iGEM 2020 — Team Aalto-Helsinki

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

# Team:Aalto-Helsinki/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

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

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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

# Team:Aalto-Helsinki/Engineering

![](https://static.igem.org/mediawiki/2020/5/50/T--Aalto-Helsinki--sinisens_black.png)

- [HOME](https://2020.igem.org/Team%3AAalto-Helsinki)
- PROJECT
  * [Description](https://2020.igem.org/Team%3AAalto-Helsinki/Description)
  * [Design](https://2020.igem.org/Team%3AAalto-Helsinki/Design)
  * [Engineering](https://2020.igem.org/Team%3AAalto-Helsinki/Engineering)
  * [Proof of Concept](https://2020.igem.org/Team%3AAalto-Helsinki/Proof_Of_Concept)* [Poster](https://2020.igem.org/Team%3AAalto-Helsinki/Poster)
- LAB
  * Wet-Lab
    + [Experiments](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments)
    + [Parts](https://2020.igem.org/Team%3AAalto-Helsinki/Parts)
    + [Safety](https://2020.igem.org/Team%3AAalto-Helsinki/Safety)
  * Dry-Lab
    + [Model](https://2020.igem.org/Team%3AAalto-Helsinki/Model)
  * [Results](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
- HUMAN PRACTICES
  * [Integrated HP](https://2020.igem.org/Team%3AAalto-Helsinki/Human_Practices)
  * [Education](https://2020.igem.org/Team%3AAalto-Helsinki/Education)
  * [Collaborations](https://2020.igem.org/Team%3AAalto-Helsinki/Collaborations)
  * [Partnership](https://2020.igem.org/Team%3AAalto-Helsinki/Partnership)
  * [Campaign](https://2020.igem.org/Team%3AAalto-Helsinki/Campaign)
- FUTURE
  * [Contribution](https://2020.igem.org/Team%3AAalto-Helsinki/Contribution)
  * [Implementation](https://2020.igem.org/Team%3AAalto-Helsinki/Implementation)
- PEOPLE
  * [Our Team](https://2020.igem.org/Team%3AAalto-Helsinki/Team)
  * [Attributions](https://2020.igem.org/Team%3AAalto-Helsinki/Attributions)
- [ACHIEVEMENTS](https://2020.igem.org/Team%3AAalto-Helsinki/Achievements)

Aalto-Helsinki 2020

![](https://static.igem.org/mediawiki/2020/3/32/T--Aalto-Helsinki--engineering.jpeg)

ENGINEERING

* [ENGINEERING DESIGN](#s1)
* [MAIN GOALS - TESTING KEY ELEMENTS](#s2)
* [OPTICAL DEVICE TROUBLESHOOTING](#s3)
* [REFERENCES](#s4)

# ENGINEERING DESIGN

Our main goal has been to construct a biosensor to detect and quantify macrolide antibiotics meant for
use in wastewater treatment plants. Engineering success has been demonstrated in **the overall
project** by following the engineering design cycle. As an overview, we gathered information about
environmental issues and wastewater treatment plant needs. We thoroughly discussed which kind of device
could be beneficial. We **confirmed the functioning of five basic parts** and the **full
biosensor**. Additionally, after observing unsatisfactory performance, we **improved
and adapted our design** (see [Optical Device Troubleshooting](#optical_device_troubleshooting) below).

## Project Design - General Overview

RESEARCH

We gathered information about the main environmental issues facing Europe. Our attention was caught by
issues related to water pollution. We wanted the device developed during our project to be of use in
real life, so it was crucial to maintain communication with wastewater treatment plants and make sure
all their needs are met.

IMAGINE

We were considering several ways to use synthetic biology to tackle problems currently being faced by
wastewater treatment plants. Some of them included designing bacteria capable of degrading or recycling
microplastics, as well as microbial systems detecting certain pollutants, such as pharmaceuticals or
perfluoroalkyl chemicals, more efficiently.

ASK

In order to evaluate these ideas, we asked [experts](https://2020.igem.org/Team%3AAalto-Helsinki/Human_Practices)
from several fields, including, among others, wastewater treatment plant employees, microplastics
experts and environmental institutions. The key questions we had for the interviewees concerned
feasibility and applicability of our device: we wanted to be able to develop a promising tool within the
3 months that we had access to the lab, and we wanted it to be useful for wastewater treatment plants.

DESIGN

Based on our research and experts' advice, we decided to design a biosensor to detect and quantify
macrolide antibiotics.This seemed to be the most appropriate choice since (i) these compounds have been
in the ‘watch list’ of pharmaceuticals for EU-wide monitoring in aquatic environments for several years
[1], (ii) wastewater treatment plants confirmed that a device like ours would be useful to monitor the
pharmaceutical removal process on-site as current detection methods are quite expensive [2] and (iii)
clarithromycin is one of the marker compounds used for monitoring the removal efficiency in Switzerland,
which is currently leading the research in this area [3].

# MAIN GOALS - TESTING KEY ELEMENTS

We set three main goals for our project: (i) assure the cells used for the biosensor survived in
wastewater, (ii) construct the optical device for macrolide detection and quantification, (iii)
increase the sensitivity of the biosensor and concentrate macrolides in the cells or in the sample if
necessary, since macrolides are present in wastewater in very low amounts [4].

## Cell Viability

To achieve our first goal, we performed cell viability tests, where cells were grown with various media,
including wastewater. More detailed description can be found [here](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments). These
experiments were initially conducted using a plate-reader and later additionally confirmed with a flow
cytometer. The [results](https://2020.igem.org/Team%3AAalto-Helsinki/Results) confirmed that cells can survive in wastewater.

## Optical Device Construction

ASK

**Macrolide Detection - Necessary Sensitivity**

While constructing the biosensor, our main concern was the low concentration of macrolides in wastewater
[4]. Therefore, we decided to model the expected range of the biosensor. The results indicated that the
biosensor may need to be 10-100 times more sensitive to detect the low concentrations of macrolides
found in wastewaters (Fig. 1). To solve this issue, we decided to use Rosetta to predict mutations in
the binding site of MphR that would increase the binding affinity of MphR to macrolide antibiotics. More
about mathematical and protein modelling can be found [here](https://2020.igem.org/Team%3AAalto-Helsinki/Model). In addition,
to further increase the detection of macrolide antibiotics, we decided on studying ways of concentrating
them inside our biosensor cells.

![](https://static.igem.org/mediawiki/2020/c/c8/T--Aalto-Helsinki--model_hill_equation.png)

**Figure 1.** Plot of the Hill function showing at which macrolide concentration MphR
binds to these antibiotics. Blue line shows the result for the wildtype MphR and red line for a
previously modified MphR [5].

**Selecting the right Antibiotic Resistance Gene**

Since our biosensor aims to detect macrolide antibiotics, it is fundamental the cells survive in their
presence. However, many products of antibiotic resistance genes act by pumping the compound out from the
cell or by modifying its chemical structure. This would greatly affect the detection and reliability of
the results. That is why we decided ermC is a good candidate, since it encodes a protein that methylates
selected residues of 23S rRNA, thus making the cells immune to the effect of macrolides and licosamines
[9].

DESIGN

Our genetic circuit (Fig. 2) was constructed, so that when there are no macrolides present in the sample,
**MphR, a repressor protein** [6], binds to **pMphR** (naturally a promoter of macrolide
resistance genes) [7]
preventing the production of enhanced green fluorescent protein [EGFP]. In case there are macrolides,
these compounds bind to MphR, preventing the protein from DNA binding. This allows *egfp* to be
expressed. Although for the proof-of-concept studies we are using a fluorescent output, the final
product used in wastewater treatment plants may be an electrochemical biosensor, since in many cases
there are thought to be more sensitive [8] and the output is easier to be interpreted (see our [implementation](https://2020.igem.org/Team%3AAalto-Helsinki/Implementation) page). The constitutive promoter we selected is a medium
strength promoter (BBa\_J23106) to avoid production of too many repressor proteins and *egfp* being
silenced regardless of macrolide concentrations. All proteins expressed in our genetic circuit contain
the same ribosomal binding site, with medium strength 0,764 (BBa\_B0029) to decrease the effects of
metabolic burden.

In our lab experiments we also included ***ermC***, which is regulated by the same promoter as
mphR. This gene ensures the biosensor survives even in high concentrations of macrolides [9]. It was
placed under a constitutive promoter (BBa\_J23106), so we can test the biosensor in a wide range of
macrolide concentrations. In our genetic circuit it would be expressed together with mphr (Fig. 2).
However, in our final biosensor this may not be necessary, since the concentrations of macrolide
antibiotics in wastewater are rather low [4]. This would make the biosensor safer to use, since there
would be no risk of releasing an antibiotic resistance gene into the environment.

![](https://static.igem.org/mediawiki/2020/c/cb/T--Aalto-Helsinki--genetic_circuit.jpeg)

**Figure 2.** Schematic overview of our genetic circuit. MphR and *ermC* are
expressed together under a weak constitutive promoter. When MphR is unbound by macrolides, it
remains bound to pMphR, preventing *egfp* transcription. In presence of macrolides MphR
disassociates from pMphR and *egfp* can be expressed.

BUILD

In order to build a biosensor, we decided to use Modular Cloning protocol [10], since it is modular, it
allows for addition of more parts than RFC10 at the same time and is compatible with iGEM standards. Our
final optical device has been divided into two level 1 assemblies: called a **repressor cassette**
and an
**output cassette**, so it is easier to spot which part has malfunctioned in case we face any issues
(Fig. 3). We have constructed our repressor cassette using Modular Cloning protocol (see our [experiments](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments) page). We sequenced the assembly to make sure it is correct
before assembling the full optical device.

![](https://static.igem.org/mediawiki/2020/6/66/T--Aalto-Helsinki--experiments_plasmids_lv1.png)

**Figure 3.** Construction of level 1 repressor cassette (level 1 MphR) and level 1
output cassette (level 1 EGFP).

TEST

***ermC* functionality**

We have grown cells transformed with a repressor cassette (Fig. 4) in 1, 10, 50 and 100 µg/ml of
erythromycin, clarithromycin and, as a control, spectinomycin. The results showed that ***ermC* is
expressed and provides resistance to macrolide antibiotics**. The part can be found in [iGEM’s registry](https://igem.org/Registry). More
about the test can be read on our [experiments](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments)
page.

![](https://static.igem.org/mediawiki/2020/1/16/T--Aalto-Helsinki--engineering_ermc_part.png)

**Figure 4.** Survival of cells transformed with a repressor cassette expressing
ermC. OD - optical density. SPE - spectinomycin. CLA - clarithromycin. ERY - erythromycin.

**Full Optical Device Functionality**

We conducted a plate-reader experiment, where we grew cells transformed with the optical device in
various erythromycin concentrations for 20 h. Non-transformed *Escherichia coli* cells grown in LB
with 100 µl/ml
erythromycin were a positive control. A negative control comprised non-transformed cells grown in LB
with no antibiotic. More details about the experiment, as well as the protocol can be found on our [experiments](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments) page. Although **there seems to be a correlation between
erythromycin correlation and the fluorescent output, the differences are too insignificant for the
biosensor to be practical** (Fig. 5). It was apparent our current design requires an improvement.
See the
[Optical Design Troubleshooting](#optical_device_troubleshooting)
section below. **However, the test confirmed that pMphR and MphR are functional**.

![](https://static.igem.org/mediawiki/2020/a/a5/T--Aalto-Helsinki--engineering_pconst_average.png)

**Figure 5.** Normalized fluorescence values of cells containing our optical device
grown in various erythromycin concentrations, as well as cells transformed with an output
cassette expressing *egfp* without a repressor.

# OPTICAL DEVICE TROUBLESHOOTING

ASK

We have sequenced the optical device to see whether, despite seeing a correct length band, there has been
a mistake that results with little *egfp* being expressed. The results show no differences between
what we assembled and what we expected.

Our initial hypothesis was that the codon optimization of natural *egfp* sequences may have
affected the secondary structure of the mRNA and **decrease or block the translation of the
protein**. We
examined the probability of this happening with an online tool (<https://rna.urmc.rochester.edu/RNAstructure.html>) [11] and decided this
issue is unlikely to be a source of the issue. Another problem we considered was a suboptimal strength
of the promoter: **MphR may be overexpressed**, thus always being bound to pMphR with erythromycin
having
little effect, causing such low EGFP production.

LEARN

**Is the output signal sufficient?**

To investigate what is the issue, we performed several tests. First, we constructed a new level 1 output
cassette, but with *egfp* replaced by *sfgfp*. Then we performed a plate reader experiment,
where we measured fluorescence produced in absence of the repressor and compared the intensity of their
fluorescent signal. For more details see our [experiments](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments)
page. **sfGFP turned out to produce much stronger fluorescence** (Fig. 6). We decided to use sfGFP
instead as it may decrease the time needed for the measurement.

![](https://static.igem.org/mediawiki/2020/1/13/T--Aalto-Helsinki--engineering_sfgfp_egfp.png)

**Figure 6.** Normalized fluorescence of output cells transformed with output
cassettes consisting of either *sfgfp* or *egfp*. The graph includes data from one
biological repeat (comprising 3 technical repeats).

To see the effect of MphR on *egfp* expression, we performed a plate-reader experiment, where we
measured the fluorescence of level 1 output cassette, which does not express MphR gene so there is no
repressor present. We also measured fluorescence of the optical device in various erythromycin
concentrations for comparison. For more details see our [experiments](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments)
page. We did observe that MphR represses the *egfp*, but the difference between the output of cells
expressing and not expressing the repressor is not significant (more on our [results](https://2020.igem.org/Team%3AAalto-Helsinki/Results) page.
Worth mentioning, we were
already expecting such results since in our mathematical model
it can be seen that the increase in macrolide concentrations result only in slight changes of the
fluorescence (see our [modelling](https://2020.igem.org/Team%3AAalto-Helsinki/Model) page). Nonetheless, we wanted to test our system in the
laboratory to confirm the model
and the experiment were in accordance.

**Can a weaker promoter improve the output signal?**

To assess whether *mphR* overexpression can be responsible for low sensitivity, we run a MATLAB
scan with five different promoter strengths with constant intervals between 0 and 0.02
polymerase/second. As expected, the output signal was higher when less repressor was being produced
(Fig. 7). For that reason we decided to construct a new optical device, where we replaced a constitutive
promoter with an inducible one.

![](https://static.igem.org/mediawiki/2020/1/1c/T--Aalto-Helsinki--model_scan_egfp.png)

**Figure 7.** Scan of EGFP production in a constant erythromycin concentration.
Different lines correspond to different promoter strengths. The lowest line represents the
strength of the constitutive promoter used in the initial design, the highest line represents
the output with no repressor present.

IMPROVE

We had four potential ways to improve the range and sensitivity of our biosensor:

1. Constructing a new optical device, where the constitutive promoter was replaced by an **inducible
   pBAD
   promoter** (BBa\_I0500) [12]. This would allow us to test various induction strengths and
   select the
   most optimal one. The results showed improved sensitivity, but it was still not sufficient for the
   needs of wastewater treatment plants (Fig. 8).

![](https://static.igem.org/mediawiki/2020/8/8d/T--Aalto-Helsinki--pBAD_genetic_circuit.png)

**Figure 8.** **A.** New genetic circuit for the inducible optical device.
**B.** OD normalized fluorescence values produced by TOP10 cells transformed with an
inducible optical device grown in different erythromycin (ERY) concentrations (0, 0.1, 1 and
100 µg/ml) and 0.1% of arabinose. The graph includes data from one repeat (comprising 3
technical repeats).

2. Constructing alternative inducible optical devices with MphR mutants with changes to the ligand
   binding sites. We expect these changes to improve the binding affinity of MphR to erythromycin (to
   know more, see our [modelling](https://2020.igem.org/Team%3AAalto-Helsinki/Model) page). One of the ordered mutants (third best score for
   erythromycin, see our [modelling](https://2020.igem.org/Team%3AAalto-Helsinki/Model) page) seemed to improve biosensor’s sensitivity and produce
   better separation between fluorescence measured in samples containing different macrolide
   concentrations (Fig. 9).

![](https://static.igem.org/mediawiki/2020/f/f1/T--Aalto-Helsinki--mutants.png)

**Figure 9.** Fluorescence produced by cells transformed with the optical device
comprising a MphR mutant in different erythromycin (ERY) concentrations [µg/ml].
Fluorescence values were OD normalized. The data includes an average of two experiments
(each comprising 3 technical repeats).

3. Concentrating macrolides inside the biosensor cells by using a **modified strain** with removed
   element of several multidrug resistance pumps and **altered FhuA pore** with a constantly open
   conformation, which leads to their hyperporination [13]. The strain showed improved sensitivity
   compared to TOP10, since fluorescence produced in samples containing different macrolide
   concentrations showed greater and quicker separation (Fig. 10).

![](https://static.igem.org/mediawiki/2020/6/68/T--Aalto-Helsinki--mta_results.png)

**Figure 10.** OD normalized fluorescence values produced by GKCW104 cells
transformed with an constitutive optical device grown in different erythromycin (ERY)
concentrations (0, 0.1, 1 and 100 µg/ml) and 0.1% of arabinose. The graph includes data from
two repeats (each comprising 3 technical repeats).

4. Concentrating macrolides in the sample using isoelectric focusing on a gel [14] as a proof of
   concept for concentrating using paper-based microfluidics. Even though the initial tests were
   inconclusive (see our [results](https://2020.igem.org/Team%3AAalto-Helsinki/Results) page), this technique may work with a more specialized device
   or gel optimized for small analytes. More information on our concentration attempts can be found on
   our [design](https://2020.igem.org/Team%3AAalto-Helsinki/Design) page.

IMAGINE

Although we found many potential ways of improving the sensitivity of our optical device, there is still
a lot of research to be done before it can be used for the purpose of optimizing pharmaceutical removal
in wastewater treatment plants. The next steps would include:

1. Improving reporter gene expression by using a stronger ribosomal binding site, changing the distance
   between ribosomal binding site and the initiation codon or using an enhancer sequence [15].
2. Testing improved design with MphR mutant in the modified strain (after confirming it can survive in
   wastewater).
3. Constructing the optical device in a low copy number plasmid or integrating the optical device into
   a bacterial chromosome. This may prevent plasmid loss and improve sensitivity of the biosensor [16].
4. Constructing a biosensor with a more sensitive signal than an optical output, such as
   electrochemical biosensor based on the Mtr pathway. More can be read on our [contributions](https://2020.igem.org/Team%3AAalto-Helsinki/Contribution) page in section *Mtr pathway in Escherichia coli*.
5. Further research methods for concentrating macrolides in the samples.

# REFERENCES

**1.** Agency for Healthcare Research and Quality. (2018). 2017 national healthcare quality and
disparities report (Report No. 18-0033-EF). U.S. Department of Health and Human Services. https://
www.ahrq.gov/research/findings/nhqrdr/nhqdr17/index.html

**2.** Schafhauser, B. H., Kristofco, L. A., de Oliveira, Cíntia Mara Ribas, & Brooks, B. W. (2018).
Global review and analysis of erythromycin in the environment: Occurrence, bioaccumulation and
antibiotic resistance hazards. Environmental Pollution, 238, 440-451. doi:10.1016/j.envpol.2018.03.052

**3.** Verordnung des UVEK zur Überprüfung des Reinigungseffekts von Massnahmen zur Elimination von
organischen Spurenstoffen bei Abwasserreinigungsanlagen, 814.201.231 § 2 (2016).
https://www.admin.ch/opc/de/classified-compilation/20160123/index.html#a2

**4.** Kanoh, S., & Rubin, B. K. (2010). Mechanisms of action and clinical application of macrolides
as immunomodulatory medications. Clinical Microbiology Reviews, 23(3), 590-615. doi:10.1128/cmr.00078-09

**5.** Kasey, C. M., Zerrad, M., Li, Y., Cropp, T. A., & Williams, G. J. (2018). Development of
Transcription Factor-Based Designer Macrolide Biosensors for Metabolic Engineering and Synthetic
Biology. ACS Synthetic Biology, 7(1), 227–239. https://doi.org/10.1021/acssynbio.7b00287

**6.** Zheng, J., Sagar, V., Smolinsky, A., Bourke, C., LaRonde-LeBlanc, N., & Cropp, T. A. (2009).
Structure and function of the macrolide biosensor protein, MphR(A), with and without erythromycin.
Journal of Molecular Biology, 387(5), 1250-1260. doi:10.1016/j.jmb.2009.02.058

**7.** Gardner, L., Zou, Y., Mara, A., Cropp, T., & Deiters, A. (2011). Photochemical control of
bacterial signal processing using a light-activated erythromycin. Molecular Biosystems, 7(9), 2554. doi:
10.1039/c1mb05166k

**8.** Yi, H., Li, M., Huo, X., Zeng, G., Lai, C., & Huang, D. et al. (2019). Recent development of
advanced biotechnology for wastewater treatment. Critical Reviews In Biotechnology, 40(1), 99-118. doi:
10.1080/07388551.2019.1682964

**9.** UniProt: a worldwide hub of protein knowledge. (2018). Nucleic Acids Research, 47(D1),
D506-D515. doi: 10.1093/nar/gky1049

**10.** Haddock, Traci & Densmore, Douglas & Appleton, Evan & Carr, Swati & Iverson, Sonya & Freitas,
Monique & Jin, S. & Awtry, Jake & Desai, Devina & Lozanoski, Thomas & Shah, Pooja & Agarwal, Yash &
Lewis, Kathleen & Pacheco, Alan. (2015). BBF RFC 94: Type IIS Assembly for Bacterial Transcriptional
Units: A Standardized Assembly Method for Building Bacterial Transcriptional Units Using the Type IIS
Restriction Enzymes BsaI and BbsI.

**11.** Reuter, J. S., & Watson, R. M. (2017). Welcome to the Predict a Secondary Structure Web
Server. Predict a Secondary Structure Web Server.
https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html

**12.** Lee, N., Gielow, W., & Wallace, R. (1981). Mechanism of araC autoregulation and the domains
of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli.
Proceedings Of The National Academy Of Sciences, 78(2), 752-756. doi: 10.1073/pnas.78.2.752

**13.** Krishnamoorthy, G., Wolloscheck, D., Weeks, J., Croft, C., Rybenkov, V., & Zgurskaya, H.
(2016). Breaking the permeability barrier of Escherichia coli by controlled hyperporination of the outer
membrane. Antimicrobial Agents And Chemotherapy, AAC.01882-16. doi: 10.1128/aac.01882-16

**14.** Zhao, C., Ge, Z., & Yang, C. (2017). Microfluidic Techniques for Analytes Concentration.
Micromachines, 8(1), 28. https://doi.org/10.3390/mi8010028

**15.** Kondo, T., & Yumura, S. (2019). Translation enhancement by a Dictyostelium gene sequence in
Escherichia coli. Applied Microbiology And Biotechnology, 103(8), 3501-3510. doi:
10.1007/s00253-019-09746-7

**16.** Hicks, M., Bachmann, T., & Wang, B. (2019). Synthetic Biology Enables Programmable Cell‐Based
Biosensors. Chemphyschem, 21(2), 132-144. doi: 10.1002/cphc.201900739

[![](https://static.igem.org/mediawiki/2020/4/44/T--Aalto-Helsinki--logo_aalto_biz.png)](https://www.aalto.fi/en/school-of-business)
[![](https://static.igem.org/mediawiki/2020/9/9e/T--Aalto-Helsinki--logo_aalto_chem_en.png)](https://www.aalto.fi/en/school-of-chemical-engineering)
[![](https://static.igem.org/mediawiki/2020/a/af/T--Aalto-Helsinki--logo_aalto_elec_en.png)](https://www.aalto.fi/en/school-of-electrical-engineering)
[![](https://static.igem.org/mediawiki/2020/8/85/T--Aalto-Helsinki--logo_aalto_eng.png)](https://www.aalto.fi/en/school-of-engineering)
[![](https://static.igem.org/mediawiki/2020/f/f6/T--Aalto-Helsinki--logo_aalto_sci.png)](https://www.aalto.fi/en/school-of-science)

[![](https://static.igem.org/mediawiki/2020/a/a3/T--Aalto-Helsinki--logo_helsinki.png)](https://www.helsinki.fi/en)
[![](https://static.igem.org/mediawiki/2020/8/8b/T--Aalto-Helsinki--logo_hilife.jpg)](https://www.helsinki.fi/en/helsinki-institute-of-life-science)

[![](https://static.igem.org/mediawiki/2020/4/4c/T--Aalto-Helsinki--logo_teollisuuden_vesi.png)](https://www.teollisuudenvesi.fi/en/)
[![](https://static.igem.org/mediawiki/2020/9/9f/T--Aalto-Helsinki--logo_thermofisher.png)](https://www.thermofisher.com/fi/en/home.html)

[![](https://static.igem.org/mediawiki/2020/1/14/T--Aalto-Helsinki--logo_kemianteollisuus.png)](https://www.kemianteollisuus.fi/en/)

[![](https://static.igem.org/mediawiki/2020/1/1a/T--Aalto-Helsinki--logo_andritz.jpg)](https://www.andritz.com/group-en)

[![](https://static.igem.org/mediawiki/2020/3/3c/T--Aalto-Helsinki--logo_tek.png)](https://www.tek.fi/en)

[![](https://static.igem.org/mediawiki/2020/3/3c/T--Aalto-Helsinki--logo_vtt.png)](https://www.vttresearch.com/en)

Special thanks to HSY for all their support

[![](https://static.igem.org/mediawiki/2020/b/b0/T--Aalto-Helsinki--logo_HSY.jpg)](https://www.hsy.fi/)

Kemistintie 1, Espoo, Finland

team@aaltohelsinki.com

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

# Team:Aalto-Helsinki/Experiments

![](https://static.igem.org/mediawiki/2020/5/50/T--Aalto-Helsinki--sinisens_black.png)

- [HOME](https://2020.igem.org/Team%3AAalto-Helsinki)
- PROJECT
  * [Description](https://2020.igem.org/Team%3AAalto-Helsinki/Description)
  * [Design](https://2020.igem.org/Team%3AAalto-Helsinki/Design)
  * [Engineering](https://2020.igem.org/Team%3AAalto-Helsinki/Engineering)
  * [Proof of Concept](https://2020.igem.org/Team%3AAalto-Helsinki/Proof_Of_Concept)* [Poster](https://2020.igem.org/Team%3AAalto-Helsinki/Poster)
- LAB
  * Wet-Lab
    + [Experiments](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments)
    + [Parts](https://2020.igem.org/Team%3AAalto-Helsinki/Parts)
    + [Safety](https://2020.igem.org/Team%3AAalto-Helsinki/Safety)
  * Dry-Lab
    + [Model](https://2020.igem.org/Team%3AAalto-Helsinki/Model)
  * [Results](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
- HUMAN PRACTICES
  * [Integrated HP](https://2020.igem.org/Team%3AAalto-Helsinki/Human_Practices)
  * [Education](https://2020.igem.org/Team%3AAalto-Helsinki/Education)
  * [Collaborations](https://2020.igem.org/Team%3AAalto-Helsinki/Collaborations)
  * [Partnership](https://2020.igem.org/Team%3AAalto-Helsinki/Partnership)
  * [Campaign](https://2020.igem.org/Team%3AAalto-Helsinki/Campaign)
- FUTURE
  * [Contribution](https://2020.igem.org/Team%3AAalto-Helsinki/Contribution)
  * [Implementation](https://2020.igem.org/Team%3AAalto-Helsinki/Implementation)
- PEOPLE
  * [Our Team](https://2020.igem.org/Team%3AAalto-Helsinki/Team)
  * [Attributions](https://2020.igem.org/Team%3AAalto-Helsinki/Attributions)
- [ACHIEVEMENTS](https://2020.igem.org/Team%3AAalto-Helsinki/Achievements)

Aalto-Helsinki 2020

![](https://static.igem.org/mediawiki/2020/2/2c/T--Aalto-Helsinki--experiments.jpeg)

EXPERIMENTS

* [GOALS OF THE EXPERIMENTS](#s1)
* [VIABILITY TESTS](#s2)
* [CONSTRUCTING AND TESTING THE GENETIC CIRCUIT](#s3)
* [OPTICAL DEVICE FINE-TUNING](#s4)
* [IMPROVING SENSITIVITY - MphR MUTANTS](#s5)
* [IMMOBILIZATION TESTS](#s6)
* [CONCENTRATION EXPERIMENTS](#s7)
* [REFERENCES](#s8)

# GOALS OF THE EXPERIMENTS

The goals of our experiments were the following:

* Confirm the strain chosen for the biosensor can survive in wastewater
* Assemble and test functionality of the genetic circuit

+ Ensure *ermC* provides resistance to macrolide antibiotics
+ Correlate amount of erythromycin to GFP expression
+ Improve the sensitivity of the biosensor

* Find potential immobilization methods for our biosensor
* Assess whether isoelectric focusing is a suitable method for concentrating macrolides in a
  sample

Detailed protocols can be found [here](https://static.igem.org/mediawiki/2020/0/06/T--Aalto-Helsinki--experiments_protocols.pdf).

Our lab notebook is available [here](https://static.igem.org/mediawiki/2020/e/e6/T--Aalto-Helsinki--lab_notebook.pdf).

All sequences used can be downloaded [here](https://2020.igem.org/File%3AT--Aalto-Helsinki--sequences.zip).

# VIABILITY TESTS

## Plate-Reader Experiment

In order for our biosensor to work, it is crucial that the organism we selected is capable of
surviving in wastewater. For that, BL21 cells were transformed with GFP-expression plasmid
(pET28a-sfGFP, Addgene) and the fluorescence produced by them was measured for 20 h in different
growth media: Luria Bretani (LB) as a positive control, milli-Q as a negative control, wastewater,
LB and wastewater 1:1, 1:3 and 3:1. The GFP values were normalized using OD600 measurements.
Protocol: Plate-reader experiment for cell viability.

## Flow Cytometry Experiment

To confirm the plate-reader experiment results and obtain more precise measurements concerning cell
survival and viability, we have performed flow cytometry for cells that have been grown in following
experimental set-ups for 3 h: LB as a positive control, milli-Q as a negative control, wastewater,
LB and wastewater 1:1, 1:3 and 3:1. The cells were induced with IPTG 1.5 h before the measurement. A
viability dye was added to distinguish dead from living cells. Protocol: Flow cytometry experiment for cell viability.

# CONSTRUCTING AND TESTING THE GENETIC CIRCUIT

## Modular Cloning System

MoClo is a Golden Gate based cloning system utilizing BsaI and BbsI restriction enzymes [1]. This
method was chosen, because it allows for addition of more parts than RFC10 in a single reaction and
is compatible with iGEM standards. Another advantage is its modularity. This feature was very
relevant for this project, since it is easier to troubleshoot if one part is not functional, as well
as replace elements with altered sequences. More about MoClo can be found [here](https://dspace.mit.edu/handle/1721.1/98267). All of the
constructed assemblies were sequences to confirm the sequence is correct (See: Sequencing protocol).

## Constructing Backbones for MoClo

Since most of the backbones available from iGEM distribution kits were compatible for RFC10 cloning,
it has been decided that RFC10 backbones (pBS1C3 containing chloramphenicol resistance gene and
pSB1K3 with kanamycin resistance gene) will be adjusted for MoClo by inserting a sequence,
containing RFC94 suffix and prefix, as well as a red fluorescence protein gene (RFP) for easy
selection of successful transformants (BBa\_K2560001). Since we are cloning two level 1 assemblies
and one level 2 assembly, we needed to construct three different backbones (Fig. 1). Protocols:
Digestion & Ligation. The enzymes used for digestion of pSB1C3
backbone and RFP insert were XbaI and SpeI. These enzymes have the same sticky ends: initially
targeted cut sites were PstI and EcoRI, but due to insufficient overhang length we had to choose
XbaI and SpeI (see Plasmid design troubleshooting on our [contribution](https://2020.igem.org/Team%3AAalto-Helsinki/Contribution) page). Phosphatase was used to limit backbone
re-ligation.

![](https://static.igem.org/mediawiki/2020/e/e1/T--Aalto-Helsinki--experiments_plasmid_backbones.png)

**Figure 1.** Construction of backbones suitable for MoClo experiments. Level 1A
and 1B selection backbones (based on pSB1C3) contained chloramphenicol resistance gene (CHL)
and level 2 backbone (based on pSB1K3) encoded for kanamycin resistance (KAN). All of the
constructed backbones have a gene for red fluorescence protein (RFP) for easier selection.

To initially assess whether the ligation was successful, we performed colony PCR (protocol: Colony PCR) with FW2 and RV primers from selected colonies that
expressed RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel (protocol: Agarose gel electrophoresis). The bands were visualized
with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected lengths of successfully
assembled backbones were around 3000 bases. Re-ligation of backbone would result in around 2000 bp
band for pSB1C3 and 2200 bp for pSB1K3 backbone.

## Level 1 Assemblies

Since MoClo allows for reliable insertion of up to 5 fragments, our final optical device has been
divided into two level 1 assemblies: called level 1 repressor cassette (or level 1 MphR) and level 1
output cassette (or level 1 EGFP). The inserts required to construct the level 1 repressor cassette
included: MphR (BBa\_K3386001) [2] and *ermC* (BBa\_K3386000) [3] genes under a constitutive
promoter (BBa\_J23106) and a terminator (BBa\_B0029). Level 1 output cassette comprises EGFP
(BBa\_K3386003) gene under an inducible promoter (pMphR - BBa\_K3386002) negatively controlled by MphR
and a terminator (BBa\_B0029). All sequences containing proteins also included ribosomal binding
sites (BBa\_B0015) in order to decrease the number of elements in one MoClo reaction, except for
*egfp* which was preceded by BBa\_K3386005. Both of level one assemblies were ligated into a
level 1A and level 1B chloramphenicol selection backbones containing different suffixes with
different overhangs (Fig. 2). Protocol: Modular Cloning. The
enzyme used for digestion of inserts and backbones were BbsI.

![](https://static.igem.org/mediawiki/2020/6/66/T--Aalto-Helsinki--experiments_plasmids_lv1.png)

**Figure 2.** Construction of level 1 repressor cassette (level 1 MphR) and level
1 output cassette (level 1 EGFP).

To initially assess whether the cloning was successful, we performed colony PCR (protocol: Colony PCR) with FW2 and RV primers from selected colonies that
did not express RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel. The bands were
visualized with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected length of successful
assembly was around 3000 bp.

### Functionality Test

In order to confirm the *ermC* gene is functional, TOP10 cells transformed with level 1 MphR
assembly were grown in LB with various concentrations of erythromycin (10, 50 and 100 μg/ml).
Optical density was measured after 20 h of growth. The cells were also grown in clarithromycin,
which is structurally similar to erythromycin. The aim was to assess the resistance that *ermC*
provides to other macrolide antibiotics. As a negative control, cells were grown in media containing
spectinomycin, which belongs to a different class of antibiotics and has a different mechanism of
action. A positive control comprised cells grown in LB. A similar experiment was performed using
non-transformed TOP10 cells, to ensure the difference in growth rates and survival was not a result
of metabolic burden due to transformation with high copy number plasmid. All values were normalized.
We did not perform western-blotting to confirm MphR is expressed, but since *mphr* gene was
expressed under the same promoter as *ermC* and had the same RBS, it is very likely this
protein is properly produced as well.

## Optical Device Assembly

When two level 1 assemblies (containing MphR and EGFP constructs) were correctly ligated, a level 2
assembly could be cloned into a level 2 kanamycin selection backbone (Fig. 3). Protocol: Modular Cloning. The enzymes used for digestion of inserts and the
backbone was BsaI.

![](https://static.igem.org/mediawiki/2020/2/22/T--Aalto-Helsinki--experiments_plasmids_lv2.png)

**Figure 3.** Construction of the optical device. MphR construct includes
*mphr*, *ermC*, a constitutive promoter and a terminator. EGFP construct includes
pMpR, *egfp* and a terminator. Level 2 backbone encodes for kanamycin resistance (KAN).

To initially assess whether the cloning was successful, we performed colony PCR (protocol: Colony PCR) with F\_kan and R\_kan primers primers from selected
colonies that did not express RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel. The
bands were visualized with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected length of
successful assembly was around 4500 bp.

### Functionality Test

We performed a plate-reader experiment, where we grew cells transformed with the optical device in
various erythromycin concentrations. Non-transformed *E. coli*, as well as cells transformed
with the optical device grown in LB with 100 µl kanamycin were positive controls. A negative control
comprised non-transformed cells grown in LB with no antibiotic. Optical density and fluorescence
were measured every 15 minutes over 15 h. Experiments were performed at 37°C. The GFP intensity was
normalized using OD600 measurements. Protocol: Plate-reader
experiment for the optical device.

# OPTICAL DEVICE FINE-TUNING

## Stronger Output Signal

We performed a plate-reader experiment, where we compare fluorescence signal obtained from cells
transformed with the optical device grown for 20 h in various erythromycin concentrations to cells
transformed with level 1 output cassettes (both the original one containing *egfp*, as well as
newly assembled one where *egfp* was replaced with *sfgfp*) grown in lack of repressor (no
MphR present). Non-transformed *E. coli* grown in LB with 100 µl erythromycin were a positive
control. A negative control comprised non-transformed cells grown in LB with no antibiotic. Optical
density and fluorescence were measured every 15 minutes over 15 h. Experiments were performed at
37°C. The GFP intensity was normalized using OD600 measurements. Protocol: Plate-reader experiment for the output signal comparison.

## Altered Optical Device Assembly with Inducible Promoter

The altered optical device was assembled analogically to the initial optical device (protocol: Modular Cloning), but constitutive promoter was replaced with pBAD
promoter. See section: Optical device assembly. In addition to an inducible optical device
containing the output cassette with EGFP, another assembly was constructed with sfGFP. See section:
Optical Device Assembly. To initially assess whether the cloning was successful, we performed colony
PCR (protocol: Colony PCR) with FW2, RV primers (for level 1
assembly), as well as F\_kan and R\_kan primers (for level 2 assembly) from selected colonies that did
not express RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel. The bands were
visualized with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected length of successful
assembly was around 3700 bp.

### Functionality Test

WWe performed a plate-reader experiment, where we grew cells transformed with the optical device in
various combinations of erythromycin and arabinose concentrations (Table 1). Non-transformed *E.
coli*, as well as cells transformed with the inducible optical device grown in LB with 100 µl
erythromycin were positive controls. A negative control comprised non-transformed TOP10 cells grown
in LB with no antibiotic. Optical density and fluorescence were measured every 15 minutes over 15 h.
Experiments were performed at 37°C. The GFP intensity was normalized using OD600 measurements. More
detailed protocol: Plate-reader experiment for the inducible optical
device.

**Table 1.** Tested variations of erythromycin and arabinose concentrations to optimize
the promoter strength.

| Arabinose by weight [%] | 0,1µg/ml Erythromycin | 1µg/ml Erythromycin | 10µg/ml Erythromycin | 50µg/ml Erythromycin | 100µg/ml Erythromycin |
| 0,005 | x | x | x | x | x |
| 0,01 | x | x | x | x | x |
| 0,05 | x | x | x | x | x |
| 0,1 | x | x | x | x | x |

# Improving Sensitivity - MphR Mutants

In order to see if alterations to MphR modelled by our team improve sensitivity of the biosensor, we
have used MoClo to construct new optical devices (analogically to the original optical device, see
section: Optical device assembly), using 5 different MphR mutants (Table 2). More about our
modelling can be found [here](https://2020.igem.org/Team%3AAalto-Helsinki/Model).

**Table 2.** MphR mutants ordered for testing.

| MphR Mutant | Chosen characteristic |
| MphR common mutations Erythromycin and Clarithromycin | M60A, M94V, V127A, T155I |
| MphR Erythromycin 1 | V67A, V127A, T155A, M156I |
| MphR Erythromycin 2 | V67A, M94V, V127A, T155I, M156I |
| MphR Clarithromycin 1 | M60A, M94V, V127A, T155I |
| MphR Clarithromycin 2 | M60A, Y104Q, V127A, T155Y, M156A |

To initially assess whether the cloning of level 1 assemblies was successful, we performed colony PCR
(protocol: Colony PCRColony PCR) with F\_kan and R\_kan primers from selected
colonies that did not express RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel. The
bands were visualized with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected length of
successful assembly was around 3700 bp.

### Functionality Test

We performed a plate-reader experiment, where cells transformed with the inducible optical device
comprising a modified MphR sequence were grown in various erythromycin concentrations (0 µg/ml, 1
µg/ml, 10 µg/ml and 100 µg/ml) and 0.1% of arabinose. Non-transformed TOP10 cells, as well as cells
transformed with the optical device grown in LB with 50 µg/ml kanamycin were positive controls. A
negative control comprised non-transformed TOP10 cells grown in LB with no antibiotic. Optical
density and fluorescence were measured every 15 minutes over 15 h. Experiments were performed at
37°C. The GFP intensity was normalized using OD600 measurements. More detailed protocol: Plate-reader experiment for MphR mutants. An analogical experiment
was performed with clarithromycin instead of erythromycin.

# IMMOBILIZATION TESTS

In order to evaluate what immobilization technique we should use for our biosensor, we performed
imaging using confocal microscopy. Immobilization can be achieved with different approaches from
which we decided to try two: attachment and entrapment. For attachment, we had two matrices:
Poly-D-Lysine and Poly-L-Lysine. For entrapment, we used three different matrices: alginate, egg
white and Cultrex. As in our previous viability experiments, we used BL21 cells with and without
pET28a-sfGFP (Addgene). Non-transformed BL21 cells were used to assure that fluorescence was due to
GFP and not autofluorescence. Conditions were observed with 20X Live Preview to see movement of the
cells and pictures were taken from each condition during a 10 minute time-frame for documentation.
For each condition, viability dye was added to gain insight on the different matrices. The dye
penetrates compromised plasma membranes and binds to nucleic acid with high-affinity, which exposes
the less healthy cells.

## Poly-D & L-lysine Immobilization

We prepared the polylysine plates one day in advance by pipetting 200 µL of 1 % polylysine solution
into a 24-well plate. The plate was left to incubate overnight at room temperature. After this,
wells were washed twice with PBS, and 50 µL of cells were added and left to settle for 30 minutes.
0.5 µL of viability dye was added 20 minutes before imaging. More detailed protocol: Immobilization experiments.

## Alginate Immobilization

Alginate beads for cell entrapment were prepared using a 2 % sodium alginate solution. After the
beads had hardened, they were transferred into a 96-well plate. Viability dye was added 30 minutes
before imaging. More detailed protocol: Immobilization
experiments.

## Egg White & Cultrex Immobilization

A 96-well plate was prepared for cell entrapment experiments with egg white and Cultrex. We tested
two different conditions: matrix on bottom and matrix on top. We prepared the plate by adding 10 µl
of cells and 20 µl of matrix on top of the cells. For the other condition, only 20 µl of matrix was
added to the wells. The plate was dried in 37°C for 30 minutes for the matrices to harden. After,
100 µl of cells were added to the wells with only matrix. Viability dye was added 20 mins before
imaging. More detailed protocol: Immobilization experiments.

# CONCENTRATION EXPERIMENTS

## Isoelectric focusing for Macrolide Concentration

As a proof of concept for concentrating macrolide antibiotics in the sample, we performed an
isoelectric focusing (IEF) experiment. The protocol can be found [here](https://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006164B.pdf). Specifications used were: for rehydration with the sample, 500
µl rehydration buffer was added; 17 cm IPT pH 7-10 gel was run at 10000V for a total of 50000 Vh.
Two ReadyStrip immobilized pH gradient (IPG) pH7-10 gel strips were rehydrated utilizing sample
rehydration with 1 mg of erythromycin and 1mg thymol blue (later replaced with cresol red, since it
is only charged in high pH, while thymol blue is charged in the whole pH range of the IPG gel).
Thymol blue is coloured and has a near identical pKa to erythromycin, enabling the localization of
the invisible erythromycin band on the gel [4]. The rehydrated gels were positioned in the Biorad
Protean IEF device and up to 10 000 volts was applied to move the analytes toward the pH of the gel
matching their pKa. More detailed protocol: Isoelectric focusing for
erythromycin.

## Modified *E. coli* Strain

We performed a plate-reader experiment, where we grow TOP10 and GKCW104 modified E.coli strain cells
transformed with the constitutive optical device in various erythromycin concentrations for 20 h.
GKCW104 has deleted tolC genes, which are proteins forming an outer membrane channel in several
multidrug resistance efflux pumps. Moreover, this strain has open FhuA proteins, which leads to
hyperporination of the cell. GKCW104 strain has to be induced with arabinose for the cells to start
producing the modifications to cell membrane. It is also worth noting, that the strain is
intrinsically resistant to kanamycin [5].

GKCW104 transformed with the constitutive optical device were grown in LB containing 0.1% arabinose
and various erythromycin concentrations (0 µg/ml, 1 µg/ml, 100 µg/ml). TOP10 cells grown in LB with
50 µg/ml kanamycin were a positive control. Negative controls comprised TOP10 cells grown in LB with
no antibiotic and GKCW104 transformed with the constitutive optical device grown with LB, with no
erythromycin and arabinose added. Optical density and fluorescence were measured every 15 minutes
over 15 h. Experiment was performed at 37°C. The GFP intensity was normalized using OD600
measurements. Protocol: Plate-reader experiment with GKCW104.

# REFERENCES

**1.** Haddock, Traci & Densmore, Douglas & Appleton, Evan & Carr, Swati & Iverson, Sonya &
Freitas, Monique & Jin, S. & Awtry, Jake & Desai, Devina & Lozanoski, Thomas & Shah, Pooja &
Agarwal, Yash & Lewis, Kathleen & Pacheco, Alan. (2015). BBF RFC 94: Type IIS Assembly for Bacterial
Transcriptional Units: A Standardized Assembly Method for Building Bacterial Transcriptional Units
Using the Type IIS Restriction Enzymes BsaI and BbsI.

**2.** Noguchi, N., Emura, A., Matsuyama, H., O'Hara, K., Sasatsu, M., & Kono, M. (1995).
Nucleotide sequence and characterization of erythromycin resistance determinant that encodes
macrolide 2'-phosphotransferase I in Escherichia coli. Antimicrobial Agents And Chemotherapy,
39(10), 2359-2363. doi: 10.1128/aac.39.10.2359

**3.** - Gene - NCBI. (2020). ermC 23S RNA methylase [Staphylococcus aureus]. Bethesda (MD):
National Library of Medicine (US), National Center for Biotechnology Information; Retrieved 2
October 2020, from https://www.ncbi.nlm.nih.gov/gene/1263245

**4.** Balderas-Hernández, P., Ramı́rez, M., Rojas-Hernández, A., & Gutiérrez, A. (1998).
Determination of p K a 's for thymol blue in aqueous medium: evidence of dimer formation. Talanta,
46(6), 1439-1452. doi: 10.1016/s0039-9140(98)00015-0

**5.** Krishnamoorthy, G., Wolloscheck, D., Weeks, J., Croft, C., Rybenkov, V., & Zgurskaya, H.
(2016). Breaking the permeability barrier of Escherichia coli by controlled hyperporination of the
outer membrane. Antimicrobial Agents And Chemotherapy, AAC.01882-16. doi: 10.1128/aac.01882-16

[![](https://static.igem.org/mediawiki/2020/4/44/T--Aalto-Helsinki--logo_aalto_biz.png)](https://www.aalto.fi/en/school-of-business)
[![](https://static.igem.org/mediawiki/2020/9/9e/T--Aalto-Helsinki--logo_aalto_chem_en.png)](https://www.aalto.fi/en/school-of-chemical-engineering)
[![](https://static.igem.org/mediawiki/2020/a/af/T--Aalto-Helsinki--logo_aalto_elec_en.png)](https://www.aalto.fi/en/school-of-electrical-engineering)
[![](https://static.igem.org/mediawiki/2020/8/85/T--Aalto-Helsinki--logo_aalto_eng.png)](https://www.aalto.fi/en/school-of-engineering)
[![](https://static.igem.org/mediawiki/2020/f/f6/T--Aalto-Helsinki--logo_aalto_sci.png)](https://www.aalto.fi/en/school-of-science)

[![](https://static.igem.org/mediawiki/2020/a/a3/T--Aalto-Helsinki--logo_helsinki.png)](https://www.helsinki.fi/en)
[![](https://static.igem.org/mediawiki/2020/8/8b/T--Aalto-Helsinki--logo_hilife.jpg)](https://www.helsinki.fi/en/helsinki-institute-of-life-science)

[![](https://static.igem.org/mediawiki/2020/4/4c/T--Aalto-Helsinki--logo_teollisuuden_vesi.png)](https://www.teollisuudenvesi.fi/en/)
[![](https://static.igem.org/mediawiki/2020/9/9f/T--Aalto-Helsinki--logo_thermofisher.png)](https://www.thermofisher.com/fi/en/home.html)

[![](https://static.igem.org/mediawiki/2020/1/14/T--Aalto-Helsinki--logo_kemianteollisuus.png)](https://www.kemianteollisuus.fi/en/)

[![](https://static.igem.org/mediawiki/2020/1/1a/T--Aalto-Helsinki--logo_andritz.jpg)](https://www.andritz.com/group-en)

[![](https://static.igem.org/mediawiki/2020/3/3c/T--Aalto-Helsinki--logo_tek.png)](https://www.tek.fi/en)

[![](https://static.igem.org/mediawiki/2020/3/3c/T--Aalto-Helsinki--logo_vtt.png)](https://www.vttresearch.com/en)

Special thanks to HSY for all their support

[![](https://static.igem.org/mediawiki/2020/b/b0/T--Aalto-Helsinki--logo_HSY.jpg)](https://www.hsy.fi/)

Kemistintie 1, Espoo, Finland

team@aaltohelsinki.com

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

# Team:Aalto-Helsinki/Results

![](https://static.igem.org/mediawiki/2020/5/50/T--Aalto-Helsinki--sinisens_black.png)

- [HOME](https://2020.igem.org/Team%3AAalto-Helsinki)
- PROJECT
  * [Description](https://2020.igem.org/Team%3AAalto-Helsinki/Description)
  * [Design](https://2020.igem.org/Team%3AAalto-Helsinki/Design)
  * [Engineering](https://2020.igem.org/Team%3AAalto-Helsinki/Engineering)
  * [Proof of Concept](https://2020.igem.org/Team%3AAalto-Helsinki/Proof_Of_Concept)* [Poster](https://2020.igem.org/Team%3AAalto-Helsinki/Poster)
- LAB
  * Wet-Lab
    + [Experiments](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments)
    + [Parts](https://2020.igem.org/Team%3AAalto-Helsinki/Parts)
    + [Safety](https://2020.igem.org/Team%3AAalto-Helsinki/Safety)
  * Dry-Lab
    + [Model](https://2020.igem.org/Team%3AAalto-Helsinki/Model)
  * [Results](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
- HUMAN PRACTICES
  * [Integrated HP](https://2020.igem.org/Team%3AAalto-Helsinki/Human_Practices)
  * [Education](https://2020.igem.org/Team%3AAalto-Helsinki/Education)
  * [Collaborations](https://2020.igem.org/Team%3AAalto-Helsinki/Collaborations)
  * [Partnership](https://2020.igem.org/Team%3AAalto-Helsinki/Partnership)
  * [Campaign](https://2020.igem.org/Team%3AAalto-Helsinki/Campaign)
- FUTURE
  * [Contribution](https://2020.igem.org/Team%3AAalto-Helsinki/Contribution)
  * [Implementation](https://2020.igem.org/Team%3AAalto-Helsinki/Implementation)
- PEOPLE
  * [Our Team](https://2020.igem.org/Team%3AAalto-Helsinki/Team)
  * [Attributions](https://2020.igem.org/Team%3AAalto-Helsinki/Attributions)
- [ACHIEVEMENTS](https://2020.igem.org/Team%3AAalto-Helsinki/Achievements)

Aalto-Helsinki 2020

![](https://static.igem.org/mediawiki/2020/b/bb/T--Aalto-Helsinki--proof.jpeg)

RESULTS

* [ACCOMPLISHMENTS](#s1)
* [CELL VIABILITY TESTS RESULTS](#s2)
* [ESTIMATING THE SENSITIVITY](#s3)
* [ASSESSING THE QUANTIFICATION](#s4)
* [CONFIRMING FUNCTIONALITY OF CLONED ASSEMBLIES](#s5)
* [CONFIRMING THE NECESSITY OF PROMOTER CHANGE](#s6)
* [BIOSENSOR FINE-TUNING](#s7)
* [ASSESSING MODIFICATIONS OF FhuA TRANSMEMBRANE PROTEIN
  AND SENSITIVITY OF GKCW104 STRAIN](#s8)
* [IMPROVING SENSITIVITY WITH PROTEIN MODELLING](#s9)
* [TESTING MODIFIED MphRs](#s10)
* [IMMOBILIZATION](#s11)
* [ISOELECTRIC FOCUSING](#s12)
* [FUTURE TESTS](#s13)
* [REFERENCES](#s14)

# ACCOMPLISHMENTS

## Wet-Lab

1. We were able to confirm BL21 cells survive in wastewater.
2. We assessed functionality of the *ermC* gene.
3. We succeeded in building our genetic circuit and showing that it gives an output.
4. We improved the sensitivity of our genetic circuit by changing to an inducible promoter, testing a strain
   of *E. coli* with modified outer membrane and using an altered transcription factor.
5. We tested several immobilization methods and found that egg white, matrigel and alginate might
   be suitable for our biosensor.

## Dry-Lab: Modelling

1. We estimated the range of our biosensor.
2. We were able to build a mathematical model of the biosensor pathway.
3. We designed improved ligand binding sites.
4. We assessed the modified structure of FhuA with homology-modelling.

# CELL VIABILITY TESTS RESULTS

## Plate-Reader Experiment

We have confirmed that the cells were able to survive in all experimental set-ups except for the
negative control (milliQ). Surprisingly, we observed the best growth for samples that contained a
mix of LB and wastewater (1:1 WW:LB and 3:1 WW:LB). Cells grown in LB and 1:3 WW:LB were also able
to proliferate. As expected, wastewater was not an optimal medium: cells were able to survive, but
we do not observe proliferation (Fig. 1).

![](https://static.igem.org/mediawiki/2020/f/f4/T--Aalto-Helsinki--proof_cell_viability.png)

**Figure 1.** Plate-reader experiments results.

**Conclusion:** *E. coli* cells seem to be suitable as an organism of-choice for a biosensor
construction. We suspect the efficient proliferation in samples containing half or more wastewater
may be a result of stimulation by some compounds present in wastewater.

## Flow Cytometry Experiment for Cell Viability

To confirm results obtained from the plate-reader experiment, we performed a similar test using flow
cytometry. Most viable cells were found in 1:1 WW:LB samples. This was followed by pure LB and WW
samples. Pure milliQ had the least amount of viable cells (Fig. 2).

![](https://static.igem.org/mediawiki/2020/3/32/T--Aalto-Helsinki--engineering_flow_cytometry.png)

**Figure 2.** Flow cytometry experiments results.

**Conclusion:** The flow cytometry results confirmed that *E. coli* cells were capable of
growing in media containing wastewater at least for the period of three hours.

# ESTIMATING THE SENSITIVITY

As the concentration of macrolides in the influent and effluent wastewater is low, we used
experimental data from literature to determine the sensitivity of our MphR-based biosensor.
Experimental data from wildtype and best performing mutated *E. coli* was used to examine
current range (Fig. 3).

![](https://static.igem.org/mediawiki/2020/c/c8/T--Aalto-Helsinki--model_hill_equation.png)

**Figure 3.** MatLab model estimating the range of our biosensor.

**Conclusion:** Macrolide concentration in wastewater was found to be too low for the current
MphR-based biosensor found in literature. To address this problem, we started looking into different
methods of concentrating the sample. More about our concentrating research can be found in our [design](https://2020.igem.org/Team%3AAalto-Helsinki/Design) page.

# ASSESSING THE QUANTIFICATION

Due to the fact that the goal of the biosensor was not only to detect the macrolides from wastewater
but also quantify them, we used our mathematical model to predict how different concentrations
affect the optical output. Scan of 10 different concentrations of macrolide in range: 0.1 nM-10μM
was plotted to assess the behaviour of our biosensor in various concentrations (Fig. 4).

![](https://static.igem.org/mediawiki/2020/1/1c/T--Aalto-Helsinki--model_scan_egfp.png)

**Figure 4.** Output of the SINISENS biosensor with different macrolide
concentration in wastewater.

**Conclusion:** Very little variation was found between outputs of different concentrations,
especially in the designed working time-frame of the biosensor.

# CONFIRMING FUNCTIONALITY OF CLONED ASSEMBLIES

## Level 1 MphR Assembly - *ermC* Functionality

Level 1 MphR (repressor cassette) and level 1 EGFP (output cassette) assemblies were constructed
using MoClo protocol. Successful colonies were identified by colony PCR (Fig. 5). The assemblies
were confirmed with sequencing.

![](https://static.igem.org/mediawiki/2020/9/9d/T--Aalto-Helsinki--LEVEL_1_EGFP_and_MPHR.png)

**Figure 5.** Gel run of samples from colony PCR. Correct colonies were marked
with ‘x’. First gel contains the output cassette with egfp. Second gel contains the
repressor cassette.

When growing *E. coli* cells transformed with the repressor cassette, the lowest concentration
of all of the antibiotics (10 ⲙg/ml) seem to not have a major effect on the cell growth. When it
comes to cells grown in 50 and 100 ⲙg/ml, both transformed and non-transformed cells grown in
spectinomycin showed no resistance towards this antibiotic. Media containing macrolide antibiotics
seemed to only inhibit the growth of non-transformed cells (Fig. 7).

![](https://static.igem.org/mediawiki/2020/1/16/T--Aalto-Helsinki--engineering_ermc_part.png)

**Figure 6.** Survival of cells transformed with a repressor cassette expressing
ermC. OD - optical density. SPE - spectinomycin. CLA - clarithromycin. ERY - erythromycin.

**Conclusion:** *ermC* gene is functional and provides resistance for both erythromycin and
clarithromycin. Since both *ermC* and MphR are expressed under the same promoter and have RBS
of the same strength, we suspect that MphR is also produced. However, final confirmation of MphR
functionality can be proved by testing the full optical device (level 2 assembly).

## Level 2 Optical Device Assembly

Level 2 assembly (optical device) was constructed using MoClo protocol. Successful colonies were
identified by colony PCR (Fig. 7). The assembly was confirmed with sequencing.

![](https://static.igem.org/mediawiki/2020/1/16/T--Aalto-Helsinki--level_2_const.png)

**Figure 7.** Gel run of samples from colony PCR containing the assembled optical
device. Correct colonies were marked with ‘x’.

Cells transformed with the optical device were grown in various erythromycin concentrations. The
amount of erythromycin present in the media seemed to correlate with the amount of fluorescence
produced. (Fig 8). However, the differences between fluorescence produced in different
concentrations were not distinguishable enough for the biosensor to be useful for detection of very
small macrolide amounts.

![](https://static.igem.org/mediawiki/2020/a/a5/T--Aalto-Helsinki--engineering_pconst_average.png)

**Figure 8.** OD normalized fluorescence values produced by cells grown in
different erythromycin (ERY) concentrations (0, 0.1, 0.5, 1, 10, 50, 100 µg/ml). The graph
includes data from three repeats (each comprising 3 technical repeats).

**Conclusion:** It is apparent that the current design requires further fine-tuning. Initially it
was considered that egfp is not expressed due to codon optimization. We suspected our changes might
have led to formation of a secondary structure of mRNA. However, these results may also indicate
that in spite of what our model previously suggested, the strength of the constitutive promoter was
too high and there was always a MphR protein bound to DNA, regardless of the amount of antibiotic.
For that reason we attempted to construct the full device again, replacing the constitutive promoter
with an inducible one. This would allow us to select an optimal promoter strength.

# CONFIRMING THE NECESSITY OF PROMOTER CHANGE

The initial biosensor was designed with an constitutive promoter which was later suspected to be
producing too much repressor protein, MphR. Scan of five different promoter strengths in range:
0-0.02 polymerase/second was plotted to visualise the behaviour of our biosensor with various
promoter strengths (Fig. 10).

![](https://static.igem.org/mediawiki/2020/c/c2/T--Aalto-Helsinki--model_pbad_scan.png)

**Figure 9.** Scan of EGFP production in a constant erythromycin concentration.
Different lines correspond to different promoter strengths. The lowest line represents the
strength of the constitutive promoter used in our initial design, the highest line
represents the output with no repressor present.

**Conclusion:** Promoter strength was found to have strong correlation with the output of the
biosensor. As expected, less repressor present resulted in higher concentration of EGFP. As a
result, we decided to swap our initial promoter to an inducible promoter, pBAD.

# BIOSENSOR FINE-TUNING

Before constructing the genetic circuit with the pBAD promoter, we decided to test different GFP
options. Level 1 sfGFP assembly (sfGFP output cassette), comprising *sfgfp* reporter gene
instead of egfp, was constructed using MoClo protocol. Successful colonies were identified by colony
PCR (Fig. 10). The assembly was confirmed with sequencing.

![](https://static.igem.org/mediawiki/2020/a/a5/T--Aalto-Helsinki--lvl1_sfGFP.png)

**Figure 10.** Gel run of samples from colony PCR. Correct colonies were marked
with ‘x’. The contains the output cassette with *sfgfp*.

To see if the output cassetete is capable of producing enough fluorescence, we have grown the cells
transformed with output cassettes containing either sfGFP (Addgene) or EGFP were grown without a
repressor. It was clear sfGFP produces stronger output (Fig. 11).

![](https://static.igem.org/mediawiki/2020/1/13/T--Aalto-Helsinki--engineering_sfgfp_egfp.png)

**Figure 11.** Comparison of fluorescent output produced by cells expressing two
output cassettes (assemblies containing a reporter protein driven by an inducible pMphR
promoter, see our [experiments](https://2020.igem.org/Team%3AAalto-Helsinki/Experiments) page), one containing sfGFP, second EGFP, grown
in absence of the repressor. Fluorescence values were OD normalized. The graph includes data
from one biological repeat (comprising 3 technical repeats).

**Conclusion:** The *sfgfp* seems to be a more suitable reporter protein for the biosensor as
it produces stronger signals.

## Inducible Optical Device Assembly

Level 2 inducible assembly (inducible optical device), comprising a pBAD promoter instead of the
constitutive one, as well as sfgfp instead of egfp, was constructed using MoClo protocol. Successful
colonies were identified by colony PCR (Fig. 12).

![](https://static.igem.org/mediawiki/2020/0/0d/T--Aalto-Helsinki--pBAD_level_2_both.png)

**Figure 12.** Gel run of samples from colony PCR. A. Colonies containing the
inducible optical device. The assembly was confirmed with sequencing.

## Optimizing Promoter Strength

The goals of initial experiments after construction of the inducible device were: **(i) finding the
necessary induction time before resuspending cells to experimental set-ups and (ii) selecting
the optimal arabinose concentration in the experimental set-ups that would produce the best
sensitivity**. Since the macrolide resistance gene, *ermC* is driven by the same promoter
as MphR (pBAD), we wanted to assess whether cells will survive being placed in media containing
erythromycin or if they require prior induction. We have performed an experiment with cells that
were not induced and induced with 1% arabinose 30 or 10 minutes prior to being placed in media
containing 100 µg/m erythromycin and various concentrations of arabinose (0.1%, 0.5% and 1%). We
observed the cell growth in all of the samples. We concluded that the induction prior to placing
cells in erythromycin is not necessary.

In order to find the optimal promoter strength and achieve the most visible separation between
fluorescence produced in different concentrations, the cells transformed with the inducible optical
device were grown in three different arabinose concentrations. The observed results indicate that
0.1% is the most optimal induction (Fig. 13). It is worth noting that the separation between
fluorescence produced in different erythromycin concentrations was also more distinguishable than in
the optical device containing the constitutive promoter.

![](https://static.igem.org/mediawiki/2020/2/2a/T--Aalto-Helsinki--pBAD_optimization.png)

**Figure 13.** Selecting the optimal arabinose concentration. OD normalized
fluorescence produced by cells transformed with an inducible optical device grown in
different erythromycin (ERY) concentrations (0, 0.1, 1 and 100 µg/ml). A . 0.1% arabinose B.
0.5% arabinose C. 1% arabinose.

**Conclusion:** Construction of a new optical device containing a weaker promoter may improve
sensitivity of the biosensor.

# ASSESSING MODIFICATIONS OF FhuA TRANSMEMBRANE PROTEIN AND SENSITIVITY OF GKCW104 STRAIN

One of the methods we investigated to improve sensitivity was using transmembrane protein FhuA. We
found proposed modifications to this protein in literature that would allow more molecules through
it and wanted to assess their feasibility. The results from the SWISS-MODEL scoring were very
promising and all the values of the modified FhuA protein were relatively close to the ones of the
unmodified. More details can be found on our Modelling page.

In addition to that, we performed tests with GKCW104 strain obtained from Krishnamorthy and the
colleagues [1], which has removed *tolC* gene (an element of many multidrug resistance pumps)
and modified FhuA protein, leading to cell’s hyperporination. We transformed GKCW104 with the
constitutive optical device (as the cells require arabinose induction to introduce above mentioned
changes to the membrane and thus, using a pBAD promoter is not advisable). The results showed
improved separation between fluorescence intensity produced in different erythromycin concentrations
in comparison to TOP10 cells (Fig. 14). However, due to time constraint, we only replicated the
experiment twice.

![](https://static.igem.org/mediawiki/2020/6/68/T--Aalto-Helsinki--mta_results.png)

**Figure 14.** OD normalized fluorescence values produced by GKCW104 cells
transformed with an constitutive optical device grown in different erythromycin (ERY)
concentrations (0, 0.1, 1 and 100 µg/ml) and 0.1% of arabinose. The graph includes data from
two repeats (each comprising 3 technical repeats).

**Conclusion:** The stability and protein structure were estimated with SWISS-MODEL and the
obtained results suggest that these modifications might be feasible to work in practice.
Additionally, using GKCW104 strain for the biosensor could potentially improve its sensitivity.

# IMPROVING SENSITIVITY WITH PROTEIN MODELLING

An improvement of the sensitivity was needed in order to detect macrolides, as the low concentration
was found to be a problem. To address it, we decided to improve the binding affinity of MphR with
macrolide molecules. Improved binding affinity would result in quicker and stronger binding of these
molecules, thus initiating EGFP transcription earlier. Rosetta’s ligand binding site designing
protocol was used to generate an improved MphR molecule. We decided to test the most promising
mutants experimentally (Fig. 15).

![](https://static.igem.org/mediawiki/2020/6/6f/T--Aalto-Helsinki--model_ordered_sequences.png)

**Figure 15.** MphR sequences with the mutations that were ordered to be tested
experimentally.

# TESTING MODIFIED MphRs

We replaced **natural MphR with *new MphR sequences modelled in Rosetta***. These
modifications were likely to improve binding affinity of MphR to macrolides. We experimentally
tested five of the sequences that showed best scores for erythromycin, clarithromycin or both. Level
1 and 2 assemblies containing MphR mutants were constructed using MoClo protocol. Successful
colonies were identified by colony PCR (Fig. 16). The assemblies were confirmed with sequencing.

![](https://static.igem.org/mediawiki/2020/e/e9/T--Aalto-Helsinki--mutants_gels.png)

**Figure 16.** **A.** Level 1 assembly comprising pBAD and mphr mutants.
**B**. Level 2 assembly comprising pBAD, MphR mutants and *sfgfp*. Bands were
visualized on 0.7% agarose gel using SybrSafe. The samples with bands at correct lengths
were marked with the x symbol.

One of them (third best score for erythromycin, see our [modelling](https://2020.igem.org/Team%3AAalto-Helsinki/Model)
page) seemed to improve biosensor’s sensitivity and produce better separation between fluorescence
measured in samples containing different macrolide concentrations (Fig. 17). We have only performed
two repeats and these results should be replicated, but the initial data seems promising.

![](https://static.igem.org/mediawiki/2020/f/f1/T--Aalto-Helsinki--mutants.png)

**Figure 17.** Fluorescence produced by cells transformed with the optical device
comprising a MphR mutant in different erythromycin (ERY) concentrations [µg/ml].
Fluorescence values were OD normalized. The data includes an average of two biological
repeats (each comprising three technical ones).

# IMMOBILIZATION

All the entrapment matrices; alginate beads, egg white and Cultrex, were able to immobilize the cells
(Fig. 19-21). Viability dye was able to penetrate all of the matrices and the dyed cells are shown
in the figures as magenta color. Viability of the cells was not significantly decreased during the
incubation time based on the low amount of magenta colored cells. It was confirmed that the
non-transformed cells showed magenta colored cells, but no fluorescence. When trying egg white and
Cultrex entrapment by adding the cells on top of the matrix, immobilization could not be seen.

[

](https://static.igem.org/mediawiki/2020/f/f3/T--Aalto-Helsinki--alginate.mp4)

**Figure 19.** *E. coli* cells expressing sfGFP immobilized on alginate
beads.

[

](https://static.igem.org/mediawiki/2020/b/b3/T--Aalto-Helsinki--egg_white.mp4)

**Figure 20.** *E. coli* cells expressing sfGFP immobilized on egg white.

[

](https://static.igem.org/mediawiki/2020/e/ea/T--Aalto-Helsinki--culturex.mp4)

**Figure 21.** *E. coli* cells expressing sfGFP immobilized on Cultrex.

[

](https://static.igem.org/mediawiki/2020/8/86/T--Aalto-Helsinki--poly_L_lysine.mp4)

**Figure 22.** *E. coli* cells expressing sfGFP immobilized on
Poly-L-Lysine.

**Conclusion:** When comparing these three different matrices, egg white has a non-uniform
consistency, which is why it might not be the best option for our final product. Disadvantage of
Cultrex is its more expensive price compared to alginate. We found a disadvantage of alginate beads
to be that when measuring fluorescence, the light is reflected from the spheres, which makes it
impossible to get fluorescence reads.

Polylysine immobilization experiments were unsuccessful as we saw movement of the cells in all of
the samples (Fig. 22). In literature, research groups have documented successful immobilization.
Thus, polylysine immobilization should be repeated.

# ISOELECTRIC FOCUSING

We investigated using isoelectric focusing for concentrating the macrolide antibiotics from the waste
water sample. The experiment was performed with erythromycin, thymol blue and cresol red. As
expected, erythromycin was not visible on the gel. Thymol blue was chosen since it has the same pKa
as erythromycin (8.9), but this did not give better results, most likely due to it being negatively
charged both above and below its pKa. Finally, we repeated the test with cresol red, which has a pKa
of 8.3. Cresol red did produce a small band close to the cathode, however the band did not have
clear borders and was located very close to the end of the IPG gel, so it is questionable if it
actually worked (Fig. 23).

![](https://static.igem.org/mediawiki/2020/4/4d/T--Aalto-Helsinki--ief.jpg)

**Figure 23.** Results of isoelectric focusing with cresol red. A small band
without clear borders was visible next to the cathode.

**Conclusion:** Even though the initial tests were not successful, this technique may work with a
more specialized device or gel optimized for small analytes.

# FUTURE TESTS

1. Performing viability tests with GKCW104 to confirm it can survive in wastewater and it is
   suitable microorganism for our biosensor.
2. Transformation of GKCW104 with an optical device containing a MphR mutant in order to assess
   whether it may have a synergistic effect on the sensitivity.
3. Testing sensitivity of the optical device containing MphR mutants to other macrolide antibiotics
   (clarithromycin and azithromycin) to assure initial results are replicable.
4. Constructing a new optical device with a weaker promoter with similar strength that was achieved
   by pBAD induced by 0.1% arabinose.
5. Improving the design of the genetic circuit to increase the strength of the output signal. It
   may include using a stronger RBS, changing the distance between RBS and initiation codon or
   adding an enhancer sequence, such as the mlcR gene from Dictyostelium discoideum, which has been
   shown to significantly increase protein expression levels. Additionally, it is easy to adjust
   and inexpensive [2].
6. Constructing the optical device in a low copy number plasmid [3].
7. Integrating the optical device into a bacterial chromosome to avoid plasmid loss and possibly
   improve the sensitivity.
8. Further investigation of possibilities of concentrating the macrolide in the sample.
9. Repeating immobilization tests with poly-lysine. Assessing suitability of other immobilization
   techniques, such as latex or silica layers [4,5].
10. Constructing a biosensor with a more sensitive signal than an optical output. One option is
    using the Mtr pathway to build an electrochemical biosensor. More details about an
    electrochemical biosensor for macrolide detection can be found on our [contributions](https://2020.igem.org/Team%3AAalto-Helsinki/Contribution) page in the section *Mtr pathway in Escherichia
    coli*.
11. Testing the biosensor’s performance in wastewater. The concentration of macrolides could be
    measured with liquid chromatography–mass spectrometry beforehand.
12. Testing the biosensor at lower temperatures than 37°C, such as room temperature, to assure that
    it can function in wastewater treatment plant conditions.

# REFERENCES

**1.** Krishnamoorthy, G., Wolloscheck, D., Weeks, J., Croft, C., Rybenkov, V., & Zgurskaya, H.
(2016). Breaking the permeability barrier of Escherichia coli by controlled hyperporination of the
outer membrane. Antimicrobial Agents And Chemotherapy, 60(12), 7372-7381. doi: 10.1128/aac.01882-16

**2.** Kondo, T., & Yumura, S. (2019). Translation enhancement by a Dictyostelium gene sequence
in Escherichia coli. Applied Microbiology And Biotechnology, 103(8), 3501-3510. doi:
10.1007/s00253-019-09746-7

**3.** Hicks, M., Bachmann, T., & Wang, B. (2019). Synthetic Biology Enables Programmable
Cell‐Based Biosensors. Chemphyschem, 21(2), 132-144. doi: 10.1002/cphc.201900739

**4.** Asal, M., Özen, Ö, Şahinler, M., Baysal, H. T., & Polatoğlu, I. (2019). An overview of
biomolecules, immobilization methods and support materials of biosensors. Sensor Review, 39(3),
377-386. doi:10.1108/SR-04-2018-0084

**5.** Bhardwaj, T. (2014). A Review on Immobilization Techniques of Biosensors. International
journal of engineering research and technology, 3.

[![](https://static.igem.org/mediawiki/2020/4/44/T--Aalto-Helsinki--logo_aalto_biz.png)](https://www.aalto.fi/en/school-of-business)
[![](https://static.igem.org/mediawiki/2020/9/9e/T--Aalto-Helsinki--logo_aalto_chem_en.png)](https://www.aalto.fi/en/school-of-chemical-engineering)
[![](https://static.igem.org/mediawiki/2020/a/af/T--Aalto-Helsinki--logo_aalto_elec_en.png)](https://www.aalto.fi/en/school-of-electrical-engineering)
[![](https://static.igem.org/mediawiki/2020/8/85/T--Aalto-Helsinki--logo_aalto_eng.png)](https://www.aalto.fi/en/school-of-engineering)
[![](https://static.igem.org/mediawiki/2020/f/f6/T--Aalto-Helsinki--logo_aalto_sci.png)](https://www.aalto.fi/en/school-of-science)

[![](https://static.igem.org/mediawiki/2020/a/a3/T--Aalto-Helsinki--logo_helsinki.png)](https://www.helsinki.fi/en)
[![](https://static.igem.org/mediawiki/2020/8/8b/T--Aalto-Helsinki--logo_hilife.jpg)](https://www.helsinki.fi/en/helsinki-institute-of-life-science)

[![](https://static.igem.org/mediawiki/2020/4/4c/T--Aalto-Helsinki--logo_teollisuuden_vesi.png)](https://www.teollisuudenvesi.fi/en/)
[![](https://static.igem.org/mediawiki/2020/9/9f/T--Aalto-Helsinki--logo_thermofisher.png)](https://www.thermofisher.com/fi/en/home.html)

[![](https://static.igem.org/mediawiki/2020/1/14/T--Aalto-Helsinki--logo_kemianteollisuus.png)](https://www.kemianteollisuus.fi/en/)

[![](https://static.igem.org/mediawiki/2020/1/1a/T--Aalto-Helsinki--logo_andritz.jpg)](https://www.andritz.com/group-en)

[![](https://static.igem.org/mediawiki/2020/3/3c/T--Aalto-Helsinki--logo_tek.png)](https://www.tek.fi/en)

[![](https://static.igem.org/mediawiki/2020/3/3c/T--Aalto-Helsinki--logo_vtt.png)](https://www.vttresearch.com/en)

Special thanks to HSY for all their support

[![](https://static.igem.org/mediawiki/2020/b/b0/T--Aalto-Helsinki--logo_HSY.jpg)](https://www.hsy.fi/)

Kemistintie 1, Espoo, Finland

team@aaltohelsinki.com