iGEM 2022 — Team iiser-tvm

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{% block title %}Notebook{% endblock %}
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![](https://static.igem.wiki/teams/4359/wiki/notebook/banner-10.png)

Throughout the duration of our project, each lab member maintained a lab notebook for their personal reference. We collated the main experiments of each day into a single document for internal reference and as a record of work. This lab notebook is written in chronological order. Detailed protocols for each experiment can be found in the experiments page.

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{% extends "layout-pages.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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![](https://static.igem.wiki/teams/4359/wiki/engineering/banner-4.png)

+ [Overview](#head1)
+ [Engineering Cycle #1](#head2)
+ [Engineering Cycle #2](#head3)
+ [Engineering Cycle #3](#head4)
+ [References](#ref)

---

# Introduction

---

The Design–Build–Test–Learn (DBTL) cycle is a framework used to derive designs that can be integrated into a feedback loop. The recursive nature of the DBTL cycle enables repeated improvements and is a popular approach to building parts in synthetic biology.
Each stage in the cycle has a distinct objective:

* The design is a conceptualization of the engineered system.
* The build is the execution of the design, in the form of new DNA parts or engineered organisms.
* The test is an objective and unbiased measure of the performance of the build.
* The results of the test enable learning, which facilitates improved design.

Our DBTL cycles focussed on (1) finding an appropriate *E. coli* chassis for OMV production (2) attenuating ClyA for usage in synthetic biology (3) finding epitope tags for recombinant protein detection.

---

# Selecting a hypervesiculating *E. coli* strain for OMV production

##### Engineering Cycle #1

---

*E. coli* K-12 lab strains naturally produce OMVs, however the low rate of production makes OMV isolation from *E. coli* K-12 cultures very resource intensive. Utilizing *E. coli* strains optimized for OMV isolation reduces the culture volume needed as well as the total time and equipment required for downstream processing.

### Design

In order to improve OMV yield, certain gene mutations can be introduced, which increase OMV production. Deletions in the following genes have been shown to increase yield:

**ΔtolR**

In *E. coli*, the Tol-Pal system comprises five proteins, including three inner membrane proteins (tolA, tolQ, and tolR), one periplasmic protein tolB, and outer membrane protein Pal. The Tol-Pal system tethers the outer membrane to the peptidoglycan layer and the inner membrane. Deletion of tolR produces defects in the Tol-Pal system, causing the dissociation of outer membrane from the underlying peptidoglycan, inducing membrane curvature and blebbing [[1]](#ref).

**ΔtolA**

tolA is another protein in the Tol-Pal system. Deleting tolA creates a hypervesiculating phenotype in a manner similar to deleting tolR [[2]](#ref).

**ΔdegP**

degP is a periplasmic protease/ chaperone that processes misfolded/unfolded proteins in the periplasm. Deleting degP enhances OMV production facilitates the removal of misfolded proteins from the cell [[3]](#ref).

**Δnlp1**

nlp1 is an outer membrane-anchored lipoprotein that has been associated with cell division, but its precise function is unknown. Deleting nlp1 is thought to induce instability in the peptidoglycan-outer membrane cross-links, hence increasing OMV production. [[4]](#ref)

### Build

Four *E. coli* K12 BW25113 strains, each with a single knockout of the genes (ΔtolR, ΔtolA, ΔdegP and Δnlp1) were obtained from the Keio collection [[5]](#ref). These knockout strains contain a kanamycin resistance cassette in place of the genes.

### Test

**Comparing fitness**

Given that our chosen *E. coli* chassis would experience a metabolic burden during heterologous protein production, we compared growth curves and OD600 at the final time point (final OD600) of the strains to gain a rough estimate of the fitness cost of the gene knockouts. All cultures were standardized to intital OD600 = 0.001 and grown for 18h with orbital shaking in a 96-well plate. The OD600 was measured every 15 min by a microplate spectrophotometer.

![picture](https://static.igem.wiki/teams/4359/wiki/results/1.png)

***Fig 1:**
Comparative analysis of growth curves of four hypervesiculating E.coli strains.*

![picture](https://static.igem.wiki/teams/4359/wiki/results/2.png)

***Fig 2:**
Individual growth curves of (A) ΔtolA (B) ΔtolR (C) ΔdegP and (D) Δnlp1. Error bars indicate mean ± standard error of the mean of three biological replicates, each having two technical replicates.*

**Table 1:** Final OD600 of four hypervesiculating strains. Values are reported as mean ± standard error of the mean of three biological replicates, each having two technical replicates.

| Strain | Mean final OD600 ± standard error of mean |
| --- | --- |
| ΔtolA | 0.71 ± 0.015 |
| ΔtolR | 0.71± 0.022 |
| Δnlp1 | 0.70 ± 0.015 |
| ΔdegP | 0.67 ± 0.012 |

**Comparing total OMV yield**

The concentration of OMVs in a solution can be indirectly quantified using protein concentration as a proxy measure [[2][6]](#ref). Protein quantification was performed using a Bradford Assay. A standard curve was constructed using Bovine Serum Albumin (BSA), and the regression line was used to compute protein concentrations of OMV samples (Table 2, Fig 3). .

**Table 2:** Protein concentration of OMVs fractions isolated from ΔtolR and Δnlp1. Higher total protein was noted for the OMV fraction isolated from Δnlp1. Measurements were carried out in duplicate.

| Sample | Mean adjusted absorbance at 595nm (A) | Protein concentration (ug/ml) (C) | Total volume (ml) (T) | Total protein (P = C\*T) (ug) |
| --- | --- | --- | --- | --- |
| ΔtolR OMV | 0.028 | 100.64 | 0.11 | 11.06993 |
| Δnlp1 OMV | 0.011 | 48.18 | 0.31 | 14.93495 |

![picture](https://static.igem.wiki/teams/4359/wiki/engineering/eng2.png)

***Fig 3:**
Comparative analysis of the OMV yield from ΔtolR and Δnlp1. OMV yield is measured as total protein in the sample.*

**Comparing the size distribution of OMVs**

The size distribution of nanoparticles such as OMVs can be measured via dynamic light scattering. We compared the size distributions of OMV isolated from *E. coli* ΔtolR and Δnlp1 (Fig 4)

![picture](https://static.igem.wiki/teams/4359/wiki/engineering/eng3.png)

***Fig 4:**
Comparison of the size distribution of OMVs isolated from *E. coli* ΔtolR and *E. coli* Δnlp1. The X axis indicates diameter (in nm) and the Y axis indicates the percentage. There is a strong correspondence between the two distributions.*

Average diameter of *E. coli* ΔtolR OMVs = 40 nm

Average diameter of *E. coli* Δnlp1 OMVs = 39 nm

### Learn

Among the four strains, ΔdegP showed delayed growth kinetics and lowest final OD600 (Fig 1, 2, Table 1) and was considered unsuitable for heterologous protein expression. Literature suggests that ΔtolR produces more OMVs than ΔtolA [[7]](#ref). Therefore, further analysis of OMV yield and size distribution was restricted to ΔtolR and Δnlp1. We found no significant difference between the sizes of OMVs derived from both strains (Fig 4), but found that Δnlp1 produced more OMVs than ΔtolR when subjected to the same culture conditions and isolation protocol (Fig 3). From our observations, we chose Δnlp1 to produce OMVs for the remainder of the project.

---

# Attenuating Cytolysin A

##### Engineering Cycle #2

---

Cytolysin A (ClyA) is a 34 kDa protein coded by the ClyA gene in *E. coli* genome. ClyA preferentially localizes to the outer membrane, causing it to be enriched in OMVs. We exploited this property to use ClyA to localize and display recombinant fusion partners on the surface of OMV. The existing version of ClyA on the registry, ([BBa\_K811000](http://parts.igem.org/Part%3ABBa_K811000)) is hemolytically active and toxic to mammalian cells [[8]](#ref). It has been used by teams in other composite parts, such as [BBa\_K3989010](http://parts.igem.org/Part%3ABBa_K3989010), and [BBa\_K4375019](http://parts.igem.org/Part%3ABBa_K4375019). While genetic fusions with other proteins abolishes the pore-forming ability of ClyA, we wished to create a safer alternative for future teams.

### Design

ClyA exists in three known conformations: monomeric, protomeric and oligomeric. The monomer is the soluble form of the protein that undergoes a conformational change to the membrane-inserted protomeric form. The conformational change involves rearrangement of the beta-tongue motif to a helix-turn-helix motif. Upon reaching the target membrane, ClyA protomers oligomerize to insert into the membrane to form a pore. We designed ClyA (Y181F) ([BBa\_K4359001](http://parts.igem.org/Part%3ABBa_K4359001)), a derivative of ClyA with a Y181F substitution mutation. This tyrosine residue is contained in the cholesterol binding site in the beta-tongue motif of ClyA. The substitution is speculated to hinder the transition from the beta-tongue motif to the helix-turn-helix motif accompanying protomer formation. Y181F results in a near loss of the hemolytic activity of ClyA, as determined by erythrocyte lysis assays in previous studies [[9]](#ref).

### Build

We avoided working with the unattenuated version of ClyA. We wished to evaluate the safety of ClyA (Y181F) when used as intended in a composite part like ClyA-3XFLAG-THP ([BBa\_K4359009](http://parts.igem.org/Part%3ABBa_K4359009)). We obtained the sequence for ClyA-3XFLAG-THP via gene synthesis.

### Test

The MTT assay is a colorimetric assay for assessing cell metabolic activity, and can be used to measure cell viability. In this case, we aimed to measure cell viability following treatments of different concentrations of THP-OMVs expressing ClyA-3XFLAG-THP, as compared to a control treatment of PBS. SK-BR-3 cells were utilised for this assay. The mean percentage viability is reported as the viability of THP-OMVs treated SK-B-R3 cells normalized to the mean viability following PBS treatment (Fig 5). The complete protocol can be found on the [experiments page](https://2022.igem.wiki/iiser-tvm/experiments).

![picture](https://static.igem.wiki/teams/4359/wiki/engineering/eng4.png)

***Fig 5:**
Cytotoxicity of THP-OMVs as measured using the MTT assay with SK-BR-3 cell cultures. Values are reported as mean ± standard error of the mean of three biological replicates.*

### Learn

The results of the MTT assay indicated that THP-OMVs did not significantly affect cell viability. We utilized ClyA (Y181F) for our other construct ClyA-V5-Affi ([BBa\_K4359008](http://parts.igem.org/Part%3ABBa_K4359008)).

---

# Selecting an epitope tag for detection of recombinant proteins

##### Engineering Cycle #3

---

### Design

Detecting our recombinant proteins was an important part of the project. Epitope tags can be used for this purpose as they are well characterized and antibodies are commercially available. We required two tags whose detection was orthogonal and whose antibodies would cross react minimally with mammalian proteins. We originally received the construct ClyA-Myc-Affi ([BBa\_K4359006](http://parts.igem.org/Part%3ABBa_K4359006)), containing the myc tag. We hypothesized that antibodies against the myc tag would also cross react with endogenous c-myc which is often overexpressed in many cancer cell lines.

### Build

ClyA-Myc-Affi was obtained from Dr. Vipul Gujrati, TUM Germany. It was cloned in the backbone pGEX-4T1.

### Test

We performed a Western blot of cell lysate of the breast cancer cell line SK-BR-3 (Fig 6). Endogenous c-myc is 62 kDa but is known to degrade quickly to lower molecular weights.

![picture](https://static.igem.wiki/teams/4359/wiki/engineering/eng5.png)

***Fig 6:**
Western blot of SK-BR-3 cell lysate. The membrane was probed with monoclonal anti-myc tag antibody.*

### Learn

While we were unable to ascertain whether the signal detected was from c-myc, we concluded that the anti-myc tag antibody cross-reacted with components of the cell lysate of our test cell line, SK-BR-3. This could potentially interfere with detection during cellular uptake studies. We designed an alternative version with a V5 tag in place of the myc tag, ClyA-V5-Affi ([BBa\_K4359008](http://parts.igem.org/Part%3ABBa_K4359008))

---

# References

---

1. Shrivastava, R., Jiang, X., & Chng, S.-S. (2017). Outer membrane lipid homeostasis via retrograde phospholipid transport in Escherichia coli: A physiological function for the Tol-Pal complex. Molecular Microbiology, 106(3), 395–408. <https://doi.org/10.1111/mmi.13772>

2. Reimer, S. L., Beniac, D. R., Hiebert, S. L., Booth, T. F., Chong, P. M., Westmacott, G. R., Zhanel, G. G., & Bay, D. C. (2021). Comparative Analysis of Outer Membrane Vesicle Isolation Methods With an Escherichia coli tolA Mutant Reveals a Hypervesiculating Phenotype With Outer-Inner Membrane Vesicle Content. Frontiers in Microbiology, 12, 628801. <https://doi.org/10.3389/fmicb.2021.628801>

3. Schwechheimer, C., & Kuehn, M. J. (2013). Synthetic Effect between Envelope Stress and Lack of Outer Membrane Vesicle Production in Escherichia coli. Journal of Bacteriology, 195(18), 4161–4173. <https://doi.org/10.1128/JB.02192-12>

4. Schwechheimer, C., Rodriguez, D. L., & Kuehn, M. J. (2015). NlpI‐mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli. MicrobiologyOpen, 4(3), 375–389. <https://doi.org/10.1002/mbo3.244>

5. *E. coli* Keio Knockouts. Link [here](https://horizondiscovery.com/en/non-mammalian-research-tools/products/e-coli-keio-knockouts)

6. Gujrati, V., Kim, S., Kim, S.-H., Min, J. J., Choy, H. E., Kim, S. C., & Jon, S. (2014). Bioengineered Bacterial Outer Membrane Vesicles as Cell-Specific Drug-Delivery Vehicles for Cancer Therapy. ACS Nano, 8(2), 1525–1537. https://doi.org/10.1021/nn405724x

7. Li, Q., Li, Z., Fei, X., Tian, Y., Zhou, G., Hu, Y., Wang, S., & Shi, H. (2022). The role of TolA, TolB, and TolR in cell morphology, OMVs production, and virulence of Salmonella Choleraesuis. AMB Express, 12(1), 5. https://doi.org/10.1186/s13568-022-01347-4

8. Mueller, M., Grauschopf, U., Maier, T., Glockshuber, R., & Ban, N. (2009). The structure of a cytolytic α-helical toxin pore reveals its assembly mechanism. Nature, 459(7247), 726–730. <https://doi.org/10.1038/nature08026>

9. Kulshrestha, A., Maurya, S., Gupta, T., Roy, R., Punnathanam, S., & Ayappa, K. G. (2021). Conformational flexibility is a key determinant of the lytic activity of the pore forming protein, Cytolysin A [Preprint]. Biophysics. <https://doi.org/10.1101/2021.10.23.465544>

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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![](https://static.igem.wiki/teams/4359/wiki/protocols/banner-5.png)

+ [Overview](#head1)
+ [Materials](#head2)
+ [Methods](#head3)

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# Overview

---

This page contains a list of materials (bacterial strains, plasmids, primers, antibodies) and methods for this project. Original source material(s) for the protocols are indicated where relevant.

---

# Materials

---

## Bacterial Strains

| Strain | Description | Source |
| --- | --- | --- |
| E.coli K12 Standard laboratory strain | Mutations Lab, IISER TVM | |
| E.coli DH5ɑ Strain optimized for cloning | LICB, IISER TVM | |
| E.coli Mach1 Fast growing, chemically competent cells | Host-Pathogen Interaction Lab, IISER TVM | |
| E.coli XL10 gold Ultracompetent, fast growing competent cells | Mutations Lab, IISER TVM | |
| E.coli K12 ΔtolA K12 strain with gene knockout ΔtolR::Kan | Keio collection | |
| E.coli K12 ΔTolR K12 strain with gene knockout ΔtolA::Kan | Keio collection | |
| E.coli K12 Δnlp1 K12 strain with gene knockout Δnlp1::Kan | Keio collection | |
| E.coli K12 ΔdegP K12 strain with gene knockout ΔdegP::Kan | Keio collection | |

## Plasmids

| Plasmid | Description | Source |
| --- | --- | --- |
| pGEX-4T1 Plasmid for protein expression in E.coli. pBR322 origin, IPTG inducible tac promoter | Dr. Vipul Gujrati, TUM Germany | |
| pET-28a Plasmid for protein expression in E.coli. IPTG inducible T7 promoter | Mutations Lab, IISER TVM | |

## Primers

| Primers | Description | Sequence |
| --- | --- | --- |
| ClyA\_Myc\_Affi\_FP1 Forward sequencing primer for ClyA-Myc-Affi (backbone specific) | cataattcgtgtcgctcaaggc | |
| ClyA\_Myc\_Affi\_FP2 Forward sequencing primer for ClyA-Myc-Affi (insert specific) | ccaaacagtgtatgaatggtgtgg | |
| ClyA\_Myc\_Affi\_RP1 Reverse sequencing primer for ClyA-Myc-Affi (backbone specific) | caagctgtgaccgtctcc | |
| ClyA\_THP\_FP1\_EcoNI Forward primer for ClyA-3XFLAG-THP | TTGAACCCTATACTAGGCAAATTGATGGCCT | |
| ClyA\_THP\_RP1\_BamHI Reverse primer for ClyA-3XFLAG-THP | GTTACGGATCCGATTTAACAAAACG | |
| ClyA\_Affi\_FP1\_EcoNI Forward primer for ClyA-V5-Affi | ttgaacCCTATACTAGGAaagctaatg | |
| ClyA\_Affi\_RP1\_BamHI Reverse primer for ClyA-V5-Affi | GTTACggatccAATtcattttggtg | |

## Antibodies

| Antibody | Catalog number/supplier | Dilution used |
| --- | --- | --- |
| Anti OmpA Ab *E. coli* Antibody Research Corporation, Prayaga Scientific | 1:200 (Cell culture) 1:300 (Western blot) | |
| Anti V5-Tag, rabbit mAb #13202, Cell Signaling Technologies | 1:400 (Cell culture) 1:1000 (Western blot) | |
| ANTI-FLAG M2, Clone M2, mouse mAb,  Sigma-Aldrich | 1:300 (Cell culture) | |
| Alexa Fluor™ 488 conjugated AffiniPure Goat anti-Rabbit IgG (H+L) Jackson ImmunoResearch laboratories | 1:1000 (Cell culture) | |
| Alexa Fluor™ 488 conjugated AffiniPure Goat anti-Mouse IgG (H+L) Jackson ImmunoResearch laboratories | 1:1000 (Cell culture) | |
| Cyanine Cy™ 5 conjugated AffiniPure Goat anti-Rabbit IgG (H+L) Jackson ImmunoResearch laboratories | 1:1000 (Cell culture) | |
| Anti-myc tag primary antibody, Rabbit Thermo Fisher | 1:500 (Western blot) | |
| Anti-Rabbit HRP conjugated antibody Antibody Research Corporation | 1:5000 (Western blot) | |
| V5-Tag, rabbit mAb #13202, Cell Signaling Technologies | 1:400 (Cell culture) 1:1000 (Western blot) | |

## Softwares

| Software | Use |
| --- | --- |
| R Studio Analysing growth curve and Bradford assay results | |
| Graphpad Prism | Plotting and visualising results |
| MATLAB | Math modeling |
| Python Math modelling | |
| ImageLab Imaging and analysing SDS PAGE gels and Western blots | |
| A Plasmid Editor Analysing DNA sequences | |
| SnapGene | Analysing DNA sequences, designing primers, analysing chromatograms |

---

# Methods

---

## Bacterial Cultures

## Cloning

## OMV Isolation and Characterization

## Protein Quantification and Identification

## Mammalian Cell Culture and Cellular Uptake Experiments

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{% extends "layout-pages.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Describe all the safety issues of your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4359/wiki/results/banner-14.png)

+ [Overview](#head1)
+ [Phase 1](#head2)
+ [Phase 2](#head3)
+ [Phase 3](#head4)
+ [References](#ref)

---

# Overview

---

The overall goal of the project was to produce and test two populations of modified OMVs: affi-OMVs expressing an anti-HER2 affibody and THP-OMVs expressing a tumour homing peptide (THP) against CX3CR1. Our hypothesis was that both affi-OMVs and THP-OMVs would be selectively and simultaneously internalised in HER2+ and CX3CR1+ breast cancer cells, when compared to non-cancerous cells. The project was divided into three phases with distinct goals, to enable us to track our progress and achieve important milestones. Details of particular experiments can be found in their corresponding protocols or in the lab notebook.

---

# Phase 1

---

Phase 1 of the project focused on isolating unmodified native OMVs from different hypervesiculating *E.coli* strains. This involved selecting an appropriate  *E.coli* strain for OMV production, optimising the protocol for OMV isolation, verifying the isolation of OMVs, and characterising OMV size, morphology, and concentration.

## Comparison of Growth Curves

Standard laboratory strains of *E.coli* have minimal production of OMVs in culture, and are unsuitable for purposes requiring large amounts of OMVs [[1]](#ref). Four *E.coli* K12 single gene knockout strains (ΔtolA, ΔtolR, Δnlp1, ΔdegP) were previously reported to have improved yields over the parental K12 strain [[1][2][3][4]](#ref). These strains were obtained from the Keio collection [[5]](#ref). We compared growth curves and final OD600 of the strains to gain a rough estimate of the fitness cost of the gene knockouts. All cultures were standardised to OD600 = 0.001 and grown for 18h with orbital shaking in a 96-well plate. OD600 was measured every 15 minutes by a microplate reader.

ΔdegP showed delayed growth kinetics and the lowest final OD600 (Fig 1, 2, Table 1). From our literature review, we found that ΔtolR produces more OMVs than ΔtolA. Therefore, the remainder of the project was restricted to *E.coli* Δnlp1 and *E.coli* ΔtolR.

![picture](https://static.igem.wiki/teams/4359/wiki/results/1.png)

***Fig 1:**
Comparative analysis of growth curves of four hypervesiculating *E.coli*  strains.*

![picture](https://static.igem.wiki/teams/4359/wiki/results/2.png)

***Fig 2:**
Individual growth curves of (A) ΔtolA (B) ΔtolR (C) ΔdegP and (D) Δnlp1. Error bars indicate mean ± standard error of mean of three biological replicates, measured in duplicates.*

**Table 1:** Final OD600 of four hypervesiculating strains. Values are reported as mean ± standard error of mean of three biological replicates, each measured in duplicates.

| Strain | Mean final OD600 ± standard error of mean |
| --- | --- |
| ΔtolA | 0.71 ± 0.015 |
| ΔtolR | 0.71± 0.022 |
| Δnlp1 | 0.70 ± 0.015 |
| ΔdegP | 0.67 ± 0.012 |

## Isolation of native OMVs from from *E.coli* ΔtolR and *E.coli* Δnlp1

The next step was to isolate OMVs from *E.coli* ΔtolR and *E.coli* Δnlp1. OMVs were isolated by serial centrifugation of cell-free supernatants of *E.coli* cultures, first at 48,000g and then 100,000g. The complete protocol is available on the experiments page.

We went through many iterations of optimisation where we tried different rotors, increased the duration of centrifugation, added alternate rounds of centrifugation to concentrate the sample, and switched from syringe filtration to vacuum filtration to process higher culture volumes.

OMVs were isolated from *E.coli* ΔtolR and *E.coli* Δnlp1, under similar conditions and with the same protocol. We used these samples to confirm the presence of OMVs in the isolated fraction, and to compare vesiculation between ΔtolR and Δnlp1.

## Confirmation of the presence of OMVs in the isolated fraction via Anti-OmpA Western blot

OMVs can be detected indirectly by probing for the presence of outer membrane proteins that are present in most gram negative bacteria. The most common protein used for this purpose is Outer Membrane Protein A (OmpA). OmpA is a 37kDa constitutively expressed protein.

To reliably use OmpA as a marker for OMVs, we verified that OmpA was present in all strains we utilised. We also confirmed the presence of OmpA in the OMV fraction isolated from *E.coli* ΔtolR (Fig 3).

![picture](https://static.igem.wiki/teams/4359/wiki/results/3.png)

***Fig 3:**
Anti-OmpA Western blot of OMVs isolated from ΔtolR, and bacterial pellets of parental strain *E.coli* K12 and hypervesiculating strains ΔtolA, ΔtolR, Δnlp1 and ΔdegP. Overnight cultures were standardised to OD600 = 1, and equal volumes of culture were loaded in each lane to control for cell numbers, and probed with anti-OmpA anitbody. Bands were detected at the expected size of 37 kDa. An additional lower molecular weight band was noted in the bacterial pellets, that was absent in the OMV fraction.*

Subsequently, we confirmed the presence of OmpA in the OMV fraction isolated from *E.coli* Δnlp1, and compared it to OMVs previously isolated from *E.coli* ΔtolR. OmpA was detected in both samples, indicating the presence of OMVs (Fig 4).

![picture](https://static.igem.wiki/teams/4359/wiki/results/4.png)

***Fig 4:**
Anti-OmpA Western blot of OMV fractions from *E.coli* ΔtolR and *E.coli* Δnlp1. Equal volumes were loaded for the OMV samples. Cell pellets of both strains were also analysed, but the presence of excess sample in these lanes hindered the proper resolution of proteins during electrophoresis, causing a hazy and diffused appearance (Fig 5, duplicate samples were analysed on SDS PAGE).*

## Evaluating the effectiveness of the OMV isolation protocol

During the isolation of OMVs, cell debris may co-sediment, causing inflated protein concentration values, as well as introducing unwanted bacterial cell components. Isolated OMVs were found to be free of viable cells by plating on LB agar. SDS PAGE analysis revealed that OMVs were highly enriched for proteins ~35 kDa in size, which we hypothesise are members of the outer membrane protein (Omp) family (Fig 5). Very faint bands were noted for other molecular weights, indicating the general absence of cell debris. Overall, the OMV isolation protocol was effective and primarily yielded OMVs, with minimal contamination.

![picture](https://static.igem.wiki/teams/4359/wiki/results/5.png)

***Fig 5:**
SDS PAGE of OMVs are their corresponding source strains. Excess sample for bacterial pellets caused the appearance of a smear. OMV samples had intense bands ~35 kDa. This SDS PAGE gel is a duplicate of the samples analysed by Western blot (Fig 3).*

## Confirmation of the presence of OMVs in the isolated fraction via transmission electron microscopy

To confirm the presence of OMVs, we first imaged native ΔtolR OMVs using a 120kV transmission electron microscope (Fig 6). The images clearly indicated the presence of spherical heterogeneous structures, 100-200 nm in diameter, consistent with the reported size range of OMVs [[7]](#ref).

![picture](https://static.igem.wiki/teams/4359/wiki/results/6.png)

***Fig 6:**
Transmission electron microscope image of OMVs isolated from *E.coli* ΔtolR. Spherical structures were noted but the lipid layer was not visible. Stain used was 4% phosphotungstic acid stain and the ΔtolR OMVs were resuspended in distilled water.*

To better visualise the morphology of these structures, OMV sample was imaged by cryo-TEM.

![picture](https://static.igem.wiki/teams/4359/wiki/results/7.png)

***Fig 7:**
(A, B) Cryogenic electron microscope images of *E.coli* ΔtolR OMVs suspended in 1X PBS. The lipid bilayer is distinct, confirming the presence of OMVs.*

## Comparison of OMV yields from *E.coli* ΔtolR and *E.coli* Δnlp1

OMVs can be indirectly quantified using protein concentration as a proxy measure [[2]](#ref) [[6]](#ref). Other methods of quantification are nanoparticle tracking analysis and electron microscopy. Protein quantification of the OMV samples was performed via a Bradford Assay. A standard curve was constructed using Bovine Serum Albumin (BSA) and the regression line was used to compute protein concentrations of OMV samples (Table 2, Fig 8). When subjected to the same culture conditions and isolation procedure, *E.coli* Δnlp1 yielded a higher amount of OMVs. This may be due to intrinsic differences in the physiology of the two strains, or due to differences in cell numbers.

Table 2: Protein concentration of OMVs fractions isolated from *E.coli* ΔtolR and *E.coli* Δnlp1. Measurements were carried out in duplicates.

| Sample | Mean adjusted absorbance at 595nm (A) | Protein concentration (ug/ml) (C) | Total volume (ml) (T) | Total protein (P = C\*T) (ug) |
| --- | --- | --- | --- | --- |
| ΔtolR OMV | 0.028 | 100.64 | 0.11 | 11.06993 |
| Δnlp1 OMV | 0.011 | 48.18 | 0.31 | 14.93495 |

![picture](https://static.igem.wiki/teams/4359/wiki/results/8.png)

***Fig 8:**
Comparative analysis of the OMV yield from two *E.coli* strains. OMV yield is measured as total protein in the sample, in micrograms.*

## Comparison of size distribution of OMVs from *E.coli* ΔtolR and *E.coli* Δnlp1

The size distribution of nanoparticles such as OMVs can be measured via dynamic light scattering [[9]](#ref). We compared the size distributions of OMVs isolated from *E.coli* ΔtolR and *E.coli* Δnlp1.

![picture](https://static.igem.wiki/teams/4359/wiki/results/9.png)

***Fig 9:**
Comparison of the size distribution of OMVs from *E.coli* ΔtolR and *E.coli* Δnlp1. The X axis indicates diameter (in nm) and the Y axis indicates the percentage. There is a strong correspondence between the two distributions.*

Average diameter of *E. coli* ΔtolR OMVs = 40 nm

Average diameter of *E. coli* Δnlp1 OMVs = 39 nm

**Summary**

We acquired four *E.coli* hypervesiculating strains: ΔtolA, ΔtolR, Δnlp1, ΔdegP. From our bacterial fitness experiment and literature review, we restricted our experiments to *E.coli* ΔtolR and *E.coli* Δnlp1. We verified the presence of OMVs in the isolated fractions of ΔtolR and Δnlp1 by TEM and probing for OmpA. We compared the size distribution and OMV yield from ΔtolR and Δnlp1, and found that Δnlp1 produced more OMVs, but the size distribution did not differ. We chose Δnlp1 *E.coli* to be the chassis for the project. We confirmed that the isolated OMV were free from viable cells and cell debris.

---

# Phase 2

---

The goal of phase 2 was to produce affi-OMVs and THP-OMVs. This involved designing and obtaining DNA constructs for our composite parts, cloning them into *E.coli* expression vector pGEX-4T1, inducing the expression of the proteins and isolating affi-OMVs and THP-OMVs.

## Testing the expression of the ClyA-Myc-Affi

We designed two alternate sequences for the affibody: ClyA-Myc-Affi ([BBa\_K4359006](http://parts.igem.org/Part%3ABBa_K4359006)) and ClyA-V5-Affi ([BBa\_K4359008](http://parts.igem.org/Part%3ABBa_K4359008)) . We first received ClyA-Myc-Affi cloned into a pGEX-4T1 backbone from Dr. Vipul Gujrati, TUM Germany. This backbone was chosen because the multiple cloning site (MCS) was downstream of the IPTG-inducible tac promoter, enabling control over protein expression. We verified the identity of the plasmid and the insert via a PCR with backbone-specific primers (Fig 10A) and Sanger sequencing (Fig 10B). Following confirmation, we transformed the plasmid into *E.coli* Δnlp1.

![picture](https://static.igem.wiki/teams/4359/wiki/results/10.png)

***Fig 10:**
(A) PCR amplification of ClyA-Myc-Affi produced an amplicon of the expected size (1.5kb). (B) Chromatogram of Sanger sequencing of the sample pGEX-4T1 ClyA-Myc-Affi. Three sequencing reactions were carried, two on the top strand and one on the bottom strand. On average, 300-400 bp of reads were obtained per reaction.*

Next, we tested the expression of the protein. An overnight culture was diluted 1/100 into 25 ml of LB ampicillin and grown at 37 degree celsius till mid-log. The culture was induced with IPTG (final concentration 1 mM) and grown for 18 hrs at 20 degree celsius. Cells were pelleted by centrifugation and run on SDS PAGE, followed by Coomassie Brilliant Blue staining (Fig 11). A band at 45kDa was visible, corresponding to the molecular weight of ClyA-Myc-Affi.

![picture](https://static.igem.wiki/teams/4359/wiki/results/11.png)

***Fig 11:**
SDS PAGE gel image comparing protein profiles of cells before and after IPTG induction. A band ~45 kDa in size is visible following induction, suggesting the presence of ClyA-Myc-Affi.*

## Verifying the localisation of ClyA-Myc-Affi to the OMV fraction

Affi-OMVs were isolated from *E.coli* Δnlp1 expressing ClyA-Myc-Affi. OMVs obtained were analysed by SDS PAGE to detect the presence of ClyA-Myc-Affi in the OMV fraction (Fig 12).

![picture](https://static.igem.wiki/teams/4359/wiki/results/12.png)

***Fig 12:**
(A) SDS PAGE gel image indicating expression of ClyA-Myc-Affi (red arrow) in the bacterial cells following IPTG induction. A faint band is also visible prior to induction, in line with the known leaky behaviour of the lac operon. A faint band of the same molecular weight (black arrow) is visible in Affi-OMVs but is absent in native OMVs. Lower molecular weight bands are also visible in both cells post-induction and Affi-OMV (green arrow) but absent in cells pre-induction. (B) SDS PAGE gel image of Affi-OMV sample from (A). Higher amount was loaded to visualise the bands more clearly. ClyA-Myc-Affi is visible at 45 kDa (black arrow), along with the lower molecular weight band noted earlier (green arrow).*

The presence of ClyA-Myc-Affi in affi-OMVs was definitively confirmed via Western blot (Fig 13).

![picture](https://static.igem.wiki/teams/4359/wiki/results/13.png)

***Fig 13:**
(A) Western blot showing the presence of ClyA-Myc-Affi in *E.coli* cell pellets before (0 hrs) and after (18 hrs) of IPTG induction. The membrane was probed with rabbit anti-myc tag primary antibody. A band was detected at the expected size of 45 kDa. An additional higher molecular weight band was also noted. (B) The presence of ClyA-Myc-Affi was also noted in Affi-OMVs. Signal detected was proportional to the amount of OMVs loaded.*

## Cloning ClyA-V5-Affi and ClyA-3XFLAG-THP into pGEX-4T1 backbone

As determined from our [engineering cycle 3](https://2022.igem.wiki/iiser-tvm/engineering/), we found that the myc tag is incompatible for use with mammalian cell lines due to cross-reactivity of anti-myc antibodies with the myc-tag and components of mammalian cell lysate. We improved our design to replace this tag with the V5 epitope tag instead. We obtained our composite parts ClyA-V5-Affi (BBa\_K4359008) and ClyA-3XFLAG-THP (BBa\_K4359009) by DNA synthesis. The parts were flanked by EcoN1 and BamH1 restriction sites which were used to ligate them into the plasmid pGEX-4T1. We successfully digested and ligated these parts into the backbone to produce pGEX-4T1 ClyA-V5-Affi and pGEX-4T1 ClyA-3XFLAG-THP (Fig 14). We transformed these plasmids in *E.coli* DH5a and confirmed our clones by colony PCR, restriction digestion (Fig 15) and Sanger sequencing (Fig 16).

![picture](https://static.igem.wiki/teams/4359/wiki/results/14.png)

***Fig 14:**
Agarose gel image of the pGEX-4T1 backbone digested with EcoN1 and BamH1. Linearised backbone with cohesive ends was obtained at the expected size (4.3 kb). (B) PCR amplification of gBlocks dsDNA gene fragments of ClyA-3X-FLAG (1049 bp) and ClyA-V5-Affi (1217 bp) using the Fantom high-fidelity polymerase. The PCR fragments were digested with EcoN1 and BamH1 and ligated (with a 1:7 ratio of insert:backbone) into the linearised backbone.*

![picture](https://static.igem.wiki/teams/4359/wiki/results/15.png)

***Fig 15:**
(A) Agarose gel images of colony PCR to identify the positive clones. We screened 5 colonies each for pGEX4T-1 ClyA-V5-Affi and pGEX4T-1 ClyA-3XFLAG-THP, and identified 3 positive clones for the former and 2 positive clones for the latter. (B) Restriction digestion using the EcoN1 and BamH1 restriction sites released inserts corresponding to their expected sizes: ClyA-3XFLAG-THP (1 kb) and ClyA-V5-Affi (1.2 kb).*

![picture](https://static.igem.wiki/teams/4359/wiki/results/16.png)

***Fig 16:**
Representative chromatogram of Sanger sequencing of the sample pGEX-4T1 ClyA-V5-Affi and pGEX 4T1 ClyA-3XFLAG-THP. For pGEX-4T1 ClyA-V5-Affi, three sequencing reactions were carried: two on the top strand and one on the bottom strand. For pGEX-4T1 ClyA-3X-FLAG, two sequencing reactions were carried: one on the top strand and one on the bottom strand. On average, 300-400 bp of reads were obtained per reaction.*

## Testing the expression of ClyA-V5-Affi and ClyA-3XFLAG-THP

Following successful cloning, we transformed *E.coli* Δnlp1 to produce two populations: one containing pGEX-4T1 ClyA-V5-Affi and the other containing pGEX-4T1 ClyA-3XFLAG-THP, to produce Affi-OMVs and THP-OMVs respectively. Prior to isolating OMVs, we tested the expression of the proteins, similar to Fig 11. We verified that full length ClyA-V5-Affii and ClyA\_3XFLAG\_THP were being produced, corresponding to the appropriate band sizes.

![picture](https://static.igem.wiki/teams/4359/wiki/results/17.png)

***Fig 17:**
SDS PAGE gel image indicating expression of ClyA-V5-Affi (green arrow) and ClyA-3XFLAG-THP (red arrow) in the bacterial cells following IPTG induction. Lower molecular weight bands (black), similar to the size seen in Fig (11) and Fig (12) are also visible in both cases post-induction.*

## Verifying the localisation of ClyA-V5-Affi and ClyA-3XFLAG-THP to the OMV fraction

Affi-OMVs containing ClyA-V5-Affi and THP-OMVs containing ClyA-3XFLAG-THP were isolated. We investigated the localisation of these proteins to the OMV fraction by Western blot.

![picture](https://static.igem.wiki/teams/4359/wiki/results/18.png)

***Fig 18:**
Anti-FLAG Western blot of bacterial cells (before and after induction) and THP-OMV fraction. Equal volumes were loaded for the induced and uninduced cell samples. A band corresponding to ClyA-3XFLAG-THP at 37kDa is visible in all lanes, suggesting the production and localization of ClyA-3XFLAG-THP to the OMV fraction. A lower weight band is visible in the induced cells. From earlier SDS PAGE analysis (Fig 9), we noted low levels of expression occurring prior to induction as well.*

![picture](https://static.igem.wiki/teams/4359/wiki/results/19.png)

***Fig 19:**
Anti-V5 Western blot of bacterial cells (before and after induction) and Affi-OMV fraction. Equal volumes were loaded for the induced and uninduced cell samples. A band at the appropriate corresponding to ClyA-V5-Affi is visible in all lanes, suggesting the production and localization of ClyA-V5-Affi to the OMV fraction. A lower weight band is visible in the induced cells. From earlier SDS PAGE analysis (Fig 9), we noted low levels of expression occurring prior to induction as well.*

## Testing the display of ClyA-V5-Affi on the outer surface of OMVs

Previous Western blots indicate that the recombinant proteins are present in the OMV fraction but do not provide information about the orientation of the protein. For Affi-OMVs and THP-OMVs to be internalised, it is important for the affibody and THP respectively to be present on the external surface of the OMV, and not in the lumen or embedded in the membrane.

A proteinase K test was carried out for Affi-OMVs to investigate this. Proteinase K is a non-specific protease. If the affibody and the tag are exposed on the outer surface, we expect them to be degraded or truncated following proteinase K treatment. If the affibody and tag are in the interior of the OMV, they should be protected from Proteinase K by the membrane, and should be intact. If OMVs are lysed with SDS prior to proteinase K treatment, then regardless of the orientation, we expect protein degradation. The following results were obtained for this test:

![picture](https://static.igem.wiki/teams/4359/wiki/results/20.png)

***Fig 20:**
Results of the Proteinase K test performed on Affi-OMVs. 7.5ug of OMVs were incubated with or without SDS and with or without Proteinase K, as indicated. The entire volume of the reaction was analysed by Western blot against the V5 tag.*

We did not obtain any conclusive results from this experiment.

## Investigating the safety of modified OMV

The MTT assay is a colorimetric assay for assessing cell metabolic activity, and can be used to measure cell viability. In this case, we aimed to measure cell viability following treatments of different concentrations of THP-OMVs expressing ClyA-3XFLAG-THP, as compared to a control treatment of 1X PBS. SK-BR-3 cells were utilised for this assay. The mean percentage viability is reported as the viability of THP-OMV treated SK-BR-3 cells normalised to the mean viability following PBS treatment (Fig 21).

![picture](https://static.igem.wiki/teams/4359/wiki/results/21.png)

***Fig 21:**
Cytotoxicity of THP-OMVs as measured using the MTT assay with SK-BR-3 cell cultures. The mean percentage viability is reported as the viability of THP-treated SK-BR-3 cells normalised to the mean viability following PBS treatment. Viability is reported as mean ± standard error of mean of three biological replicates. Concentrations of THP-OMVs indicated are working concentrations.*

**Summary**

In Phase 2, we successfully cloned ClyA-V5-Affi and ClyA-3XFLAG-THP into the backbone pGEX-4T1. We tested the expression of all three proteins and verified that they translocated to the OMV fraction. We investigated the safety of THP-OMVs by the MTT assay and did not find significant cytotoxicity. We performed a Proteinase K test to verify the display of ClyA-V5-Affi on the outer surface of OMVs but did not obtain any conclusive results.

---

# Phase 3

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In phase 3 of the project, we aimed to test the internalisation of Affi-OMVs and THP-OMVs in the HER2+ breast cancer cell line, SK-BR-3, compared to a control cell line HEK 293T. The results of these experiments can be found on the [proof of concept](https://2022.igem.wiki/iiser-tvm/proof-of-concept) page.

---

# References

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1. Shrivastava, R., Jiang, X., & Chng, S.-S. (2017). Outer membrane lipid homeostasis via retrograde phospholipid transport in Escherichia coli: A physiological function for the Tol-Pal complex. Molecular Microbiology, 106(3), 395–408. <https://doi.org/10.1111/mmi.13772>

2. Reimer, S. L., Beniac, D. R., Hiebert, S. L., Booth, T. F., Chong, P. M., Westmacott, G. R., Zhanel, G. G., & Bay, D. C. (2021). Comparative Analysis of Outer Membrane Vesicle Isolation Methods With an Escherichia coli tolA Mutant Reveals a Hypervesiculating Phenotype With Outer-Inner Membrane Vesicle Content. Frontiers in Microbiology, 12, 628801. <https://doi.org/10.3389/fmicb.2021.628801>

3. Schwechheimer, C., & Kuehn, M. J. (2013). Synthetic Effect between Envelope Stress and Lack of Outer Membrane Vesicle Production in Escherichia coli. Journal of Bacteriology, 195(18), 4161–4173. <https://doi.org/10.1128/JB.02192-12>

4. Schwechheimer, C., Rodriguez, D. L., & Kuehn, M. J. (2015). NlpI‐mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli. MicrobiologyOpen, 4(3), 375–389. <https://doi.org/10.1002/mbo3.244>

5. *E. coli* Keio Knockouts. Link [here](https://horizondiscovery.com/en/non-mammalian-research-tools/products/e-coli-keio-knockouts)

6. Li, Q., Li, Z., Fei, X., Tian, Y., Zhou, G., Hu, Y., Wang, S., & Shi, H. (2022). The role of TolA, TolB, and TolR in cell morphology, OMVs production, and virulence of Salmonella Choleraesuis. AMB Express, 12(1), 5. [https://doi.org/10.1186/s13568-022-01347-4](https://doi.org/10.1186/s13568-022-01347-4%3C)

7. Shen, Y., Torchia, M. L. G., Lawson, G. W., Karp, C. L., Ashwell, J. D., & Mazmanian, S. K. (2012). Outer Membrane Vesicles of a Human Commensal Mediate Immune Regulation and Disease Protection. Cell Host & Microbe, 12(4), 509–520. <https://doi.org/10.1016/j.chom.2012.08.004>

8. Gujrati, V., Kim, S., Kim, S.-H., Min, J. J., Choy, H. E., Kim, S. C., & Jon, S. (2014). Bioengineered Bacterial Outer Membrane Vesicles as Cell-Specific Drug-Delivery Vehicles for Cancer Therapy. ACS Nano, 8(2), 1525–1537. <https://doi.org/10.1021/nn405724x>

9. Balhuizen, M. D., Versluis, C. M., van Grondelle, M. O., Veldhuizen, E. J. A., & Haagsman, H. P. (2022). Modulation of outer membrane vesicle-based immune responses by cathelicidins. Vaccine, 40(16), 2399–2408 <https://doi.org/10.1016/j.vaccine.2022.03.015>

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