iGEM 2023 — Team gu-frankfurt

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "layout.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 %}
{% block page\_content %}

![Hero Image](https://static.igem.wiki/teams/4660/wiki/hero-images/hero-images/project-banner-new.svg)

# Engineering

Project

* ![](https://static.igem.wiki/teams/4660/wiki/icons/magnifying-glass-solid.svg)Index

  + [Paperstrips](#section1)
  + [Bobas](#section2)
  + [BlaR1/VbrK Expression Test](#section3)
  + [VbrR Binding Site and BlaA Promoter Prediction](#section4)
  + [Receptor Design](#section5)
* ![](https://static.igem.wiki/teams/4660/wiki/icons/book-solid.svg)References

  1. Stocker J, Balluch D, Gsell M, Harms H, Feliciano J, Daunert S, Malik KA, van der Meer JR.
     Development of a set of simple bacterial biosensors for quantitative and rapid measurements
     of arsenite and arsenate in potable water.
     Environ Sci Technol. 2003 Oct 15;37(20):4743-50.
     DOI: 10.1021/es034258b. PMID: 14594387.
  2. Wilke MS, Hills TL, Zhang HZ, Chambers HF, Strynadka NC. Crystal structures of the Apo and
     penicillin-acylated forms of the BlaR1 beta-lactam sensor of Staphylococcus aureus. J Biol
     Chem. 2004 Nov 5;279(45):47278-87. DOI: 10.1074/jbc.M407054200. Epub 2004 Aug 18. PMID:
     15322076.
  3. Yuan-Chao Lou, Hsuan-Yu Huang, Hsin-Hong Yeh, Wei-Hung Chiang, Chinpan Chen, Kuen-Phon Wu,
     Structural basis of transcriptional activation by the OmpR/PhoB-family response regulator
     PmrA,
     Nucleic Acids Research, 2023;, gkad724, https://doi.org/10.1093/nar/gkad724

![The iGEM engineering cycle.](https://static.igem.wiki/teams/4660/wiki/engineering/section0-image1.svg)

Created with BioRender.com.

Paper strips

Cycle 1

Design

We first wanted to test the paper strip approach with a positive control. As
the pOdd
plasmids that we used for molecular cloning already contain a
β-galactosidase under
control of a lac promotor, we chose them as a positive control. Here, the
protein
expression should be induced by the addition of
Isopropyl-β-D-thiogalactopyranoside
(IPTG), a substance that binds and inhibits the repressor. However, the lac
promotor is
considered to be a leaky promotor and thus there will be some background
protein
expression prior to induction. As a negative control we chose the plasmid
backbone
JUMP29-1A which contains a GFP under control of a lac promotor, but no
β-galactosidase.
Our first test of the paper strips was an approach published in literauture
by Stocker
et al. in 2003.

Build

We transformed pOdd3 into the E. coli expression strain BL21(DE3). We
subsequently
inoculated a 5 ml LB preculture supplemented with 100 µg/ml ampicillin and
let the
culture grow at 37°C and 180 rpm. The next day, we used this culture and
followed the
approach from Stocker et al 2003. Here, the overnight culture is harvested
by
centrifugation and then resuspended in 1 ml of drying protectant solution
(0.5% (w/v)
peptone, 0.3% meat extract, 10% gelatine, 1% sodium ascorbate, 5% raffinose
and 5%
sodium glutamate). After applying the samples to Whatman filter paper, the
paperstrips
are freeze-dried using lyophilization.

Test

Before testing the paperstrips, we first wanted to see if we can induce and
see the
expression of the β-galactosidase in the cell suspension we applied to our
paperstrips.
This allows us to actually verify if our samples work and is an important
control to
check wether the paperstrip approach worked. For the test we took 20 µl of
cell
suspension, added 4 µl of 100 µM IPTG and 12 µl 20 mg/ml X-Gal in DMF and
incubated them
at 37°C. The tested cultures of the BL21 cells containing pOdd3 all turned
blue and
thus, protein expression of the β-galactosidase was verified.

For testing the paper strips, we followed the procedure from Stocker et al
2003. We
placed the paper strips in 1M, 100 µM and 10 µM IPTG solution and incubated
them at 30°C
for 30 min. To prevent them from drying out, we placed them in a plastic
bag. 5 µl of 20
mg/ml X-Gal in DMF solution were then pipetted on the premarked spot. 2 h
later, there
was a color change to a greenish color.

Learn

Since the positive control for β-galactosidase expression in the cell
suspension turned
blue as expected, we were irritated by the greenish colour of the
paperstrip. After some
reading and evaluation, we concluded that the green color is due to the
reaction of
X-Gal with light and not due to cleavage by the β-galactosidase. Another
possible error
source was the treatment process of the paperstrips: we were worried, that
by placing
the paper strips in a solution, the cells might diffuse from the paper
strip. After all,
we concluded three possible error sources: either the signal intensity was
too low
because the cell density was too low, the cells diffused from the paperstrip
into the
testing solution or the reactivation process of the bacteria did not work.

Cycle 2

Design

To enhance the signal, we decided to place 2x 5µl of bacteria suspension on
the paper
strip instead of only 1 x 5µl. This time, we also did not place the whole
paper strip
into the testing solution to prevent possible diffusion of the cells into
the testing
solution.

Build

In this cycle, we only adapted the amount of bacteria which were placed on
the paper
strip but still followed the same procedure as in the first cycle.
Additionally we also
established a proper negative control by only placing LB medium or drying
protectant
solution on the paper strip. This way we can directly compare paperstrips
witha and
without X-Gal and confirm that a change in color is specific due to the
enzymatic
reaction of the β-galactosidase and not just a reaction of X-Gal with light.

Test

Instead of placing the paper strip into the testing solution, we pipetted LB
medium, the
test solution and X-Gal directly onto the paper strip. After incubation at
37°C in the
dark for about 30 minutes the color change could be observed. We also tested
to
reactivate the bacteria with water. The blue spot which appeared was less
vibrant than
with LB Medium. The negative controls with only medium or drying protectant
solution
stayed colorless. We also noticed that using 20 mg/ml X-Gal in DMF causes
only a color
change at the outer circle of the bacterial spot. We diluted this X-Gal
stock in water
to test different X-Gal concentrations and noted that 1 mg/ml X-Gal is
sufficient to
observe the color change.

![Paper strip test with different IPTG concentrations.](https://static.igem.wiki/teams/4660/wiki/engineering/section1-image1.png)

**Figure:** Paper strip test with different IPTG concentrations and 20
mg/ml X-Gal in DMF.

![Paper strip test with differnt X-Gal concentrations.](https://static.igem.wiki/teams/4660/wiki/engineering/section1-image2.png)

**Figure:** Paper strip test with differnt X-Gal concentrations.

Learn

In order to use paper strips as a platform for any kind of whole-cell
biosensor with a
β-galactosidase as reporter gene, the cells need to be reactivated by
pipetting LB
medium directly onto the dried spot. Additionally, lower concentrations of
X-Gal are
more effective to get an vibrant and round colour change.

Bobas

Cycle 1

Design

To test the bobas with GFP as a reportergene, we first wanted to test a plasmid
containing gfp under the control of a lac promotor.

Build

We adapted the protocol form the 2020 Vilnius iGEM team. A 2,5% alginate
solution is mixed with an overnight culture in the ratio of 4:1. Using a syringe
the bobas then are released in droplets into a 0,1 M CaCl2 solution.

Test

We observed the bobas with a fluorescence microscope with a GFP Filter. The
smallest magnification is 10x and for the first try, the bobas were too big to
be seen as a sphere. So we tried using a canula to obtain smaller bobas. After
creation of the bobas, they were incubated in LB Medium, water and 1M IPTG.
Fluorescence could only be observed in the 1M IPTG solution.

Learn

The bobas need to be made as small as possible to be observed with a
fluorescence microscope but can be used as a platform for biosensors with gfp as
reportergene.

BlaR1/VbrK Expression Test

Cycle 1

Design

To assemble our whole-cell biosensor for detection of β-lactam antibiotics, we
first had to clone the separate parts into expression vectors to test the
optimal conditions and sensitivity of the promotors. Two different systems were
chosen to be promising. The VbrR/VbrK system and the BlaI/BlaR1 system both
contain two components and a respective reporter gene, which is either the
expression of a β-galactosidase or the expression of GFP. VbrR/VbrK and
BlaI/BlaR1 were all tagged with a 6xHistidin-tag, to purify the proteins and to
detect the protein in western blot analysis. The GFP fluorescence should be
detected through in-gel fluorescence and the presence of the β-galactosidase can
easily be confirmed by adding IPTG and X-Gal and observing a blue color.

Build

Each gene was inserted into a plasmid vector containing a promotor under the
control of L-arabinose. The plasmid was cloned via FX-cloning and transformed
into E. coli C43 cells. A single clone was picked and grown, to purify the
inserted plasmid. The plasmid was prepped and sequenced to confirm the right
insert and to exclude mutations.

Test

To test the best conditions for protein expression in order to determine which
conditions are suitable for our biosensor and how fast it responds, we
inoculated a preculture with a single positive clone. This was used to inoculate
a 50 ml main culture, which was grown at 37°C to an OD600 of 0.6-0,8. The
protein overexpression was induced by adding 0.02% (w/v) or 0.002% (w/v)
L-arabinose. Samples of the bacteria culture were taken before induction and in
steps of 30 mins after induction, up to 3 h. All samples were diluted
respectively in SDS loading buffer to an OD600 of 10 and loaded on an SDS-PAGE,
to perform a western blot. The overexpressed protein was detected using a
primary murine antibody against the polyhistidin-tag (1:3000 dilution) and a
secondary antibody against mouse (1:10000 dilution).

Learn

The western blots of BlaR1 overexpression showed very low signal, so we decided
to increase the concentration of the primary antibody against the
polyhistidin-tag to a 1:1000 dilution and also increase the incubation time. The
membranes of the VbrK overexpression showed that 0.02% and 0.002% (w/v)
L-arabinose induced protein expression was very high. Using the higher
concentration resulted in such high protein amounts that there was no visible
difference of VbrK between the timepoints 30 min and 3 h. This showed that a
very low L-arabinose concentration is sufficient to express VbrK and that an
incubation time of 30 min is enough to expect results from the biosensor.

![Western blot of VbrK expression test.](https://static.igem.wiki/teams/4660/wiki/engineering/section3-image1.png)

**Figure:** Western blot of VbrK expression test. Induction with 0.02%
(w/v) L-Arabinose.

![Western blot of VbrK expression test.](https://static.igem.wiki/teams/4660/wiki/engineering/section3-image2.png)

**Figure:** Western blot of VbrK expression test. Induction with 0.02%
(w/v) L-Arabinose.

VbrR Binding Site and BlaA Promoter Prediction

Cycle 1

Design

We conducted a two-step analysis for predicting the VbrR binding site. For the
promoter prediction, we opted for SAPPHIRE, which is tailored for σ70 promoter
prediction in *Pseudomonas* and *Salmonella*, two closely related
genera to *Vibrio
haemolyticus*. With our insights into potential promoter sites for
σ-factors,
we were well-equipped to ascertain the binding site for VbrR. Assuming
similarity to a known 49 bp binding site in the exsC promoter of type 3
secretion systems, we performed local alignments against our 300 bp sequence
containing the putative binding site. Additionally, we decided to map two DNA
half-sites identified by Hong et al. onto our sequence to identify potential
binding sites for the VbrR DNA-binding domains.

Build

Besides using SAPPHIRE for promoter prediction, we further investigated the
region
from the BlaA gene start to 300 bp upstream to identify the VbrR binding site.
Employing local alignments, we clustered the top 1000 sequences by score,
discarding
those exceeding a limit of 20 bp on one side of the prior sequence. For DNA
half-site identification, we also employed alignments, focusing solely on
evaluating
the gap size between the two half-sites and the number of mismatches.

Test

We were able to enhance our understanding of the genetic regulation of our
reporter
genes. After generating our local alignments, we found about 90% of all local
alignments for the binding site prediction to be potential binding sites. We
further
found no perfect alignment for the mapping of the two DNA half-site motifs to
our
sequence, but selected two sequences as promising binding sites, favoring fewer
mismatches and accepting larger gap sizes. The exact interaction between VbrR
and
the σ70 factor remains unknown. However, by integrating several studies
connected
to VbrR's superfamily, and predicting potential promoter sequences using
SAPPHIRE,
we were able to determine VbrR's binding sites and their potential function. We
predicted that the promoter region is located within sequence positions 477560 ←
477668 (108 bp), and the enhancing region is positioned at 477707 ← 477761 (54
bp).

Learn

We were able to predict the promoter region of BlaA and the binding site of
VbrR.
However, experimental work is required to validate the predicted binding sites,
promoter regions, and their function. Nevertheless, we are confident in the
reliability of our results.

Receptor Design

Cycle 1

Design

For in silico mutation of our receptor, we employed the Rosetta interface design
protocol
to automatically design the protein interface interacting with our antibiotics.
The
antibiotics, in their closed β-lactam ring state, were placed in the binding
pocket.
A rotamer library was created and 1000 mutated BlaR1 receptors were generated.
Afterward,
the top 3 mutants determined by Rosetta's total score were evaluated using MOE
and PLIP.
Thereby, the interaction between the antibiotics and the receptor was assessed
after
docking them covalently back into the optimized receptors. The approach was
driven by
the intricate process of the antibiotic's interaction with the protein.
Specifically,
the antibiotic must initially reach the serine residue while still in its active
state,
before subsequently undergoing a reaction and forming a covalent connection with
it.

Build

Molecular dockings were conducted in the Molecular Operating Environment (MOE).
The binding
site for β-lactam antibiotics was chosen based on Wilke et al.'s publication.
For noncovalent
docking, we identified the general binding pocket, for covalent docking, we
further specified the
preferred Serin as the reactive site, employing an alkylester reaction with ring
opening as
the reaction mechanism. However, MOE does not complete covalent docking
autonomously. Hence,
a manual intervention was necessary to establish a truly covalent bond between
the
macromolecule and its ligand.

Test

The approach with Penicillin G failed to yield a receptor with significantly
increased binding
affinity. However, for Ampicillin, one mutant displayed higher binding affinity,
indicated by
more hydrogen bonds and hydrophobic interactions, than the covalent Penicillin G
binding state.
Compared to the naive receptor, the mutant receptor exhibited a noticeable
reduction of critical
interactions, notably the high-energy ionic bond of the carboxylate group and
specific hydrogen
bonds. However, it did manage to establish a distinctive T-shaped π-stacking
interaction between
the benzyl ring and a phenylalanine residue.

Learn

Although we found a BlaR1 mutant for Ampicillin, we did not manage to find major
differences in
the interactions between BlaR1, BlaR1 mutants and the considered antibiotics. In
the non-covalent
approach, we optimized the protein's interface considering potential
conformations of the
antibiotics. This approach could lead to changes in amino acids that do not
interact with
the antibiotic in its inactive state (open β-lactam ring) and therefore make it
difficult
for Rosetta to generate suitable mutants of BlaR1. Hence, we chose a second
approach for
generating the BlaR1 mutants.

Cycle 2

Design

To address the limitations of our non-covalent approach, we considered
incorporating
covalent dockings into the Rosetta workflow. However, the protocol proposed by
Rosetta
was not suitable for dealing with covalent binding. As an alternative, we
pursued
optimization of the protein interface using the inactive state of the
antibiotics
without actually forming a covalent bond with the protein. The rest of the
pipeline
remained intact, constituting what we term the pseudo-covalent approach.

Build

Instead of handing over noncovalent dockings to Rosetta, we generated covalent
dockings
in MOE without manually linking the ligand to the receptor at the end. We then
passed
these pseudo-covalent dockings to Rosetta. The key difference lies in the fact,
that
Rosetta could now optimize the receptor for the inactivated antibiotics with the
opened β-lactam ring.

Test

The pseudo-covalent approach, in a similar vein, did not yield a structure with
markedly
increased estimated binding affinities for Penicillin G. Surprisingly, the
optimization
consistently leaned towards Ampicillin, regardless of the intended antibiotic in
the
interface design. However, upon closer inspection of the top three results for
Penicillin G, we identified mutants that exhibited notably exceptional estimated
binding affinities for Ampicillin.

Learn

In total, we successfully developed two mutants of BlaR1 with significantly
increased
estimated binding affinities for Ampicillin. However, the preferable BlaR1
mutant
structure for Ampicillin was found during optimization for Penicillin G. This
suggests
the potential benefit of employing similar antibiotics in the optimization
process to
attain globally optimal results for the desired antibiotic, though it also
stresses
the difficulty to make the receptor discriminate between very similar molecule
structures.

{% endblock %}

{% 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 %}

![Hero Image](https://static.igem.wiki/teams/4660/wiki/banner-images/wetlab-banner.jpg)

# Experiments

Wetlab

* ![](https://static.igem.wiki/teams/4660/wiki/icons/magnifying-glass-solid.svg)Index

  + [Chemically Competent Cells](#section1)
  + [Golden Gate Cloning](#section2)
  + [Site Directed Mutagenesis](#section3)
  + [Plasmid Preparation](#section4)
  + [Sequencing](#section5)
  + [General Protocols](#section6)

Chemically Competent Cells

Chemically Competent Cells

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/chemically-competent-cells.pdf)
instead.

Golden Gate Cloning

Golden Gate Cloning

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/golden-gate-cloning.pdf)
instead.

Blue White Screening

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/blue-white-screening.pdf)
instead.

Transformation of E.coli

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/transformation-of-ecoli.pdf)
instead.

FX-Cloning

FX-Cloning

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/fx-cloning.pdf)
instead.

Site Directed Mutagenesis

Site Directed Mutagenesis

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/pcr-site-directed-mutagenesis.pdf)
instead.

Plasmid Preparation

Plasmi Preparation

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/plasmid-preparation.pdf)
instead.

Sequencing

Sequencing

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/sequencing.pdf)
instead.

General Protocols

Expression Test

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/expression-test.pdf)
instead.

Western Blotting

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/western-blotting.pdf)
instead.

Western Blotting using BioRad Trans-Blot Turbo System

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/western-blotting-biorad-system.pdf)
instead.

Casting SDS gel

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/casting-sds-gel.pdf)
instead.

Agarose Gel

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/agarose-gel.pdf)
instead.

VbrK purification

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/vbrk-purification.pdf)
instead.

BlaR1 purification

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/blar1-purification.pdf)
instead.

Paper strips

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/paperstrips.pdf)
instead.

Bobas

Unable to display PDF file. [Download](https://static.igem.wiki/teams/4660/wiki/experiments/bobas.pdf)
instead.

{% endblock %}

{% 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 %}

![Hero Image](https://static.igem.wiki/teams/4660/wiki/banner-images/wetlab-banner.jpg)

# Results

Wetlab

* ![](https://static.igem.wiki/teams/4660/wiki/icons/magnifying-glass-solid.svg)Index

  + [Introduction](#section1)
  + [AMR a Concern?](#section2)
  + [Our Biosensor - The Prototype](#section3)
  + [Protein Production and Purification](#section4)
  + [Final Results](#section5)
* ![](https://static.igem.wiki/teams/4660/wiki/icons/book-solid.svg)References

  1. Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I.; Zhou, Xiaohui (2016):
     Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam
     antibiotics.
     In: Proceedings of the National Academy of Sciences of the United States of America 113 (6),
     S.
     1648-1653.
     DOI: 10.1073/pnas.1520300113
  2. Geertsma ER, Dutzler R. (2011):
     A versatile and efficient high-throughput cloning tool for structural biology.
     In: Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25. 10.1021/bi200178z PubMed
     21410291
  3. Dvorak P, Chrast L, Nikel PI, Fedr R, Soucek K, Sedlackova M, Chaloupkova R, deLorenzo V,
     Prokop Z, Damborsky J (2015): Exacerbation of substrate toxicity by IPTG in Escherichia coli
     BL21(DE3) carrying a synthetic metabolic pathway. In Microb Cell Fact 14:201
     10.1186/s12934-015-0393-3

Introduction

B.L.I.S.S. aims to detect antibiotic contaminations in our environment to fight the global threat
that antimicrobial resistance poses. We established the design of a biosensor that utilizes
endogenous resitance mechanisms of bacteria, to detect contaminations by β-lactam antibiotics in
water samples. Here, we are especially interested in our waste- and groundwater, as they suffer the
most pollution and are evidently involved in the development and spread of antimicrobial resistance.

On this page, we present the main results we obtained and guide you through our iGEM journey!

Wastewater Treamtent Plant

BLISS Biosensor

Proteins

Tests

Is AMR really of Concern in Wastewater Treatment Plants?

In our modern society, antibiotics are everywhere. They are regularly used in hospitals,
research
facilites, industry and argiculture. But everytime antibiotics are used, they end up and
accumulate
in one meeting point: our wastewater. Therefore, wastewater treatment plants play a
crucial role in
the spread of antibiotic resistant pathogens. Currently, there are no regulations in
Germany that
obligate wastewater treatment plants to filter, or even monitor antibiotic
concentrations in the
water. Therefore, we wanted to see how big the burden of antibiotic resistant
microorganisms in the
wasterwater treatment pools actually is.

![Schematic of all antibiotics ending up in the wastewater.](https://static.igem.wiki/teams/4660/wiki/results/section1-image1.png)

**Figure 1:** All the antibiotics used in hospitals, agriculture, research
facilities and
industry all end up in wastewater and thus in wastewater treatment plants. This
figure was
created with BioRender.com.

So we designed a simple experiment: We were able to obtain samples of the nitrifaction
and
denitrification pools of the wastewater treatment plant in Heusenstamm, Germany. In
these pools, the
two essential steps of the biological part of water treatment take place. Here,
microorganisms like
bacteria are used to first metabolize ammonia to nitrate and subsequently the nitrate to
nitrogen.

We were wondering if those bacteria evolved over the years to gain resistance against
commonly used
antibiotics.

The experimental set-up was very simple: we gathered a collection of eleven different
antibiotics
and prepared LB-agar plates containing these different antibiotics. Afterwards, we
plated 100 µl of
the samples and just watched if bacteria were able to grow despite the antibiotics.

![Schematic of the experimental set-up to test for resistant microorganisms.](https://static.igem.wiki/teams/4660/wiki/results/section1-image2.png)

**Figure 2:** Experimental set-up to test wastewater samples for resistant
microorganisms.
This figure was created with BioRender.com.

*And we got concerning results: **Every plate showed
bacterial growth!***

![Results from the antimicrobial resistance screening.](https://static.igem.wiki/teams/4660/wiki/results/section1-image3.png)

**Figure 3:** LB-agar plates with eleven different antibiotics and samples of the
nitrification and denitrification pools of the wastewater treatment plant
Heusenstamm, Germany.
Every plate showed bacterial growth.

Our Biosensor - The Prototype

The BLISS biosensor utilizes the bacterial two-component system VbrK/VbrR which
originates from the
gram-negative bacterium *Vibrio parahaemolyticus*. This system has been shown in
literature to
grant bacteria broad-spectrum resistance against beta-lactam antibiotics (Li et al.
2016). In our
case, we wanted to use the sensing properties and functionalize them for a biosensor by
replacing
the endogenous expression cassette with reporter proteins.

Since *Vibrio parahaemolyticus* is a organism of the biosafety level 2, we decided
to use the
biosafety level 1 gram-negative bacterium *Escherischia coli* for our biosensor.
Therefore, our
first step was to design the codon-optimized coding sequences for gene expression in
*E. coli*
[(Parts BBa\_K4660000 -
BBa\_K4660005)](https://2023.igem.wiki/gu-frankfurt/parts).

Initially, we planned to use Golden Gate cloning for the assembly of our biosensor
(Figure 4).

![Schematic of the Golden Gate cloning setup.](https://static.igem.wiki/teams/4660/wiki/results/section2-image1.png)

**Figure 4:** Plasmid design for the Golden Gate assembly of the VbrK/VbrR
biosensor.
The upper panel shows the level 1 plasmids, the pOdd1 vector containing the
transcriptional unit
(TU) for VbrK, pOdd2 containing the TU for VbrR and pOdd3 containing the promotor
region of VbrR
with the β-galactosidase gene. The pOdd4-spacer plasmid also needed for assembly is
not shown
here.

But we ran into some problems with this cloning approach and had to eventually change
our cloning
set-up to build our biosensor.

Therefore we changed our cloning approach and used the [fragment exchange
(FX)-cloning](https://2023.igem.wiki/gu-frankfurt/experiments) method
established by Geerstma et al. in 2011 to generate our protein constructs. FX-cloning
allows the
simple cloning of protein coding sequences into a variety of different protein
expression vectors.
In our case, we used the pBxC3H vector (Figure 5), which utilizes an L-arabinose
inducible promotor
(araBAD) for protein expression.

![Plasmid map of the pBxC3H backbone used for FX-cloning.](https://static.igem.wiki/teams/4660/wiki/results/section2-image2.png)

**Figure 5:** Plasmid map of the pBxC3H backbone we used for FX-cloning.

To implement our coding sequences into pBxC3H vector, we first had to make our basic
parts
compatible for FX-cloning using specially designed primer pairs (Table 1) and performing
a
Polymerase Chain Reaction (PCR) to amplify the FX-cloning compatible gene fragment.
Afterwards, the
PCR product was digested using the DpnI restriction enzyme, which specifically cleaves
the template
DNA used in the PCR reaction. To proceed with the restriction cloning more efficiently,
the PCR
product was purified using the NucleoSpin Gel and PCR Clean-up kit by Macherey-Nagel and
applied to
a 1% agarose gel and gel electrophoresis was performed (Figure 6).

Table 1: Sequences of the PCR primers used to generate the FX-cloning compatible
gene fragments.

| Construct | Forward primer (5' → 3') | Reverse primer (5' → 3') |
| --- | --- | --- |
| FX-VbrK | atatatgctcttctagtattaaacaatttctgcttggggtggca | tatatagctcttcatgcccgggaagctgtatcagtctcgcaggg |
| FX-VbrR | atatatgctcttctagtaggtctcaaatgaaacaaacactttta | tatatagctcttcatgctggtctcaacctctgccttcatcttgt |
| FX-BlaI | atatatgctcttctagtgcaaacaaacaagtcgagatcagtatg | tatatagctcttcatgccttctttgagatatcgttaaggatat |
| FX-BlaR1 | actgacgctcttctagtgcaaagcttctgatcatgagcatcgtg | tatatagctcttcatgcttggccgttcaaaacacccatctccttFigure x, Finished plasmid map for protein production and future purification: A. pBxC3H-VbrK. B. pBxC3H-VbrR. |

![DNA gel electrophoresis of the FX-cloning.](https://static.igem.wiki/teams/4660/wiki/results/section2-image3.png)

**Figure 6:** DNA gel electrophoresis of the FX-cloning compatible PCR products
in a 1%
agarose gel. The ladder used is the GeneRuler 1 kb DNA Ladder by ThermoScientific.

In the agarose-gel it is clearly visible that the PCR amplification of the FX-cloning
compatible
coding sequences for VbrK (~1470 bp), VbrR (~ 700 bp), BlaR1 (~ 1800 bp) and BlaI (~400
bp). Some
remains of the template plasmids that were used are also visible, but with noticably
smaller
intensitiy.

Lastly, 250 ng of the respective PCR fragments (”inserts”) were used for the restriction
digest with
SapI, together with 50 ng of the vector backbone pBxC3H. After ligation with the T4 DNA
ligase, the
reaction products were transformed in E. coli TOP10 cells to amplify and subsequently
isolate the
plasmid.

To verify the sequece of the coding sequence and check succesful incooperation of the
insert into
the pBxC3H backbone, we used the sequencing service of Microsynth Seqlab. Our samples
were sequenced
with the standard primer pBAD-for (3'-ATGCCATAGCATTTTTATCC-5'). The sequencing result
confirmed the
correct sequence of our plasmid constructs (an exemplary cutout of the sequencing result
for
pBxC3H-VbrR as well as the sequence alignment performed is shown in Figure 7 and 8).

![Cutout of the sequencing chromatogram from pBxC3H-VbrR.](https://static.igem.wiki/teams/4660/wiki/results/section2-image4.png)

**Figure 7:** Cutout of the sequencing chromatogram from pBxC3H-VbrR.

![Cutout of the sequence alignment of the sequencing result of pBxC3H-VbrR.](https://static.igem.wiki/teams/4660/wiki/results/section2-image5.png)

**Figure 8:** Cutout of the sequence alignment of the sequencing result of
pBxC3H-VbrR.

In conclusion, we successfully cloned our designed coding sequence for VbrK and VbrR
into the pBxC3H
expression vector (Figure 9). Since we also wanted to try the two-component system
BlaR1/BlaI from
*Staphylococcus aureus* for a biosensor, we cloned these coding sequences into the
pBxC3H
vector as well.

![Finished plasmid map for protein production and purification: A. pBxC3H-VbrK. B. pBxC3H-VbrR.](https://static.igem.wiki/teams/4660/wiki/results/section2-image6.png)

**Figure 9:** Finished plasmid map for protein production and purification: A.
pBxC3H-VbrK.
B. pBxC3H-VbrR.

Protein Production and Purification

After cloning the sequences of VbrK, VbrR, BlaR1 and BlaI into pBxC3H, succesfully
transformed E. coli BL21 colonies were picked for each of the protein constructs to
innoculate a 50 ml LB bacteria culture. The culture was incubated at 37°C, while shaking
at 180 rpm, until the cells reached an OD600 of 0.6 - 0.8. To check the protein yield of
the expression test, a 1 ml sample was taken before induction with L-arabinose and the
OD600 was measured. After induction, further samples were taken at 30 min intervals and
the respective OD600 was measured in order to bring all smaples to an OD600 of 10. The
cell samples were pelleted, resuspended in 2x laemmli buffer and loaded an an SDS-PAGE,
to then perform a Western Blot. Figure 10 shows the results of VbrK overexpression,
induced with 0.002% (w/v) L-arabinose. It clearly shows that no protein could be
detected before the induction (0 h), however only after 30 min two protein bands are
noticeable, of which the band at 55 kDa is highly suspected to be our 6xHis-tagged Vbrk.
The amount of protein does not increase much after 3 h.

![Expression test of pBxC3H-VbrK.](https://static.igem.wiki/teams/4660/wiki/results/section4-image1.png)

**Figure 10:** Expression test of pBxC3H-VbrK, induced with 0.002% (w/v)
L-arabinose. Cell samples were extracted every 30 min during the expression test,
harvested and respectively dilluted in 2x laemmli buffer to an OD600 of 10. The
samples were loaded a Mini-PROTEAN TGX Stain-Free Gel (BioRad). The marker used is
the PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific). The gel was run
at 90 V for 90 min and tranfered to a nitrocellulose membrane using the Trans-Blot
Turbo™ Transfer System (BioRad). Primary antibody: Monoclonal Anti-polyHistidine
antibody produced in mouse (Sigma Aldrich), 1:2000 dillution in 3% BSA (TBST).
Secondary antibody: Anti-Mouse-IgG produced in goat (Sigma Aldrich), 1:10000
dillution in 3% BSA (TBST). Amersham ECL Prime Western Blotting Detection Reagent
(Cytiva) was used for antibody detection.

Figure 11 shows the results of VbrR expression test which was performed using different
L-arabinose concentrations. In both cases, there is only a very light band in the sample
that was taken before induction at a height of 40 kDa, which is also present after
induction. Four more bands were detected in the remaining samples after induction, with
a size of ~60 kDa, 35 kDa, ~27 kDa and 24 kDa. The band at 27 kDa is suspected to be
VbrR (~25 kDa) with the additional 6xHis-tag. The higher concentration of 0.02 % (w/v)
on the left side shows a higher protein yield, in comparison to the right side where
protein expression was induced using 0.002 % (w/v) L-arabinose. The overall protein
amount decreases with time in both cases, suggesting that a longer expression time has a
negative impact on the protein yield.

![Expression test of pBxC3H-VbrR.](https://static.igem.wiki/teams/4660/wiki/results/section4-image2.png)

**Figure 11:** Expression test of pBxC3H-VbrR, induced with 0.02% (left) and
0.002% (w/v) L-arabinose (right). Cell samples were extracted every 30 min during
the expression test, harvested and respectively dilluted in 2x laemmli buffer to an
OD600 of 10. The samples were loaded a Mini-PROTEAN TGX Stain-Free Gel (BioRad). The
marker used is the PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific).
The gel was run at 90 V for 90 min and tranfered to a nitrocellulose membrane using
the Trans-Blot Turbo™ Transfer System (BioRad). Primary antibody: Monoclonal
Anti-polyHistidine antibody produced in mouse (Sigma Aldrich), 1:2000 dillution in
3% BSA (TBST). Secondary antibody: Anti-Mouse-IgG produced in goat (Sigma Aldrich),
1:10000 dillution in 3% BSA (TBST). Amersham ECL Prime Western Blotting Detection
Reagent (Cytiva) was used for antibody detection.

The expression test for BlaR1 with the according western blot (Figure 12) shows very low
signal strength in all of the collected samples. It only shows a single protein band
with a size of 55 kDa, which corresponds to the size of BlaR1. However, the band can
also be seen in the sample before protein induction, which indicates either a leaky
promotor or that a different protein was detected during western blot analysis.

![Expression test of pBxC3H-VbrR.](https://static.igem.wiki/teams/4660/wiki/results/section4-image3.png)

**Figure 12:** Expression test of pBxC3H-BlaR1, induced with 0.02% (w/v)
L-arabinose. Cell samples were extracted every 30 min during the expression test,
harvested and respectively dilluted in 2x laemmli buffer to an OD600 of 10. The
samples were loaded a Mini-PROTEAN TGX Stain-Free Gel (BioRad). The marker used is
the PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific). The gel was run
at 90 V for 90 min and tranfered to a nitrocellulose membrane using the Trans-Blot
Turbo™ Transfer System (BioRad). Primary antibody: Monoclonal Anti-polyHistidine
antibody produced in mouse (Sigma Aldrich), 1:2000 dillution in 3% BSA (TBST).
Secondary antibody: Anti-Mouse-IgG produced in goat (Sigma Aldrich), 1:10000
dillution in 3% BSA (TBST). Amersham ECL Prime Western Blotting Detection Reagent
(Cytiva) was used for antibody detection.

*These results confirmed that we were also able to
express the proteins needed for our biosensor, and the next step in building our
biosensor was accomplished.*

While whole-cell microbial biosensors have many advantages, their real-life
application is restricted due to various regulations limiting the use of genetically
modified organims. That is why we wanted to test the purification of VbrK as well as
BlaR1, to see whether we could use them in an in vitro approach for direct detection
of β-lactam antibiotics.

Therefore, we tried to establish the purification of both membrane proteins. We
adapted protocols published in literature and adapted them for [protein purification](https://2023.igem.wiki/gu-frankfurt/experiments)
in *E. coli*.

To analyze if the purification worked, we performed an SDS-PAGE with a subsequent
Coomassie staining to visualize protein bands in the gel.
The purification of both proteins, VbrK (Figure 13) and BlaR1 (Figure 14) was
successful!

![Coomassie-stained SDS-PAGE.](https://static.igem.wiki/teams/4660/wiki/results/section4-image4.png)

**Figure 13:** Coomassie-stained SDS-PAGE (12.5 % acrylamide) with samples of the
purification of VbrK. Left to right: PageRuler™ Prestained Protein Ladder
(ThermoFisher Scientific Inc.; Waltham, USA), Cell lysate, Cytosol, Unsolubilized
fraction (SolP), Solubilized fraction (SolSN), IMAC Flowthrough (FT), IMAC Wash,
IMAC elution fractions 1-5. The IMAC elution fractions E2 and E3 show protein bands
for VbrK at 55 kDa.

![Expression test of pBxC3H-VbrR.](https://static.igem.wiki/teams/4660/wiki/results/section4-image5.png)

**Figure 14:** Coomassie-stained SDS-PAGE (12.5 % acrylamide) with samples of the
purification of BlaR1. Left to right: PageRuler™ Prestained Protein Ladder
(ThermoFisher Scientific Inc.; Waltham, USA), Cell lysate, Cytosol, Unsolubilized
fraction (SolP), Solubilized fraction (SolSN), IMAC Flowthrough (FT), IMAC Wash,
IMAC elution fractions 1-5. The IMAC elution fractions E2-E4 all show protein bands
for BlaR1 at 70 kDa.

Conclusion

![](https://static.igem.wiki/teams/4660/wiki/icons/check-solid.svg)

While we did not finish the final prototype of our biosensor yet, our FX-cloning
strategy was sucessful and we were able to express VbrK, VbrR as well as BlaR1
in E. coli cells. Additionally, first purification attempts of VbrK and BlaR1
were successful. These results are the perfect basis to continue with building
our biosensor!

Final Results

Paper Strips
The aim of our project was not only to establish a whole-cell biosensor, but to design
reliable and easily adaptable detection systems, that can be used with every whole-cell
biosensor. The aim of our project was not only to establish a whole-cell biosensor, but
to design reliable and easily adaptable detection systems, that can be used with every
whole-cell biosensor.

For the positive control we tranformed the plasmid backbone pOdd3 into E. coli BL21
cells. The plasmid contains a β-galactosidase under control of a lac promotor and can be
tested by induction with IPTG. On the premarked spot, 10 µl LB Medium, 10 µl IPTG and 10
µl of 1 mg/ml X-Gal in water were added. After incubation time, the spot where the
samples were applied turned blue (Figure 15) while a negative control stayed colorless
(not shown here). As the lac promotor itself is leaky, a color change without the
addition of IPTG can be observed.

![Expression test of pBxC3H-VbrR.](https://static.igem.wiki/teams/4660/wiki/results/section5-image1.png)

**Figure 15:** Paperstrip test with different IPTG concentrations and 1 mg/ml
X-Gal in water differnt X-gal concentrations.

*We were able to establish a method to functionalize our
paperstrip approach.*

Paper Strips
To establish the alginate encapsulation of a whole-cell biosensor, or here called boba
approch, we also used a positive control. First, a GFP under the control of a lac promotor
was transformed into \*E. coli\* BL21 cells and an expression test was conducted. Protein
expression of GFP can be induced by the addition of IPTG. A culture of these cells was
incubated with 250 µM, 500 µM and 1000 µM IPTG and over the course after the first hour
samples were collected every 30 minutes up until 3 hours. These samples were then used for
an SDS-PAGE and imaged using in-gel fluorescence. As expected, the amount of protein present
increases gradually over the course of this time, scaling with the IPTG concentration.

![In-gel fluorescence of the GFP-positive control.](https://static.igem.wiki/teams/4660/wiki/results/section5-image2.png)

**Figure 16:** In-gel fluorescence of the GFP-positive control used to test the
boba approach. Samples were measured for 1-3 hours and after induction with either
250 µM or 500 µM IPTG.

![In-gel fluorescence of the GFP-positive control.](https://static.igem.wiki/teams/4660/wiki/results/section5-image3.png)

**Figure 17:** In-gel fluorescence of the GFP-positive control used to test the
boba approach. Samples were measured for 1-3 hours and after induction with 1000 µM
IPTG.

These cells were then encapsulated in bobas, which were then incubated with 250 µM, 500
µM and 1000 µM IPTG for 3 hours, and being imaged under a fluorescence microscope every
hour during this process. Again, an IPTG concentration dependent change in fluorescence
intensity was observed, gradually increasing over time.

The bobas containing fluorescent bacteria were imaged using an epi-fluorescence inverse
microscope (Zeiss). Bobas were subemerged in solutions with 1mM, 0.5mM and 0.25mM IPTG,
respectively, and images where taken at time-points 0h, 1h and 2h.

Images were taken with a monochromatic camera and the mean pixel intensity of each boba
was analyzed. The images were segmented using ImageJ. Mean pixel intensity was
calculated using ImageJ, as well.

The analysis shows a fast increase in fluorescence from time point 0 to time point 2
proving a fast read-out and a drop slight drop after 2 hours. The highest increase was
observed in 1000 µM IPTG solution. Still, lower concentrations are sufficient for a
reliable read-out and are less harmful to the cells. As a matter of fact, IPTG has
cytotoxic effect when applied in high concentrations. This may the reason for the drop
of fluorescence (Dvorak et al. 2015).

![Graph showing the mean pixel intensity per boba (normalized) in three different condition over time.](https://static.igem.wiki/teams/4660/wiki/results/section5-image4.png)

**Figure 18:** Graph showing the mean pixel intensity per boba (normalized) in
three different condition over time. Bobas were submerged in 250µM IPTG solution,
500µM IPTG solution or 1000µM IPTG solution. 8-bit images were used and segmentation
was performed free-handedly in ImageJ.

{% endblock %}