crRNAs

In our effort to synthesize the ribonucleoprotein SUMO-LbuCas13a-crRNA for the 3pm and 5pm crRNAs, there were
constructed two plasmids. The first one contains all the basic genetic parts required for the bacterial
transcription of crRNA 3pm and for expression of codon optimized LbuCas13a protein in fusion with small
ubiquitin-like modifier (SUMO) under the transcription control of T7 promoter fixed to pSB1C3 backbone. The second
one consists of the same parts, but the sequence for the bacterial transcription of crRNA 5p instead. For the
construction of these plasmids for the ribonucleoprotein expression, two different methods were applied: a) Golden
Gate assembly and b) 3A Assembly.

###### Golden Gate Assembly method

Firstly, PCR amplifications were performed utilizing 3 pairs of primers and then 1 % agarose gel electrophoresis
was conducted. As shown in Figures 18-19 all the PCR amplifications succeeded. In Figure 18 the SUMO-Cas13a for
ribonucleoprotein (5481 bp) is depicted. In Figure 19, crRNA 3p for ribonucleoprotein (152 bp), crRNA 5p for
ribonucleoprotein (152 bp) and the pSB1C3 backbone linearized (2052 bp) are depicted. Afterwards, Golden Gate
assembly of the PCR amplified products was performed for the efficient construction of the SUMO-LbuCas13a-crRNAs
coding sequence into PSB1C3 plasmid. The products of the Golden Gate assembly underwent transformation into E.
coli DH5a competent cells. To verify the successful Golden Gate assembly, colony PCR was performed using the
primers VR and VF2. Then the samples were loaded and run in 1 % agarose gel electrophoresis, but from none colony
the desired **SUMO-LbuCas13a-crRNA (5797bp)** part has been amplified.

| Name of amplified part | Primer 1 | Primer 2 | Template | Sequence Length (bp) |
| --- | --- | --- | --- | --- |
| SUMO-Cas13a for ribonucleoprotein | Ep | Sp | final T7-SUMO LbuCas13a [BBa\_K4170016](%7B%7B%20context.parts.BBa_K4170016.url%20%7D%7D) | 5481 |
| crRNA 3ps for Cas13a complex | Xp | Pp | Cloned crRNA-3ps [BBa\_K4170019](%7B%7B%20context.parts.BBa_K4170019.url%20%7D%7D) | 152 |
| crRNA 5p for Cas13a complex | Xp | Pp | Cloned crRNA-5ps [BBa\_K4170042](%7B%7B%20context.parts.BBa_K4170042.url%20%7D%7D) | 152 |
| pSB1C3 backbone linearized | Ev | Pv | BBa\_J36400 (2022 DNA distribution Kit) | 2052 |

[![]({{  context.figures.Figure_18.thumb  }})](%7B%7B%20%20context.figures.Figure_18.image%20%20%7D%7D)

1 % agarose gel electrophoresis of the PCR amplified SUMO-Cas13a part for ribonucleoprotein. SUMO-Cas13a for
ribonucleoprotein amplified from final T7-SUMO LbuCas13a with E and S primers (5481 bp). DNA ladder: DNA
ladder 1kb plus NEB.

[![]({{  context.figures.Figure_19.thumb  }})](%7B%7B%20%20context.figures.Figure_19.image%20%20%7D%7D)

1 % agarose gel electrophoresis of the PCR amplified linearized pSB1C3 backbone and crRNAs. A) crRNAs (Xp-Pp
primers) 3ps: crRNA 3p standard amplified from cloned 3p standard plasmid with Xp and Pp primers (152 bp).
crRNAs (Xp-Pp primers) 5ps: crRNA 5p standard amplified from cloned 5p standard plasmid with Xp and Pp
primers (152 bp). DNA ladder: 100bp DNA Marker NIPPON Genetics EUROPE. B) pSB1C3 backbone linearized: pSB1C3
backbone linearized amplified from Bba\_J36400 (2022 DNA distribution Kit) with Ev and Pv primers (2052 bp).
DNA ladder: DNA ladder 100bp DNA Marker NIPPON Genetics EUROPE.

###### 3A Assembly method

Firstly, 3 double digestions were performed utilizing 3 pairs of restriction enzymes, in order to create the two
parts that will be assembled and the linearized backbone plasmid. Then 1% agarose gel electrophoresis was
conducted to evaluate the results. As shown in Figures 20 and 21, all the digestions succeeded. In Figure 20 the
double digestion of cloned crRNA-3ps, 3pm, 3pe plasmids and cloned crRNA-5pm, 5pm, 5pe plasmids with XbaI and PstI
restriction enzymes are depicted. From the two bands (102 bp and 2044 bp) that aroused from both digestions, 102
bp part was used on the next steps. In Figure 21 the double digestion of final T7-SUMO-Lbu Cas13a and pSB1C3
plasmid are depicted. Final T7-SUMO-LbuCas13a was digested with EcoRI and SpeI restriction enzymes and two parts
aroused (2047 bp and 5422 bp). The 5422 bp part was utilized in the next step. pSB1C3 plasmid was digested with
EcoRI and PstI restriction enzymes and the 2047bp linearized part will be used in the next step. As long as the
three assembling parts were created with the appropriate ends, ligation protocol followed. The products of the 3A
assembly underwent transformation into E.coli DH5a competent cells. To verify the successful 3A assembly, colony
PCR was performed using the VR and VF2 primers. Then, the samples were loaded and run in 1% agarose gel
electrophoresis. As depicted on Figure 22 from all colonies, except colonies, the desired SUMO-LbuCas13a-crRNA
part has been amplified. For the colony PCR procedure, from the agar plate half amount of each colony was picked
and diluted on 10μl of dH2O performing the template DNA of the colony PCR. The other half amount was picked for
the overnight liquid culture.

[![]({{  context.figures.Figure_20.thumb  }})](%7B%7B%20%20context.figures.Figure_20.image%20%20%7D%7D)

Double digestion of cloned crRNA plasmids with EcoRI and PstI restriction enzymes. 1% agarose gel
electrophoresis. DNA ladder 1kb plus NEB. crRNA-3p-standard, crRNA-3p mismatch, crRNA-3p extra loop,
crRNA-5p-standard, crRNA-5p mismatch, crRNA-5p extra loop are depicted.

[![]({{  context.figures.Figure_21.thumb  }})](%7B%7B%20%20context.figures.Figure_21.image%20%20%7D%7D)

Double digestion of final T-7 SUMO-LbuCas13a and pSB1C3 plasmid with EcoRI/SpeI and EcoRI/PstI
respectively. 1% agarose gel electrophoresis DNA ladder 1kb plus NEB.

[![]({{  context.figures.Figure_22.thumb  }})](%7B%7B%20%20context.figures.Figure_22.image%20%20%7D%7D)

Colony PCR of E-coli DH5a transformants using VR and VF2 primers, after 3A assembly. 1 % agarose gel
electrophoresis. 3ps: LbuCas13a-SUMO-3p standard from two different colonies amplified with VR and VF2
primers (5797bp). 3pm: LbuCas13a-SUMO-3p mismatch from two different colonies amplified with VR and VF2
primers (5797 bp). 3pe: LbuCas13a-SUMO-3p with extra loop from two different colonies amplified with VR and
VF2 primers (5782 bp). 5ps: LbuCas13a-SUMO-5p standard from two different colonies amplified with VR and VF2
primers (5797 bp). 5pm: LbuCas13a-SUMO-5p mismatch from three different colonies amplified with VR and VF2
primers (5797 bp). 5pe: LbuCas13a-SUMO-5p with extra loop from two different colonies amplified with VR and
VF2 primers (5782 bp). DNA ladder: DNA ladder 1kb plus NEB. The band shown down of the 5pm3 is a byproduct
of the primer hybridization with another part of the bacteria.

## Protein experiments

---

#### Results from SUMO-LbuCas13a expression and purification

##### LbuCas13a protein expression procedure with 4 hours induction, at 37 oC using 1mM IPTG

Initially, a four-hour induction of 1L bacterial culture, at 37oC with addition of high IPTG induction factor, of
1mM was tested. The aim was to observe the behavior of the system and decide the course of action. 1ml of the
bacterial culture had been removed every hour (creation of timeline), and when the induction ended, the soluble
part of SUMO- LbuCas13a was separated from the insoluble part, according to the protocol, as described in
the [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page. The samples then were analyzed
using 8% gel electrophoresis, and an immunoblotting method was performed (Western Blot technique), using an
Anti-His primary antibody (Santa Cruz Biotechnology) in 1:2000 dilution ratio.

[![]({{  context.figures.Figure_23.thumb  }})](%7B%7B%20%20context.figures.Figure_23.image%20%20%7D%7D)

SUMO-Cas13a in 8% SDS PAGE gel with 4 hours induction, at 37oC and IPTG 1mM final concentration. The size of
SUMO-LbuCas13a is 155kDa. Lane1: 1st hour of induction (sample from whole bacterial culture). Lane 2: 2nd
hour of induction (sample from whole bacterial culture). Lane 3: 3rd hour of induction (sample from whole
bacterial culture). Lane 4: 4th hour of induction (sample from whole bacterial culture). Lane 5: Protein
marker (Nippon genetics blue Star). Lane 6: 4th hour of induction soluble part of SUMO-LbuCas13a. Lane 7:
4th hour of induction SUMO-LbuCas13a from Inclusion Bodies (IBs). Lane 8: Uninduced.

[![]({{  context.figures.Figure_24.thumb  }})](%7B%7B%20%20context.figures.Figure_24.image%20%20%7D%7D)

SUMO Cas13a in Western Blotting with 4 hours Induction at 37oC and IPTG 1mM final concentration.
SUMO-LbuCas13a size is 155kDa. Lane1:1st hour of induction (sample from whole bacterial culture). Lane 2:
2nd hour of induction (sample from whole bacterial culture). Lane3: 3rd hour of induction (sample from whole
bacterial culture). Lane 4: 4th hour of induction (sample from whole bacterial culture). Lane 5: Protein
marker (Nippon genetics blue Star). Lane 6: 4th hour of the induction of the soluble part SUMO of protein
SUMO-LbuCas13a. Lane 7: 4th hour of the induction of SUMO-LbuCas13a protein from Inclusion Bodies (IBs).
Lane 8: Uninduced.

Based on the visual observation of the sample analysis we concluded that, the use of high temperature and high
concentration of IPTG, were not enough to promote the protein expression in the soluble part. Therefore, the SUMO
booster sequence could not function during the 4h induction. In contrast to the above, the insoluble part
(inclusion bodies) was observed to contain the protein of interest, but not in a quantity capable enough to be
exploited in later procedures.

##### LbuCas13a protein expression procedure with overnight induction, at 25oC using 1mM IPTG

After the first inefficient attempt of the LbuCas13a protein expression, it was decided, that the best protocol
would be an overnight induction at 25oC with 1mM concentration of IPTG. For the construction of a timeline of the
first 6h this time, since the four-hour protocol did not work, a sample from the bacterial culture had been removed
every hour, and at the time of sixth hour, the sample taken was processed and separated into soluble and insoluble
part. The samples then were analyzed using 8% gel electrophoresis and a western blot technique was performed. For
the better visualization of the MW of our protein, we chose to run the gel for extended time (compared to the
Figure 23) and as shown in Figure 25, the band is illustrated exactly in the MW we waited.

[![]({{  context.figures.Figure_25.thumb  }})](%7B%7B%20%20context.figures.Figure_25.image%20%20%7D%7D)

SUMO-Cas13a in 8% SDS PAGE gel electrophoresis timeline for 6 hours induction at 25oC, from the overnight
induction protocol. IPTG 1mM final concentration. The size of SUMO-LbuCas13a is 155kDa. Lane1: 1st hour of
induction (sample from whole bacterial culture). Lane 2: 2nd hour of induction (sample from whole bacterial
culture). Lane3: 3rd hour of induction (sample from whole bacterial culture). Lane 4: 4th hour of induction
(sample from whole bacterial culture). Lane 5: 5th hour of induction (sample from whole bacterial culture).
Lane 6: Protein marker (Nippon genetics blue Star). Lane 7: 6th hour of induction, soluble part of
SUMO-LbuCas13a protein. Lane 8: 6th hour of the induction of SUMO-LbuCas13a protein from Inclusion Bodies
(IBs). Lane 9: Uninduced.

[![]({{  context.figures.Figure_26.thumb  }})](%7B%7B%20%20context.figures.Figure_26.image%20%20%7D%7D)

SUMO-Cas13a Western Blotting of 6 hours Induction at 25 ◦C from the overnight induction protocol with IPTG
1mM final concentration. SUMO-LbuCas13a size is 155kDa. Lane 1: 1st hour of induction (sample from whole
bacterial culture). Lane 2: 2nd hour of induction (sample from whole bacterial culture). Lane3: 3rd hour of
induction (sample from whole bacterial culture). Lane 4: 4th hour of induction (sample from whole bacterial
culture). Lane 5: 5th hour of induction (sample from whole bacterial culture). Lane 6: Protein marker
(Nippon genetics blue Star). Lane 7: 6th hour of induction soluble part of SUMO-LbuCas13a. Lane 8: 6th hour
of induction SUMO-LbuCas13a from Inclusion Bodies (IBs). Lane 9: Uninduced.

At the end of the six-hour time point the expression of the soluble SUMO-LbuCas13a was observed. At the same time
the quantity of the insoluble SUMO-LbuCas13a has been increased also.

##### LbuCas13a protein expression procedure with overnight induction, at 25oC using different concentrations of IPTG

After the conformation of the expression conditions, different concentrations of IPTG were tested, in order to: a)
determine the most efficient concentration that produces a satisfactory quantity of soluble SUMO-LbuCas13a, b)
investigate and understand which concentration offers the best soluble/insoluble ratio. Taking into account the
above, five different concentrations of IPTG were used: 1mM, 0.2mM, 0.4mM, 0.6mM and 0.8Mm following the same
protocol. Meaning, 1L bacterial cultures induced overnight at 25oC. The final products were analyzed using
SDS-PAGE electrophoresis and western blotting methods. The results rendered IPTG concentration among the most
important factors for LbuCas13a protein expression. The higher the concentration of IPTG was, the higher the
amount of the protein expressed, as depicted in Figures 27, 28. It is also observed that the induction procedure
with 1mM IPTG offered the best results.

[![]({{  context.figures.Figure_27.thumb  }})](%7B%7B%20%20context.figures.Figure_27.image%20%20%7D%7D)

SUMO-Cas13a in 8% SDS PAGE gel electrophoresis, at 25oC with overnight induction and different final
concentrations of IPTG. The Soluble part of SUMO-LbuCas13a protein is depicted. SUMO-LbuCas13a size is
155kDa. Lane1: Protein marker (Nippon genetics blue Star). Lane 2: SUMO-LbuCas13a soluble 1mM IPTG. Lane3:
SUMO-LbuCas13a soluble 0.2mM IPTG. Lane 4: SUMO-LbuCas13a soluble 0.4mM IPTG. Lane5: SUMO-Cas13a soluble 0.6
mM IPTG. Lane 6: SUMO-LbuCas13a soluble 0.8mM IPTG. Lane 7: Uninduced.

[![]({{  context.figures.Figure_28.thumb  }})](%7B%7B%20%20context.figures.Figure_28.image%20%20%7D%7D)

SUMO-LbuCas13a Bis acrylamide 30%, SDS PAGE electrophoresis 8% gel. 25oC overnight induction, different
final concentrations of IPTG. Soluble part. SUMO-LbCas13a size is 155kDa. Lane1: Protein marker (Nippon
genetics blue Star). Lane 2: SUMO-LbuCas13a soluble 1mM IPTG. Lane 3: SUMO-LbuCas13 soluble 0.2mM IPTG. Lane
4: SUMO-LbuCas13a soluble 0.4mM IPTG. Lane 5: SUMO-LbuCas13a soluble 0.6 mM IPTG. Lane 6: SUMO-LbuCas13a
soluble 0.8mM IPTG.

[![]({{  context.figures.Figure_29.thumb  }})](%7B%7B%20%20context.figures.Figure_29.image%20%20%7D%7D)

Insoluble part of SUMO-LbuCas13a in 8% SDS-PAGE gel electrophoresis from overnight induction, at 25◦C, using
different IPTG concentrations. Lane 1: Protein marker (Nippon genetics blue Star). Lane 2: SUMO-LbuCas13a
insoluble 1mM IPTG. Lane 3: SUMO-LbuCas13a insoluble 0.2mM IPTG. Lane 4: SUMO-LbuCas13a insoluble 0.4mM
IPTG. Lane 5: SUMO-LbuCas13a insoluble 0.6mM IPTG. Lane 6: SUMO-LbuCas13a insoluble 0.8mM IPTG. Lane 7:
Uninduced.

##### Purification procedure of SUMO-LbuCas13a protein

The last step for the LbuCas13a production constitutes the purification and digestion of SUMO-LbuCas13a, using the
cytiva His trap Ni-NTA column kit and the SUMO protease ULP1, respectively. Both protocols were applied on soluble
and insoluble SUMO-LbuCas13a as well. The results were analyzed through SDS-PAGE and western blotting methods. The
purification and the cleavage of the SUMO tag were performed successfully on soluble and on inclusion bodies
derived protein. The purification and the cleavage of the SUMO tag were performed successfully on soluble and on
inclusion bodies derived protein.

[![]({{  context.figures.Figure_30.thumb  }})](%7B%7B%20%20context.figures.Figure_30.image%20%20%7D%7D)

SUMO-LbuCas13a in 8% SDS PAGE gel electrophoresis, at 25oC from overnight induction with 1mM final
concentrations of IPTG. SUMO-LbuCas13a size is 155kDa. LbuCas13a size is 138kDa. Lane 1: Uninduced. Lane 2:
SUMO-LbuCas13a soluble. Lane 3: SUMO-LbuCas13a soluble purified with His Trap Ni-NTA column (Cytiva), 1st
elution. Lane 4: SUMO-LbuCas13a soluble from 1st elution, digested with SUMO protease ULP1, purified with His
Trap Ni-NTA column (Cytiva). Lane 5: SUMO-Cas13a soluble purified with His Trap Ni-NTA column (Cytiva), 2nd
elution. Lane 6: SUMO-LbuCas13a soluble purified with His Trap Ni-NTA column (Cytiva), 3rd elution. Lane 7:
Protein marker (NZY protein marker II). Lane 8: SUMO-LbuCas13a from IBs, purified with His Trap Ni-NTA column
(Cytiva), 2nd elution. Lane 9: SUMO-LbuCas13a IBs from 1st elution, digested with SUMO protease ULP1, purified
with His Trap Ni-NTA column (Cytiva). Lane 10: SUMO-Cas13a IBs, purified with His Trap Ni-NTA column (Cytiva),
1st elution.

## Microfluidic device manufacturing

---

One of the main objectives of our project was the manufacturing of a flow-focusing microfluidic device. The
selected manufacturing process was the 3D-printing. The microfluidic device is demonstrated in Figure 31 and so as
the channel geometry.

[![]({{  context.figures.Figure_31A.thumb  }})](%7B%7B%20%20context.figures.Figure_31A.image%20%20%7D%7D)

[![]({{  context.figures.Figure_31B.thumb  }})](%7B%7B%20%20context.figures.Figure_31B.image%20%20%7D%7D)

The selected microfluidic channel geometry (left) and the whole device (right)

The 3D printing technique selected was the Fused Deposition Modelling. After printing the whole microfluidic
device at once, we observed that the printed object was unable to perform microfluidic device behavior. The second
approach with the Fused Deposition Modelling was printing the device into 2 steps. The first step was the printing
of all the layers until the last layer of the microfluidic channels. The second step was the printing of the rest
layers of the device and finally the construction of the whole device by melting the bottom of the second part and
attaching it to the first one. This methodology was implemented also with the Digital Light Projection (DLP)
technique for comparison.

[![]({{  context.figures.Figure_32A.thumb  }})](%7B%7B%20%20context.figures.Figure_32A.image%20%20%7D%7D)

[![]({{  context.figures.Figure_32B.thumb  }})](%7B%7B%20%20context.figures.Figure_32B.image%20%20%7D%7D)

Both devices (FDM and DLP) demonstrated dysfunctionality and leak issues when they were tested with the pumping
system. Overall, we kept trying to improve the DLP device since it exhibited better surface smoothness, and the
post-processing was less time consuming. After printing devices with unwanted properties, we changed the design
into having only 1 oil and 1 water inlet, aiming to reduce the unwanted material inside the channels. This
device performed well, and generated droplets successfully tested it with Flow Rate 40 μL/min and 8 μL/min for
oil and water respectively that we were able to observe through a microscope.

For more details [Engineering Success](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) (page Cycle 3).

[![]({{  context.figures.Figure_33A.thumb  }})](%7B%7B%20%20context.figures.Figure_33A.image%20%20%7D%7D)

[![]({{  context.figures.Figure_33B.thumb  }})](%7B%7B%20%20context.figures.Figure_33B.image%20%20%7D%7D)

The function microfluidic device tested with pumping system (left) and the generated droplets after imagining with microscope (right)

## Bulk fluorescence experiments

---

#### Feasibility of LbuCas13a/crRNA system for miRNA detection.

The LbuCas13a/crRNA detection system is based on the LbuCas13 protein which processes crRNA-guided ssRNA
recognition that subsequently activates the trans cleavage of nontarget RNA molecules by the LbuCas13a/crRNA
machinery. Firstly, the crRNA assembles with the Cas13a and directs it to recognize the target miRNA that are
complementary to the spacer region of the crRNA. After target recognition, the Cas13a is activated to cleave
nearby RNA nonspecifically such as Reporter FQ5U RNA molecules which are added in the reaction. Specifically, the
LbuCas13a digests the fluorophore (FAM)- and quencher (BHQ1)-labeled poly-U reporter probe (FQ5U) getting rid of
the fluorescence resonance energy transfer (FRET) effect and releasing the fluorescence signals
([{{context.references.Liu\_2017.label}}](#Liu_2017)).

As described above, the detection system's main components are the LbuCas13a protein, the crRNA specific for the
target miRNA and the RNA reporter (FQ5U) necessary for the fluorescence signals.

* According to the SDS-PAGE electrophoresis results (figure), the LbuCas13a demonstrates the predicted MW with an
  excellent purity of about 95-98%.
* The 3% agarose gel electrophoresis of the in vitro transcribed crRNAs (figure) prove the estimated MW of the in
  IVT crRNAs as well as their purity.

To efficiently construct a well-characterized LbuCas13a/crRNA based detection system we had to investigate the
experimental conditions that affect the reaction performance. The following experiments were conducted to
investigate the reaction conditions and provide a strong proof-of-concept of our systems functionality:

* The **reaction buffer composition** for enabling LbuCas13a collateral activity.
* The **RNA reporter (FQ5U) concentration in the reaction**, necessary for the fluorescence signal. The results of
  this experiment are presented at the [engineering cycle 2](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
  of the engineering success wiki page (figure 2 of engineering success).
* **The time point of fluorescence detection since reaction initiation**. The fluorescence capture point should be in
  the linear part of the curve before the plateau phase for system's best performance. The results of this
  experiment are presented at the engineering cycle 2 of the
  [engineering success](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) wiki page.
* **Standard curve construction** which plots the fluorescence intensity of known added miRNA concentrations
  (standards) in the reaction. This calibration curve can be used for the determination of the target miRNA
  concentration in unknown samples. The results of this experiment are part of our project's proof-of-concept
  experiments and thus are presented on the [proof-of-concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)
  page of our Wiki.
* **Quantification of known added miR-17-3P concentrations in samples utilizing the constructed standard curve**.
  Based on the DIAS platform standard curve (figure) we tested the system's detection accuracy using different
  reaction samples which contain a specified known concentration of the miR-17-3P. The results of this experiment
  are part of our project's proof-of-concept experiments and thus are presented on the
  [proof-of-concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D) page of our Wiki.
* **Quantification of known added miR-17-3P concentrations in "non-selective miRNA-enriched complex environments"
  utilizing the constructed standard curve**. The aim of this study is to investigate the LbuCas13a enzyme's ability
  to efficiently bind with the crRNA and subsequently with the target miRNA in an “artificial environment” which
  contains a pool of non-specific miRNAs. The results of this experiment are part of our project's proof-of-concept
  experiments and thus are presented on the [proof-of-concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)
  page of our Wiki.
* **Relative quantification of miRNA-17-3P in total RNA extracts isolated from 2 different cell lines**. The aim of
  this experiment is to demonstrate the LbuCas13a/crRNA system's feasibility in detecting the miR-17-3P in a pool
  of total RNAs as well as investigate the target miR-17-3P expression levels in lung cancer cell compared to
  normal lung cells. The results of this assay are part of our project's proof of concept experiments and thus are
  presented on the [proof-of-concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D) page of our Wiki.

#### Reaction buffer composition enabling LbuCas13a collateral activity.

The reaction buffer composition affects the functionality of the LbuCas13a enzyme as well as the stability of the
crRNA molecules necessary for the reaction. If the reaction buffer does not contain the necessary components or
the appropriate pH, the functionality of the LbuCas13a protein can be minimized reducing the detection system's
performance ([{{context.references.Shan\_2019.label}}](#Shan_2019)).
According to an extensive literature research 2 reaction buffers were deemed suitable for use
([{{context.references.Liu\_2017.label}}](#Liu_2017)).
In addition, we decide to investigate the LbuCas13a activity on the Taq DNA polymerase standard buffer (Taq
DNA Polymerase PCR Buffer, Cat. No. 18067-017, invitrogen) to clarify if the LbuCas13a/crRNA reaction can be
performed at a standard PCR buffer.

Buffers tested for LbuCas13a activity

| Buffer A (7) | Buffer B (6) | Taq DNA Polymerase standard buffer |
| --- | --- | --- |
| Tris HCl 10mM | HEPES 20mM | Tris HCl 20mM |
| KCl 50mM | KCl 50mM | KCl 50mM |
| MgCl2 1.5mM | MgCl 6mM | MgCl2 - |
| pH 8.3 | pH 7.0 | pH 8.3 |

According to our kinetic analysis results, the Buffer B demonstrated inconsistent results which proved that this
buffer is not suitable for our LbuCas13a/crRNA reaction. This can be attributed to the low reaction pH (slightly
acidic) or to the HEPES buffer composition.

We then conducted comparative experiments between the Buffer A and the Taq DNA Polymerase standard buffer to test
the LbuCas13a enzyme's functionality. LbuCas13a (20nM) was incubated with 20nM crRNA and 1μM FQ5U RNA reporter in
reaction buffer A or in Taq DNA Polymerase standard buffer respectively. Two different concentrations of a
synthetic target miR-17-3p (1nm & 5nM) was added in each reaction tube. The total reaction volumes were 50μl. The
mixtures were then incubated in a fluorescence micropate reader (Tekan) at 37 °C for 1 hour with fluorescence
measurements (FAM channel, λex 494 nm, λem 518nm) taken every second min. The raw fluorescence intensities
generated by the efficient cleavage of the RNA reporter by the LbuCas13a enzyme in each tested reaction buffer are
presented on Figures 34 (Reaction Buffer A) and 35 (Taq DNA polymerase standard buffer).

Analyzing the results, we can conclude that the Buffer A provides consistent and reliable results while the common
Taq Polymerase standard buffer provides inconsistent results without statistical significance in the observed
fluorescence intensities of 1nM and 5nM reaction tubes. In the latter buffer, the fluorescence intensity is not
proportionally elevated according to the target miRNA concentration (1nM or 5nM) added in the reaction. These
results likely prove that the MgCl2 concentration added in Buffer A is necessary for the proper functionality of
the LbuCas13a enzyme. The effect of the Mg2+ ion on the Cas13 processing efficiency has been previously reported
on literately for the type VI-D Cas13d ribonuclease
([{{context.references.Zhang\_2018.label}}](#Zhang_2018)). In addition, since the pH
and the KCl concentrations are the same between the 2 buffers, the differences of Tris HCl buffer concentration
could probably affect the system's functionality. Improper reaction buffer could cause a significant background
signal implicated the analysis. However, to draw safe conclusions further experiments should be conducted.

[![]({{  context.figures.Figure_34.thumb  }})](%7B%7B%20%20context.figures.Figure_34.image%20%20%7D%7D)

Fluorescence intensity of CRISPR/Cas13a system with 1nm and 5nm of target miRNA in standard buffer of Taq DNA polymerase.

[![]({{  context.figures.Figure_35.thumb  }})](%7B%7B%20%20context.figures.Figure_35.image%20%20%7D%7D)

Fluorescence intensity of CRISPR/Cas13a system with 1nm and 5nm of target miRNA in buffer 7.

#### Investigation of the RNA reporter concentration required for the detection assay.

Since we concluded that the Buffer A ensures the enzyme's functionality, we had then to evaluate the RNA Reporter
concentration needed to achieve the best detection system's performance. Reaction tubes were prepared with 20nM
LbuCas13a, 20nM crRNA, 1nM target miR-17-3P and different concentrations of RNA Reporter (0.1nM, 0.25nM, 0.5 nM,
0.75nM & 1nM). The total reaction volumes were 50μl. The mixtures were then incubated in a fluorescence micropate
reader (Tekan) at 37 °C for 1 hour with fluorescence measurements (FAM channel, λex 494 nm, λem 518nm) taken every
second min as previously. After data acquisition we proceeded to a Michaelis-Menten analysis exploited the
in-silico analysis provided on the [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page.
Further information regarding the Michaelis-Menten analysis of our system is provided on the
[measurement](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D) page.

{% include 'references.html' %}
{% endblock %}