iGEM 2023 — Team iiser-kolkata


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![](https://static.igem.wiki/teams/4781/wiki/thumbnails/3.png)

# Cycles

[OMV Isolation and Purification](#2)

[TAT genetic circuit](#3)

[OMV Fusion Verification with Xanthomonas](#4)

[XOO CULTURE SYSTEM](#1)

[Machine Learning Model for Mutational Response](#5)

# XOO CULTURE SYSTEM

XOO CULTURE SYSTEM

# Design

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/design.png)

# Design

Our team embarked on an exciting journey to study the growth of two strains of Xanthomonas
bacteria: Xanthomonas oryzae pv oryzae BXO43 from the Center for DNA Fingerprinting and
Diagnostics (CDFD) and Xanthomonas oryzae pv. oryzae BXO1 from the Center for Cellular and
Molecular Biology (CCMB). The aim was to optimize their growth conditions and conduct
various assays to gain valuable insights.

# Build

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/build.png)

# Build

Our first task was to create a suitable environment for these bacterial strains. We obtained
glycerol
stocks of Xanthomonas oryzae pv. oryzae BXO43 from CDFD. To facilitate their growth, we
prepared
rifampicin PSA plates and covered them with aluminum foil, graciously borrowed from Dr.
Sreeramaiah
Gangappa's Plant Biology lab.

We streaked Xanthomonas oryzae pv. oryzae BXO43 on two rifampicin PSA plates and placed them
in a
28°C
incubator for a week. Simultaneously, we received agar stabs of Xanthomonas oryzae pv.
oryzae BXO1
from
CCMB. These were streaked onto PSA plates without any antibiotics and also incubated at
28°C.

To provide the necessary growth medium, we attempted to prepare 100ml of PSA rich media
three times,
but
each time, it got contaminated. Despite the setbacks, we persevered.

After seven days, we observed the growth of Xanthomonas BXO43 colonies . Unfortunately, one
of the
plates that originated from the rifampicin-containing colonies was contaminated. To rectify
this, we
streaked two LB agar plates from the remaining colonies and placed them in the 28°C
incubator.

In parallel, we initiated primary cultures of Xanthomonas BXO1 from CCMB and kept them in an
incubator
shaker at 28°C for 24 hours. We encountered some complications due to issues with agar and
LB
concentrations from the previous day, but we persisted in our efforts.

One of the primary cultures showed turbidity after 24 hours, indicating growth, but the
other one
showed
no signs of development. We also measured the optical density (OD) of the primary culture,
which had
reached approximately 0.3.

For further experimentation, we prepared three different growth media: LB, PS, and Nutrient
broth,
and
autoclaved them. Subsequently, we initiated primary cultures of Xanthomonas BXO43 from CCMB
in these
three different media, originating from two different Xanthomonas-streaked plates, resulting
in a
total
of six primary cultures. These were placed in the 28°C incubator at 250rpm.

# Test

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/test.png)

# Test

With our cultures in place, we began our series of tests and assays. We streaked Xanthomonas
oryzae BXO43 and BXO1 from previously streaked plates and closely monitored their growth at
various temperatures and time points to optimize their growth conditions.

We also initiated a growth assay of the primary culture of Xanthomonas BXO1 in LB media at
28°C and 250rpm, recording their progress at different time intervals.

# Learn

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/learn.png)

# Learn

As our experiments continued, we gathered valuable data on the growth patterns of
Xanthomonas oryzae BXO43 and BXO1 under various conditions. We learned about the challenges
of media contamination and the importance of meticulously maintaining culture conditions.

Through our assays, we gained insights into the optimal growth conditions for Xanthomonas
BXO43 and laid the foundation for further research and experiments aimed at better
understanding these bacterial strains.

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/cropped-xoo-growth-curve.png)

# OMV Isolation and Purification

OMV Isolation and Purification

# Design

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/design.png)

# Design

In the early stages of our project, we recognized that OMV (Outer Membrane Vesicle)
isolation was the foundational step we needed to master. With a clear goal in mind, we
embarked on a quest to find the most effective procedures for OMV isolation.

To guide us in this endeavor, we drew inspiration from the previous year's iGEM IISER TVM
Team in 2022, who had worked extensively with hyper vesiculating strains as part of their
project. We reached out to a former member of that team, Sneha PR, who kindly agreed to
mentor us and help us obtain the hyper vesiculating E. coli strain we needed.

Simultaneously, we scoured the academic landscape to identify professors with expertise in
hyper vesiculating E. coli strains and OMV isolation. Thanks to our Principal Investigator
(PI), we reached out to these professors via email. Fortunately, our efforts bore fruit, as
we secured assistance from several professors and obtained strains from their laboratories.

Our next step was to delve into the study of these strains and compare their Outer Membrane
Vesicles. To do this, we needed a robust protocol for OMV isolation and purification.
Seeking guidance, we met with Dr. Amirul Islam Mallick, who offered valuable insights into
potential characterization studies we could undertake for OMVs

# Build

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/build.png)

# Build

With guidance from Afruja, a PhD student from Dr. Amirul Islam Mallick's lab, we began the
process of isolating OMVs.

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/amirul.jpg)

## For Bacterial Secondary Culture:

1. Prepare 250ml of bacterial secondary culture and place 4-6 autoclaved
   Oakridge tubes
   inside the laminar hood.
2. Distribute the 250ml secondary culture equally among the 4-6 Oakridge
   tubes.
3. Centrifuge the Oakridge tubes at 8000 rpm at 4°C for 30 minutes to
   pellet down the
   bacteria.
4. Perform membrane filtration on the supernatant using a 0.22-micron
   filter.
5. Collect the filtered supernatant into an autoclaved conical flask for
   ultracentrifugation.
6. Be cautious with the polycarbonate tubes, which are not autoclavable;
   wash them with
   distilled water.
7. Fill the polycarbonate tubes up to the mark, ensuring approximately
   90% capacity to
   prevent tube breakage during ultracentrifugation.
8. Weigh the tubes and their caps to ensure equal weights up to two
   decimal points.
9. Repeat the weighing procedure three times for verification.
10. Perform ultracentrifugation at 25000 rpm, 4°C, for 4 hours.
11. Once the centrifugation is complete, reduce the rpm speed gradually
    and release the
    vacuum.
12. Gently open the lid and retrieve the rotor with the cassettes
    attached.
13. Discard the supernatant from each of the 6 tubes.
14. Add 0.5-1 ml of 1X PBS buffer to one tube and pipette up and down to
    dissolve the OMVs
    into the buffer.
15. Transfer the OMV-dissolved PBS buffer to the next tube.
16. Rinse with 0.5-1 ml of 1X PBS buffer and collect in another tube.
17. Store the MCTs at -20°C.

## For OMV Purification:

1. Thaw the stored cells at 4°C.
2. Use a hood to balance the chilled SW41 tubes with a maximum of 10ml of
   sample.
3. Utilize the ultracentrifuge at 30krpm for 3 hours at 4°C.
4. Discard the supernatant to reveal a transparent pellet.
5. Dissolve the pellet in 300-500ul PBS.
6. Use a sterile 0.45um / 0.22um syringe filter to filter the pellet.
7. Store the purified OMVs in a -20/-80°C refrigerator.

# Test

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/test.png)

# Test

Armed with our isolated OMVs, we proceeded to conduct various tests and characterizations.
We employed scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to
explore the OMVs' morphology and structure. These tests yielded successful results, allowing
us to gain deeper insights into our OMVs.

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/cropped-grid-26324.png)

*TEM image of E.coli Δnlp strain*

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/cropped-3-04.png)

*SEM image*

In addition to SEM and TEM, we also performed dynamic light scattering (DLS) to determine
the size of the OMVs, further enriching our understanding of these crucial structures.

# Learn

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/learn.png)

# Learn

Through meticulous planning, collaboration with mentors and professors, and hands-on
experimentation, we successfully achieved OMV isolation and purification. Our journey in the
world of OMVs had just begun, and we had gained valuable knowledge about the procedures and
characterization techniques involved.

This phase of our project had set a strong foundation for the subsequent stages, where we
would delve deeper into the study of OMVs and their potential applications. With each step,
we continued to learn, adapt, and refine our approach, eager to unravel the mysteries of
these tiny vesicles and their significance in the field of synthetic biology.

# TAT genetic circuit

TAT genetic circuit

# Design

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/design.png)

# Design

It all started when we stumbled upon a fascinating review article by Tracy Palmer titled
"Targeting of proteins to the twin-arginine translocation pathway." In this intricate world
of protein transport, we uncovered the Twin-Arginine Translocation pathway. Excited and
intrigued by its potential, we began to explore the possibility of utilizing this pathway
for our protein of interest: fnCas12a. Our goal was to transport fnCas12a to the periplasm,
a crucial step in our project.

Our research unveiled the Tat ABCE complex as the ideal candidate for this ambitious
endeavor. To dive deeper, we turned to Uniprot, where we unearthed valuable insights into
Tat A, Tat B, Tat C, and Tat E. Armed with knowledge about their sequences, structures, and
functions, we were ready to embark on the journey of designing a genetic circuit that would
utilize these components.

# Build

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/build.png)

# Build

Designing the genetic circuit was the next logical step. To construct our vision, we sought
the necessary genetic elements from the iGEM registry, including a promoter and ribosome
binding site (RBS). The circuit took shape as follows: Promoter + RBS + TatA + RBS + TatB +
RBS + TatC + RBS + TatE + Terminator. This intricate sequence needed to be carefully
assembled for it to work effectively.

With the sequence in hand, we faced the challenge of ordering gene fragments. Our chosen
suppliers, Twist Bioscience and IDT, had specific limits on the length of sequences they
could synthesize. Thus, we divided the sequence accordingly. Our assembly method of choice
was NEB Hi-fi DNA Assembly, following a protocol that ensured each sequence had a 30-base
pair overlap with its neighboring fragments. Additionally, the first and last fragments had
overlapping sequences with the plasmid pSB1C3.

We didn't leave anything to chance. Before proceeding, we rigorously verified the assembly
in silico using Snapgene. After thorough review and double-checking, we placed our order
with Twist Bioscience, setting the stage for the next phase.

# Test

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/test.png)

# Test

While waiting for the gene fragments to arrive, we prepared the pSB1C3 plasmid from the iGEM
Distribution Kit 2023. This involved reviving the plasmid, establishing a primary culture,
and conducting plasmid isolation, ensuring that the concentration was satisfactory.

Once the gene fragments arrived, we subjected them to a critical examination. Gel
electrophoresis was our chosen method for this task. We compared the bands to the NEB 1 kb
plus DNA ladder, meticulously checking for quality and integrity. With the fragments deemed
suitable, we moved forward to the next crucial step.

We needed to linearize the plasmids to facilitate the upcoming assembly. For this, we
designed primers with overlapping base pairs with the first and last fragment. Both forward
and reverse primers were carefully selected after in-silico validation, ensuring precision
in our approach.

# Learn

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/learn.png)

# Learn

The journey was not without its challenges. We initiated a gradient PCR at various
temperatures, ultimately observing bright bands at 62 and 64 degrees Celsius during gel
electrophoresis.

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/gradient-pcr-tat.jpg)

Encouraged by these results, we proceeded with PCR at 64 degrees Celsius for gel extraction,
creating three reactions of 25ul each. Gel electrophoresis confirmed the presence of bright
bands, affirming the success of our procedure.

However, we encountered unexpected obstacles during the PCR clean-up process using the NEB
Monarch Kit. Multiple attempts were made, optimizations were considered, such as extending
the elution time and increasing the incubation period, but the problem persisted. After
careful examination, we discovered that the PCR Clean-Up kit was nearing its expiration date
and had issues.

Adapting to the situation, we embraced the Qiagen Gel Extraction Protocol, with valuable
assistance from the Protein Engineering Lab at IISER Kolkata and the Plant Biology Lab under
Dr. Sreeramaiah Gangappa. The collaboration proved fruitful as we successfully completed the
gel extraction. With concentration and 260/280 ratio within the desired range, we proceeded
with the NEB Hi-fi DNA Assembly.

The assembly was followed by transformation into E. coli DH5alpha competent cells, and later
into BL21DE3 Electrocompetent cells. With successful transformation and plasmid isolation,
we ran further checks using gel electrophoresis, confirming our progress. Finally, we
performed Colony PCR for verification and sent samples for sequencing to validate our
efforts.

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/cropped-tat-plasmid.jpg)

The culmination of our rigorous work and collaboration led to the creation of our assembled
product, a vital component for the upcoming double transformation with another circuit,
marking another chapter in our scientific journey.

# OMV Fusion Verification with Xanthomonas

OMV Fusion Verification with Xanthomonas

# Design

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/design.png)

# Design

One of the most essential roadblock we had was to do outer membrane vesicles released from
gram negative bacteria show interspecies communication. The important question we had was -
Is it possible that outer membrane vesicles from E.coli can fuse or interact with
Xanthomonas.
After a deep literature review we found out studies that have shown interactions between
Outer membrane vesicles between same species, quite few papers were shown on interactions
between interspecies communication.

# Build

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/build.png)

# Build

We came across literature studies where interaction between membrane vesicles with mammalian
cell lines was shown. Hence, we decided to use Lipophilic Dye to stain Outer membrane
vesicles and study the interaction with Xanthomonas using Flow Cytometry assay and Confocal
microscopy.

The basic design layout of the experiment was built with the help of Scientific Officer
Tamal Ghosh. Further, Dr Bidisha Sinha suggested us to use FM1-43 membrane staining dye to
stain the outer membrane of Xanthomonas and to better analyze the interaction.

The Confocal microscopy experiments set the base for further future GFP localization testing
experiments as well and isolation and interaction studies.

# Test

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/test.png)

# Test

**Flow Cytometry Results**
We first used Rhodamine B dye to stain Outer membrane vesicles and unstained *Xanthomonas
oryzae.*. Then incubated them together for 1 hour in 1:1 ratio of volume. A
significant amplification curve was obtained when *Xanthomonas oryzae.* were incubated
with OMV’s suggesting that some interaction was happening.
A triplicate of the following experiments was done which showed us same results hence, we
decided to proceed and try confocal microscopy.

This cycle was crucial for us to understand that outer membrane vesicles carry the
possibility of potential gene transfer agent. We further went on to perform experiment with
GFP loaded outer membrane vesicles and positive results were obtained.

**Confocal microscopy results**
The same sample preparation method was done for confocal microscopy as well but first we
proceeded with just DAPI as the mounting medium to stain the nucleiod of *Xanthomonas
oryzae.*. And the following positive results were obtained:

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/control-sample.jpg)

Figure : Control Sample of *Xanthomonas oryzae.*. With DAPI staining

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/fig-3-xanhomonas-with-omv.jpg)

Figure 2: Interaction of Rhodamine stained OMV’s with DAPI stained Xanthomonas, but the
localisation can only be observed in membranes.

Then we discussed the results with various microscopy experts and Dr Bidisha Sinha helped us
in providing the FM1-43 dye for membrane staining which further showed the following
positive results.

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/confocal-results.png)

# Learn

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/learn.png)

# Learn

This cycle was crucial for us to understand that outer membrane vesicles carry the
possibility of potential gene transfer agent. We further went on to perform experiment with
GFP loaded outer membrane vesicles and positive results were obtained.

Imaging of just OMV’s show no signal, the aggregate of OMV’s that come together to interact
with Xanthomonas show amplification in signal obtained.

We can improve upon this experiment by understanding the size limitation transfer of these
proteins and further the above protocol can be used to perform TEM imaging which can give
higher resolution images on the interaction taking place between OMV’s and *Xanthomonas
oryzae.*.

# Machine Learning Model for Mutational Response

Machine Learning Model for Mutational Response

# Design

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/design.png)

# Design

The idea actually came up during one of our conversations with Prof Susmita Roy when she
pointed out that her lab has recently developed this new ML approach for predicting
mutational responses in viral genome recently in light of covid outbreak and whether we can
incorporate that for our bacterial pathogen. After digging up some literature, we went
onboard with the idea. In our study, we employed a feedforward backpropagation neural
network characterized by a specific model architecture denoted as 3–8–8–1. This architecture
encompasses four distinct layers. The initial layer accommodates three input features,
facilitating the input data's entry into the network.Subsequently, we integrated two hidden
layers, each containing eight neurons, which play a pivotal role in feature transformation
and representation learning. The final layer, consisting of a solitary neuron, corresponds
to the network's output and directly addresses the prediction of our target variable.

# Build

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/build.png)

# Build

To test out the validation of the model, we used the trained model to make predictions of a
validation sequence. Initially we used the Torch package in python to implement the model.
But the prediction did not come as expected. The probabilities were coming in order of
10^-08. It was time for debugging now.

# Test

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/test.png)

# Test

After a lot of debugging and switching from Torch to Keras package, we finally tested our
model for predictions. And this time we were successful. The model predictions against the
validation file were giving probabilities of the order of 0.1. With a user defined cutoff of
0.15, we checked with actual predictions and found out the model predicted a total of 57
potential positions that can mutate in the future version of XopN. Out of these 57 predicted
mutations, the model predicted 9 residues that have actually mutated in the validation
sequence.

# Learn

![](https://static.igem.wiki/teams/4781/wiki/engineering-success/learn.png)

# Learn

With the successful prediction of mutational sites, we learned that the features selected
for estimating the random mutation worked just as well in bacteria as in viruses. The
overall low mutational probability for bacteria might be a relic of the less amount of data
availability for model training as compared to the viral case. Also we learned what could be
the possible mutations for a given XopN sequence, which in turn can be very helpful for
modifying the biocontrol in near future.

![](https://static.igem.wiki/teams/4781/wiki/website-images/model/ml-pipeline.png)

{% include 'footer.html' %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

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![](https://static.igem.wiki/teams/4781/wiki/thumbnails/21.png)

# Contents

# Phase 1 - OMV isolation and characterisation

## Bradford assay

Using protein concentration as a substitute measure, OMVs can be indirectly quantified. A
Bradford Assay was used to measure the amount of protein in the OMV samples. In order to
determine the protein contents in OMV samples, a standard curve was made using bovine
serum albumin (BSA). The regression line was used to calculate concentrations of OMVs in
samples. E. coli Δnlp1 produced the most OMVs under the same growth conditions and
isolation method. This might be the result of inherent variations in the physiological makeup of
the five strains or variations in cell densities.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/omv-isolation-characterization/white-modern-mothers-day-presentation-43.jpg)

Table 1: This table shows the different protein yields present in OMVs of 5 hypervesiculating
strains.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/omv-isolation-characterization/white-modern-mothers-day-presentation-43-1.jpg)
#Figure missing

Fig1: This figure shows the comparison of OMVs yield from different hypervesicualting strains of
E.coli. The highest is from ∆NlpI. The lowest is from ∆DegP.

## DLS

A well-known, non-invasive method for determining the size and size distribution of molecules
and other particles often in the submicron range, and with the most recent technology, smaller
than 1nm, is dynamic light scattering (DLS), also known as quasi elastic light scattering (QELS).

Dynamic light scattering is frequently used to characterise particles, emulsions, or molecules
that have dispersed or dissolved in liquids. Different intensities of laser light are scattered due to
the Brownian motion of atoms or molecules in suspension. By analysing these intensity
variations, the Stokes-Einstein connection provides the Brownian motion's velocity and,
consequently, the particle size.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/1.png)

Fig2: This is the DLS data for BL21(de3) OMVs. The size of majority of OMVs that comes out of
this data is around 120nm.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/white-modern-mothers-day-presentation-43-2.jpg)

Fig3: This is the DLS plot for ∆𝑇𝑜𝑙𝐴 OMVs. The size of majority of OMVs from this strain comes
around 120nm.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/3.png)

Fig4: This is the DLS plot for ∆TolR OMVs. The size of majority of OMVs for this strain comes
around 140nm.

## SEM

The scanning electron microscope (SEM) produces a variety of signals at the surface of solid
specimens using a focussed beam of high-energy electrons. The signals produced by
electron-sample interactions include details about the sample's exterior appearance (texture),
chemical make-up, and crystalline structure and orientation of the constituent materials. In the
majority of applications, measurements are gathered over a chosen region of the sample's
surface, and a 2-dimensional image is created to show spatial variations in these qualities.

Here, we have used SEM to visualize the OMVs from different strains.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/omv-isolation-characterization/1.jpg)

Fig5: This image shows the OMVs from ∆TolR strain.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/omv-isolation-characterization/2.jpg)

Fig6: This image shows the OMVs from ∆TolA strain.

## TEM

We initially photographed native tolR, tolA, and nlpI OMVs with a 120kV transmission electron
microscope to confirm the existence of OMVs. The scans showed the presence of spherical
heterogeneous structures with a diameter of 100-200 nm, which is compatible with the stated
size range of OMVs.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/omv-isolation-characterization/4.png)

Fig10: BL21DE3 ISOLATED OMVs

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/omv-isolation-characterization/5.png)

Fig11: ∆TolR ISOLATED OMVs

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/omv-isolation-characterization/6.png)

Fig12: ∆𝑇𝑜𝑙𝐴 ISOLATED OMVs

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/omv-isolation-characterization/7.png)

Fig13: ∆NlpI ISOLATED OMVs

## Comparison of growth curves

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/table-growth.png)

Table2: This table shows the OD600 values at different time intervals for five hypervesiculating
strains

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/tola.jpg)

Fig14: This figure shows the change in OD with time for ∆TolA strain.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/tolr.jpg)

Fig15: This figure shows the change in OD with time for ∆TolR strain.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/degp.jpg)

Fig16: This figure shows the change in OD with time for ∆DegP strain.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/nlpl.jpg)

Fig17: This figure shows the change in OD with time for ∆NlpI strain.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/tolra.jpg)

Fig18: This figure shows the change in OD with time for ∆TolRA strain.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/dls-growthcurve/growth-curve.jpg)

Fig19: This figure shows the change in OD with time for all hypervesiculating strains. The fastest
growing strain is ∆NlpI. The slowest growing strain is ∆TolA.

# Phase 2 - Cas protein loading in OMV

## Overview

One crucial aspect of our project is loading FnCas12a into OMVs to develop new CRISPR
delivery methods and biocontrol agents. We've replaced the commonly used Cas9 protein,
which is large and causes off-target effects, with FnCas12a. FnCas12a is smaller, has fewer
off-target effects, and can mature crRNA from the crRNA array, enabling multiplex gene editing.

Our plan involves creating two plasmids: SpTorA-FnCas12a-6xHis and a Tat
machinery-expressing plasmid. The SpTorA-FnCas12a-6xHis plasmid tags FnCas12a with a
SpTorA signal peptide, localizing it in the periplasmic space for loading into vesicles derived
from the outer membrane with periplasmic proteins. The Tat plasmid exogenously expresses the
Tat ABCDE complex in the inner membrane, improving FnCas12a loading into the OMVs.

We also planned to perform two western blots with anti-His antibody. First on with bacterial lysate sample with induction and bacterial lysate without induction to confirm the expression of our FnCas12a circuit.
Second, We will perform a Western blot using three types of OMVs: Type 1, isolated from BL21DE3 bacteria co-transformed with SpTorA-FnCas12a-6xHis and Tat plasmid; Type 2, isolated from BL21DE3 bacteria transformed with only SpTorA-FnCas12a-6xHis; and Type 3, OMVs from BL21DE3 bacteria without any plasmid (control sample).

## Cloning of SpTorA-FnCas12a-6xHis

The p-spTorA-GFP-H6C plasmid (Addgene 168517) was linearized (4542 bp) through PCR
using pBAD-spTorA-GFP-6xHis-FP and pBAD-spTorA-GFP-6xHis-RP.

To create FnCas12a tagged with the SpTorA signal peptide, we synthesized the sequence in
two fragments from IDT: F1-SpTorA-FnCas12a (1501 bp) and F2-SpTorA-FnCas12a (2615 bp)
within pUCIDT-AMP GoldenGate plasmids. F1-SpTorA-FnCas12a was obtained via PCR using
F1-spTorA-FnCas12a-FP and F1-spTorA-FnCas12a-RP. F2-SpTorA-FnCas12a was recovered
using F2-spTorA-FnCas12a-FP and F2-spTorA-FnCas12a-RP.

The three fragments were assembled with NEB HiFi assembly master mix to generate the
AraBad-SpTorA-FnCas12a-6xHis plasmid, and the Gibson product was transformed into
DH5alpha using the heat shock method.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/cas-loading-in-sptora/2.png)
![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/cas-loading-in-sptora/4.png)

Fig 1: After recoving of each fragments was run in gel elctrophoresis and figure shows they are
in proper location. Ladder used in first well is NEB 1KB+.

For cloning confirmation, bacteria from the transformation were spread on ampicillin plates,
resulting in colony growth. Colony PCR was performed on 12 colonies using F1-spTorA-FnCas12a-FP, F1-spTorA-FnCas12a-RP, F2-spTorA-FnCas12a-FP, and
F2-spTorA-FnCas12a-RP separately. The presence of appropriate bands in gel electrophoresis
for both PCR products confirmed the successful insertion into the plasmid backbone, with the
orientation maintained by Gibson assembly.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/cas-loading-in-sptora/3.png)

Fig2: Colony PCR result of first 6 colonies.

In Fig. 2, Colony PCR bands corresponding to F1-SpTorA primers (2615 bp) and F2-SpTorA
primers (1501 bp) were observed in the correct position for every colony, while there were no
bands for the control colony uper 2nd well was loaded with PCR product of control colony with
F1-SpTorA primers and lower 2nd well was loaded with PCR product of control colony with
F2-SpTorA primers (normal DH5alpha without transformation with the Gibson assembly
product). Ladder in first well is NEB 1KB+.

## SDS-PAGE

SDS-PAGE data showing no band around 130 Kd for uninduced bacteria but every induced
bacterial lysate have a band at this position which indicates that FnCas12a protein may be
express after induction. But the induce band is faint. It could be as FnCas12a is very big protein
so we have optimise the induction and optimisation of SDS-PAGE itself needed.

![](https://static.igem.wiki/teams/4781/wiki/website-images/wetlab/cas-loading-in-sptora/sds.png)

Figure 3

## TAT - ABCE Plasmid construction

## Overview

The Tat circuit was planned so that we could hack the twin-arginine pathway of the bacteria that
exports folded proteins to the periplasm. In Escherichia coli and other Gram-negative bacteria,
TatA, TatB, and TatC are all essential for efficient translocation. The twin arginine translocase
(Tat) is a protein transport pathway that exists in archaea, bacteria, and plant chloroplasts. In
bacteria, it exports proteins across the plasma membrane and is important for many processes,
including energy metabolism, formation of the cell envelope, biofilm formation, heavy metal
resistance, nitrogen-fixing symbiosis, bacterial pathogenesis, and others. What makes this
protein transport system unusual compared to other transport systems (such as the general
secretory, or Sec pathway) is its ability to transport fully folded proteins across membranes. This
remarkable feat has no requirement for ATP as an energy source and relies solely on the proton
motive force (PMF).

• Upon receiving primers for linearizing the plasmid pSB1C3 for hi-fi DNA assembly
compatibility, gradient PCR was set at 68°C, 62°C, 64°C, 65, 66, and 67°C.
The bright band came in at 62°C and 64°C. The further PCR was done at 62°C.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-tat-gradient-12-09-2023-68-62-64-65-66-67.jpg)

• The next step was gel extraction, for which PCR was done and yielded a concentration
of 19.4ng/ul and a 260/280 ratio of 1.78.
• The next step was to do the assembly, which was then transformed into BL21DE3
competent cells and plated.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-cropped-tat-plasmid.jpg)

• The plasmid was isolated again from these cells, which gave a concentration of
123.5ng/ul and a 260/280 ratio of 1.85. This plasmid was then sent for sequencing.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-whatsapp-image-2023-10-12-at-17-42-27-fb4e9535.jpg)
![](https://static.igem.wiki/teams/4781/wiki/result-gfp-loading/cloning-results/tat-cloning.jpg)

Figure : Agarose Gel Electrophoresis to test successful assembly of TatABCE plasmid constructs with respect to pSB1C3 control. The size differences between those two shows that the assembly is successful.

## Double Transformation

A double transformation of spTorA-GFP along with the Tat circuit was performed to check if our
hypothesis that exogenous overexpression of the Tat ABCE complex will lead to greater
transport of our desired protein into the periplasm, then eventually into the outer membrane
vesicles, yielded successful results for double transformation.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-img20231009175743.jpg)

Figure: sptorA-Fncas12a-His6xTransformed in C41(DE3), plated on LB-Agar-Amp plates

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-img20231009175728.jpg)

Figure: sptorA-gfp-His6x Transformed in C41(DE3), plated on LB-Agar-Amp

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-cropped-tat-plasmid.jpg)

Figure: Tat-assembled Transformed in C41(DE3), plated on LB-Agar-Chl plates

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-img20231009175814.jpg)

Figure: sptorA-gfp+Tat-assembled Transformed in C41(DE3), plated on LB-Agar-Amp-Chl

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-img20231009175827.jpg)

Figure: sptorA-Fncas12a+Tat-assembled Transformed in C41(DE3), plated on LB-Agar-Amp-Chl plates

## GFP LOADING IN OMV and Localization of gfp into nucleoid of pathogen:

Before loading FnCas12a into OMVs we tried loading a small fluro-protein GFP into the OMVs by using GFP tagged with SpTorA signal peptide and exogenously overexpressing TAT machinery protein complexes. Confocal imaging shows that using co-transformation with plasmid TatABCE and P-spTorA-GFP-H6C, GFP localization into the periplasm increases, indicating proper functioning of the Tat pathway overexpression plasmid.

![](https://static.igem.wiki/teams/4781/wiki/result-gfp-loading/cloning-results/judging-presentation-5-mins.jpg)

Figure : Control-uninduced shows no GFP signal, E.coli with only P-spTorA-GFP-H6C shows gfp signal in whole cell, But the E.coli co-transformed with TatABCE and P-spTorA-GFP-H6C shows GFP localization in periphery.

Next, from the co-transformed E. coli BL21(DE3) strain we isolated the OMVs after appropriate induction with 1mM IPTG and 1% (w/v) Arabinose inducer. Then we incubated the isolated OMVs with our pathogenic strain Xoo and tested using Confocal Imaging. Results show that OMV fuses with the Xoo bacteria and GFP not only loads into the OMV but after incubation, loaded gfp of the OMV localize into the nucleoid of the Xoo bacteria. This result is actually very crucial for our project which indicate that in same way if we load the Cas12a-gRNA complex then that might also localize to pathogen’s nucleoid and do the double strand break.

![](https://static.igem.wiki/teams/4781/wiki/result-gfp-loading/cloning-results/gfp-loading.jpg)

Figure: Confocal Images for GFP internalized into Xoo after GFP-loaded OMVs fuse with Xoo. (a) Xoo Plasma Membrane stained with FM4-64 (b) Xoo Nucleoid stained with DAPI (c ) Internalization of GFP loaded OMVs with Xoo (d) MERGED image of all three

# Phase 3

## Detection Circuit

## Overview

The detection circuit was incorporated into the project after interaction with the farmers and
understanding their lack of knowledge about the disease. The detection kit works as a reporter
after the DSF sensor. It uses the two-component regulator system of RpfC- RpfG to sense the
domain. The RpfG converts cyclic-di-GMP into cyclic -GMP once it senses DSF. The Vc2
riboswitch linearizes with low levels of cyclic diGMP. This leads to transcriptional activation of
gfaspurple which produces the purple color.

• Upon receiving the DNA fragments for the circuit, they were run on 1% gel
electrophoresis to check if the synthesis led to the correct fragment size.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-det-1-2-3.jpg)

The fragments were synthesized correctly.
• We then performed gradient PCR to check the optimum temperature of melting for the
primers we ordered to linearize the plasmid and make it compatible with Hi-Fi DNA
Assembly. To make it compatible, we ordered primers such that it had 30 bp overlapping
regions with the first fragment and the last fragment.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-det-tat-23-8-23-58-60-62-64-66.jpg)

The most bright band was at 62 °C. Hence, we decided to perform the PCR for linearizing the
plasmid at 62°C.
• We then performed PCR and did the gel extraction, which yielded a concentration of
6.798ng/ul and a 260/280 ratio of 1.8.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-det-gel-extraction-conc-1.jpg)

• We then moved on to NEB Hi-Fi DNA Assembly. After assembly, we transformed it
directly into chemically competent DH5alpha cells and plated it in LB-Chloramphenicol
plates. After 12-14 hours of incubation at 37°C, we noticed colonies:

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-whatsapp-image-2023-10-12-at-15-42-34-dce8aac5.jpg)

• After this, we picked a colony from the plate and gave primary culture for plasmid
isolation. The plasmid isolation resulted in a concentration of 168.5ng/ul. It was loaded in
well B1.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-det-plasmid-isolation-conc.jpg)

• The plasmid isolated again went through PCR with the forward primer of the first
fragment and the reverse primer of the last fragment to check if our circuit was cloned
successfully. Then it was run in 0.8% agarose gel. The band was supposed to come
around 4578bp.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-det-26-9-23-pcr.jpg)

This proved that our cloning was successful. Further checking was to be done by sequencing the sample.
• The plasmid was further transformed into BL21DE3 cells for protein expression and
testing of the circuit.

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cropped-det-bl21de3.jpg)

## DSF ISOLATION(HRMS Analysis)

After isolating Diffusible Signal Factor (DSF), we used High-Resolution Mass
Spectrometry to confirm the isolation of the compound. The total yield obtained after
rotavapor and vacuum was 40mg. The expected mass of isolated DSF was
approximately 211kDa, as determined through various protocols and theoretical
calculations. However, our measured mass was around 209kDa ±1.

![](https://static.igem.wiki/teams/4781/wiki/reults/1.png)

Fig - Isolated DSF peak obtained from HRMS.

![](https://static.igem.wiki/teams/4781/wiki/reults/2.png)

Fig - Impurities and noise found in HRMS peak.

Although we successfully isolated DSF, we encountered a significant amount of
impurities. This could be attributed to the LB medium and analytical grade reagents used
in the process. To address this issue, we consulted with Postdoctoral Fellow Abhishek
Singh from the Department of Chemical Sciences. Abhishek recommended employing
LC-MS for better verification and noise reduction. He also highlighted potential limitations
associated with column chromatography.

## DETECTION TESTING

The detection circuit needed 2 inducers to function- DSF and IPTG. Of these, IPTG could be
easily prepared by mixing appropriate amounts of IPTG powder in distilled water. On the other
hand, we planned to isolate DSF from Xoo Culture itself.

The Xoo cells were first pelleted down and the supernatant was lyophilized. The powdered
supernatant contained DSF. The supernatant was then resuspended in methanol to generate
different concentrations of the lyophilized stock.

Then E.coli BL21DE3 containing Detection circuit was cultured and at OD600 value between
0.6-0.8, DSF solution was added along with IPTG.

We expected to get purple color due to the chromoprotein gFas-Purple, but unfortunately we did
not see any color change.

Possible reason could be that since lyophilized stock had a lot of impurities (as shown by HRMS
data), DSF concentration may not have been adequate enough to induce the chromoprotein
expression. Furthermore, methanol and its oxidized product are cytotoxic to cells, so we are not
sure if it had any effect on the system [2].

## FUSION

It is widely established that Gram-negative bacteria utilize outer membrane vesicles (OMVs) for
both intercellular and intracellular communication [3]. Given that our target pathogen,
Xanthomonas, is also a Gram-negative bacterium, we proposed the fusion of E. coli vesicles
with Xanthomonas. However, there is a lack of direct evidence for this interaction in the existing
literature, prompting us to pursue experimental validation.
The first step in fusion verification was performed using FACS and following results were
obtained:

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cloning-results/control.png)

The control Xanthomonas unstained showed relatively less Mean Fluorescent Intensity

![](https://static.igem.wiki/teams/4781/wiki/cloning-results/cloning-results/omv-fusion.png)

A 56-fold median rise was seen when OMVs were incubated in a 2:1 volume ratio with
Xanthomonas. Conclusion the Mean Fluorescent increase in the fusion sample suggests that
there is some kind of interaction taking place between OMV’s and Xanthomonas bacteria.

Our initial step involved isolating and purifying OMVs from E. coli. Subsequently, we labelled
these OMVs with a suitable dye and incubated them with Xanthomonas cells. If fusion occurs
between the vesicles and the Xanthomonas cell membrane, the dye will diffuse throughout the
membrane. The resulting membrane signal can then be detected using Confocal Microscopy.

An extremely crucial part of our project is the internalization of OMV constituents into the target
pathogen. To test the feasibility of this process, we expressed GFP in E.coli and loaded it into
OMVs via SpTorA signal peptide. We expected that if this loading was successful and E.coli
OMVs did fuse with Xanthomonas, then GFP would be internalized and we would get GFP
signals from Xanthomonas cells. And we did obtain a positive result.

With this we have proven our hypothesis that bacterial OMVs can indeed act as a delivery
system for functional proteins into other microbial systems. This is true for any protein that
comes within the size limit of Tat secretary pathway I.e. around 150kDa [4]

# Proof of Concept

## 1. Fusion of Outer Membrane Vesicles with Xoo

Our project was completely based on the assumption that the outer membrane vesicles will
naturally fuse with the Xanthomonas. This assumption came from several literature surveys
where they mentioned about gram negative bacteria communicating with each other through
outer membrane vesicles , the communication can be intra-species or inter-species. [1]

To prove our hypothesis, we designed an experiment where we exogenously expressed GFP in
E.coli BL21DE3. We then translocated this GFP into periplasm by hijacking the TAT secretary
pathway of Gram-negative bacteria, which transport folded proteins from cytosol to periplasm.
This GFP would then be internalized into OMVs.

These OMVs were then isolated from E.coli culture and purified. Parallely, we set up a
Xanthomonas culture and incubated the OMVs with it. This would allow the OMVs to fuse with
Xanthomonas plasma membrane and the GFP to be internalized. The GFP signal would then
be recorded via Fluorescence Microscopy.

## 2. Protein loading in OMV using SpTorA signal peptide and Exogenously expression of Tat

GFP loading in OMV (phase-2 result) proves that SpTorA signal peptide and Tat machinery can
be used to load protein. In future we will do experiment to test the Cas (bigger protein) is loading
in OMV by Tor signal peptide and exogenous expression of Tat machinery by western blot
method mention in Phase-2 result.

## Future prospects:

Positive results from fusion experiments opened up new possibilities for research in this arena
as we have verified that OMVs can act as delivery systems for functional proteins in bacterial
systems. In place of GFP, there can be other important proteins such as FnCas12a which can
be loaded and delivered via OMVs. Loading of Cas protein may be difficult owing to its large
size, but as smaller Cas proteins get discovered, our results may open door to new
opportunities of CRISPR/Cas delivery across biological systems. This can therefore have
enormous implications in the therapeutic sector.

Delivery of vesicular content into mammalian systems is well known. Now, we have also verified
the same across bacterial systems. Superiority of functional protein delivery DNA delivery lies in
the fact that there is no issue of lack of protein expression (since we are already introducing
functional proteins).

Further, we can also express target specific ligands on the surface of OMVs for targeted
delivery.
If we do any further work related to this project, we'll update it here: [Link](https://drive.google.com/drive/folders/1_2MmruTX6EpR_K_AV9dl1w_7j96bBMbV?usp=sharing)

# References

1. 1. Kesty NC, Kuehn MJ. Incorporation of heterologous outer membrane and periplasmic proteins
   into Escherichia coli outer membrane vesicles. Journal of Biological Chemistry. 2004 Jan
   16;279(3):2069-76.
2. 2. Patel R, Smith SM, Robinson C. Protein transport by the bacterial Tat pathway.
   Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2014 Aug
   1;1843(8):1620-8
3. 3. Zhou, Lian, et al. "Extraction, purification and quantification of diffusible signal factor family
   quorum-sensing signal molecules in Xanthomonas oryzae pv. oryzae." Bio-protocol 7.6 (2017):
   e2190-e2190
4. 4. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904680/#:~:text=coli%...](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904680/#:~:text=coli%3A%20methanol%20toxicity.,              with%20proteins%20and%20nucleic%20acids)
5. 5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308417/
6. 6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6708511/
7. 7. Berleman, James, and Manfred Auer. "The role of bacterial outer membrane vesicles for intra‐and
   interspecies delivery." Environmental microbiology 15.2 (2013): 347-354.

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