iGEM 2020 — Team Hannover

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

# Team:Hannover/Notebook

iGEM Hannover 2020

# InToSens

## IGEM Team Hannover

[Home](https://2020.igem.org/Team%3AHannover)

Team

<Team>
<Attributions>
<Collaborations>
<Sponsors>

Project

<Contribution>
<Description>
<Design>
<Engineering>
<Experiments>
<Notebook>
[Proposed Implementation](Implementation)
[Proof of Concept](Proof_Of_Concept)
<Results>

Parts

[Parts Overview](Parts)
[Registry: Our best new basic part](http://parts.igem.org/Part%3ABBa_K3338002)
[Registry: Our best new composite part](http://parts.igem.org/Part%3ABBa_K3338010)

<Safety>

[Human Practices](Human_Practices)

Awards

<Education>
<Model>
<Software>

[Judging Forms](https://igem.org/2020_Judging_Form?id=3338)

☰

# Notebook

Here, we present a general overview of our timeline, with major meeting events. If you like a detailed review of all our
experiments, please feel free to contact us to obtain our lab notebooks as PDF.

November 2019

In November we started to recruite team members and had our first meeting. We
got to know each other and decided roughly on a topic.- **11/2020:** First meetings for team building and planning.

December 2019

We decided more about our project and came up with the name InToSens. The logo
was designed and everyone was divided into subgroups, so everyone got a special topic to work on
his own.- **12/04/2019:** Sketches for logo created, work breakdown structure
  plan/bar chart created
- **12/05/2019:** Topic better defined and first abstract written
- **12/11/2019:** Abstract for scientific target group/ general public
  discussed, logo revised
- **12/18/2019:** Responsibilities divided, initial planning of sponsors
- **12/18/2019:** Christmas party
- **12/27/2019:** Topic specified

January 2020

We planed our goals for the whole year for all subgroups and especially for
the lab work.- **01/08/2020:** First evaluation of cloning strategies and parts
- **01/15/2020:** Public relations, Girls/Boysday planned, Famelab planned
- **01/22/2020:** Registration fees acquired, iGEM Group for social media
  created
- **01/29/2020:** Public relations, new group photo

February 2020

We organized the first group activity, which was much fun. Our social media
accounts were created and we started informing other scientists and non-scientists about our
project.- **02/15/2020:** First planning of the measuring chamber
- **02/19/2020:** Socializing survey, anything blunt won. Abstract revised.

March 2020

In March, we contacted the first sponsors. In the middle of March the
Covid-19-pandemic reached Germany and we could not start working in the lab, as we planned.
After a few weeks, we decided to meet virtually on Skype and to continue the project anyway.- **03/04/2020:** Registration: Team registered but not yet approved (funding; countercheck by IGEM)
  Cloning strategy with BioBrick requirements by iGEM compared, laboratory access authorization
  organized
- **02/19/2020:** Socializing survey, anything blunt won. Abstract revised.

April 2020

We have officially registered at IGEM and could start working in the lab. We connected with the iGem
Team Darmstadt and started our biggest Collaboration. Furthermore we got our first sponsors.- **04/02/2020:** Corona, project continuation and how to react to the pandemic and emerging difficulties
  (difficult working conditions, sponsors etc)
- **04/22/2020:** First interview plans for Human Practices

May 2020

In May was the offically opening weekend. It was much fun to join the events. The lab work group
cloned 4 constructs and sequenced magA with eGFP, hGluc and both with flag tag. The measuring chamber
was designed.- **05/19/2020:** Wiki started
- **05/23/2020:** First collaboration meeting
- **05/27/2020:**  4 Constructs cloned and sequenced magA with eGFP, hGluc and both with flag tag, for 3
  constructs bacteria cultures are prepared

June 2020

The measuring chamber has been printed and the first interviews were conducted.- **06/03/2020:** Safety-form and furhter finances planned
- **06/10/2020:** First interview performed, initial cell experiments running

July 2020

The constructions are ready and working, the lab work group continued cell experiments- **07/17/2020:** Measuring chamber printed, Collaboration with TU Darmstadt running

August 2020

The constructs are working as expected in HeLa cells. We participated at the Global Meet-Up with our own workshop
“How to bake an iGem role“ and joined other workshops, which was much fun.- **08/26/2020:** Cloning completed, cell experiments are running, modeling in progress, additional focus
  on quantitative results

September 2020

Magnetic particles can be detected with our measuring chamber. We have made the project
promotion video and our team shirts got printed.- **09/18/2020:** Meeting for video planning
- **09/25/2020:** First part of Team Promotion Video recorded

October 2020

Deadlines are approaching fast...

* Say Hi:
* [Facebook](https://de-de.facebook.com/iGEMTeamHannover/)
* [Instagram](https://www.instagram.com/igem_teamhannover/)
* [Twitter](https://twitter.com/igem_hannover)
* Email
* [Github](https://github.com/igemsoftwareadmin/Hannover)
* 👋

* We are grateful to all of our
  [Sponsors!](https://2020.igem.org/Team%3AHannover/Sponsors)

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

# Team:Hannover/Engineering

iGEM Hannover 2020

# InToSens

## IGEM Team Hannover

[Home](https://2020.igem.org/Team%3AHannover)

Team

<Team>
<Attributions>
<Collaborations>
<Sponsors>

Project

<Contribution>
<Description>
<Design>
<Engineering>
<Experiments>
<Notebook>
[Proposed Implementation](Implementation)
[Proof of Concept](Proof_Of_Concept)
<Results>

Parts

[Parts Overview](Parts)
[Registry: Our best new basic part](http://parts.igem.org/Part%3ABBa_K3338002)
[Registry: Our best new composite part](http://parts.igem.org/Part%3ABBa_K3338010)[Registry: Improvement of an existing part](http://parts.igem.org/Part%3ABBa_K3338002)

<Safety>

[Human Practices](Human_Practices)

Awards

<Education>
<Model>
<Software>

[Judging Forms](https://igem.org/2020_Judging_Form?id=3338)

☰

# Engineering

**Summary:** We first describe the [genetic
engineering](#biological_engineering) of our sensor. After that, we give more details on the engineering of the [microfluidic measuring
chamber](#chamber_engineering) to identify magnetized cells. Most used parts are listed within this site as tooltip or link. For more information visit the <Parts> link and Registry for a detailed list.
Please follow us to [Proposed Implementation](Implementation) and [Proof of Concept](Proof_Of_Concept) to learn how we utilized both concepts or
back to <Design> to understand the initial design ideas.

## Engineering of our constructs

In order to successfully implement our idea of the biological sensor and the selected design, a lot
of literature research was conducted at the beginning of the project. As we had no experience with
MagAcreated part:
[BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) and LPS-dependent gene expression, we used different papers to get a first well-founded
impression. Furthermore, the right software for planning of the cloning had to be found. We decided
to use and learn the software of SnapGene, which was kindly provided to us by SnapGene. After a
short training period the first constructs could be designed. Here we proceeded schematically step
by step.

First the individual constructs were designed. For this purpose, the sequences were searched from
Addgene, assembled via SnapGene and prepared for the DNA HIFI Assembly reaction of NEB. In order to
obtain the individual sequences, which should be present in the constructs, the plasmids were
ordered from Addgene. The individual plasmids we have chosen can be seen in the table below.

Table 1: Used Plasmid with information about the manufacturer and the fragment of interest.

| Addgene number of plasmid | Fragment | Material information |
| 21751 | MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) | MS-1 MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) was a gift from Elliot Meyerowitz   (Addgene plasmid # 21751 ; [RRID:Addgene\_21751](http://n2t.net/addgene%3A21751)) |
| 22522 | Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) | phGluc was a gift from Christopher Buck   (Addgene plasmid # 22522 ; [RRID:Addgene\_22522](http://n2t.net/addgene%3A22522) ) |
| 79491 | IL6-promotercreated part: [BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008) | pENTR221-IL6.Pro was a gift from Peter Jon Nelson   (Addgene plasmid # 79491 ; [RRID:Addgene\_79491](http://n2t.net/addgene%3A79491) ) |
| 45350 | mCherryused part: [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450), P2Acreated part: [BBA\_K3338003](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338003) | AmCyan-P2Acreated part: [BBA\_K3338003](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338003)-mCherry was a gift from Ilpo Huhtaniemi   (Addgene plasmid # 45350 ; [RRID:Addgene\_45350](http://n2t.net/addgene%3A45350%20) ) |
| 21041 | IREScreated part: [BBA\_K3338004](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338004) | pmRFP\_a\_tubulin\_IRES\_puro2b was a gift from Daniel Gerlich   (Addgene plasmid # 21041 ; [RRID:Addgene\_21041](http://n2t.net/addgene%3A21041%20) ) |
| - | pEGFP-C2created part: [BBA\_K3338020](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338020) | avaiable in the lab |

The plasmids were multiplied, isolated and cut via restriction digestions. This should create the
right backbones for our constructs. The fragments, which should be arranged in different ways within
the backbone, were amplified by PCR and suitable primers. In order to perform the reaction with the
HIFI DNA Assembly Kit, the compatibility of the fragments to each other and to the backbone must be
ensured.

To verify the correct incorporation of the individual fragments and the success of the reaction, test
digestions were carried out. Only the correctly cloned plasmid could lead to the correct band length
and arrangement. After this was validated, test PCRs were performed. Also, at this point only
correct constructs could lead to an amplification of the desired length. Only when these steps were
reached, the finished plasmid was sent for sequencing to check the correct base sequence, the
correct frame and any mutations.

Unfortunately, this was sometimes not the case. In theses cases, the fragment was not inserted, or
not in the correct order. This was due to errors in planning and could then be tracked via SnapGene.
As soon as the source of the error was found, the sequence was changed, new primers were ordered and
the cloning was performed again. Possible error sources were mutations inserted via SnagGene and
mutation primers, wrong restriction sites or errors in the normal primer sequences.

After the plasmids had passed all test sections, they were multiplied by a MaxiPrep to be used for
transfection experiments.

From the individual plasmids and fragments, the first step was to start with MagAcreated part:
[BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000). The transmembrane
transporter [[1]](#lit_1) cannot be displayed by any simple
visualization methods. To check the cloning and transfection efficiency, a fluorescent protein
(EGFPused part:
[BBA\_K1123017](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K1123017))
was cloned in fusion to MagAcreated
part:
[BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000). To be more precise, the sequence of the MagAcreated part:
[BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) was placed in the
multiple cloning site of a pEGFP-C2CREATED
PART:
[BBA\_K3338020](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338020)
backbone vector. This was assembled in the composite part [BBa\_K3338012](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338012). This should result in a fusion protein that can
be visualized by fluorescence microscopy. Unfortunately, the first attempt was unsuccessful. In the
test restriction digestion described above, the correct bands could not be detected in any of the
eight clones used. Thus, the approach was changed in such a way that the backbone would be cut via a
different restriction enzyme. The basic procedure and the finally successful transfection attempt
are shown in the figure below.

![](https://static.igem.org/mediawiki/2020/8/88/T--Hannover--File_engeneering_1.png)

**Figure 1:**  The fragment MagAcreated
part:
[BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) was cloned into the pEGFP-C2CREATED
PART:
[BBA\_K3338020](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338020)
backbone via a single
restriction digest. This resulted in a fusion protein of MagAcreated part:
[BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) and EGFPused part:
[BBA\_K1123017](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K1123017), whose localization and
expression could be shown correctly in the cell membrane by fluorescence microscopy.

Since Gaussia Luciferasecreated part:
[BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) can be indicated by a simple coelenterazine reaction in medium, there was no
need to clone a fusion protein. For better comparability, the gene of luciferase, hGluccreated part:
[BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001), was also
cloned into the same backbone of the pEGFP-C2created part:
[BBA\_K3338020](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338020)
vector (see [BBa\_K3338017](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338017)).
For this purpose, the plasmid was cut at
different sites to remove the fluorescent protein EGFPused part:
[BBA\_K1123017](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K1123017).
Only the gene of Gaussia Luciferasecreated
part:
[BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) was
obtained under the control of the CMV-promoterused
part:
[BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004). The basic procedure and the successful luminescence
measurement are shown in the figure below. The signal proves the correct cloning, which has already
been validated by sequencing. In addition, the figure also shows that the functionality of Gaussia Luciferasecreated part:
[BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) is maintained and that it successfully leads to luminescence via the conversion of the
substrate coelenterazine [[2]](#lit_2).

![](https://static.igem.org/mediawiki/2020/c/c2/T--Hannover--File_engeneering_2.png)

**Figure 2:**  The gene of Gaussia Luciferasecreated part:
[BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001), hGluc, was cloned behind the CMV-promoterused part:
[BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004) in
the pEGFP backbone. Successfully transfected cells secreted the luciferase into the medium,
whereby luminescence could be successfully detected via the substrate of the luciferase, the
coelenterazine.

In order to get not only a visual proof of the expression of the two proteins, both sequences, MagAcreated part:
[BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000)
and hGluc, were labelled with a FLAG tag using appropriate primers (see [BBa\_K3338016](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338016) and [BBa\_K3338018](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338018)). This should also allow the
successful expression to be detected by a Western blot. The constructs could be successfully cloned
and sequenced . Due to the time constraints and the closure of the laboratory due to the corona
pandemic, this experiment was postponed and could not be performed successfully so far.
Nevertheless, the basic plan is shown in the figure below using the example of Gaussia Luciferasecreated part:
[BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001).

![FLAG](https://static.igem.org/mediawiki/2020/c/c0/T--Hannover--File_engeneering_3.png)

**Figure 3:**  Schematic representation of the planned procedure to check the expression
using an additional method with the example of the Gaussia Luciferasecreated part:
[BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001). In addition to
visualization by luminescence or fluorescence microscopy, the expression of both reporter
proteins should also be shown by a Western blot.

For the further progress of the project it was necessary to find a suitable combination of the two
reporter proteins. Therefore, an internal ribosome entry site (IREScreated part:
[BBA\_K3338004](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338004))
and a P2Acreated part:
[BBA\_K3338003](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338003)
peptide were used.
More details on the biological background can be found in the [design chapter](Design).
To validate the two
separation possibilities, two fluorescent proteins, namely EGFPused part:
[BBA\_K1123017](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K1123017)
and mCherryused part:
[BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450) were combined
once via
an IREScreated part:
[BBA\_K3338004](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338004)
and once via a P2Acreated part:
[BBA\_K3338003](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338003)
sequence in a pEGFP-C2created part:
[BBA\_K3338020](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338020)
backbone (see [BBa\_K3338013](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338013) and [BBa\_K3338014](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338014)). The extent of expression was reproduced
by fluorescence microscopy. These images as well as sections of both constructs with the pEGFP-C2created part:
[BBA\_K3338020](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338020)
backbone are shown in the figure below.

![FLAG](https://static.igem.org/mediawiki/2020/f/f3/T--Hannover--File_engeneering_4.png)

**Figure 4:** Fluorescence microscope images to validate the further used connection of the
two reporter proteins.

From our previous results we have concluded that the part [BBa\_K3338013](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338013), which contains a P2Acreated part:
[BBA\_K3338003](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338003)
sequence leads to a higher simultaneous
expression of both reporter proteins.

Our indication of a biofilm was thus represented by the following amplification sequence:

![](https://static.igem.org/mediawiki/2020/f/f0/T--Hannover--File_engeneering_5.PNG)

**Figure 5:** Reporter construct of our sensor under the control of a CMV-promoterused part:
[BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004) with high
basal expression. The simultaneous expression of both reporter proteins is mediated by a P2Acreated part:
[BBA\_K3338003](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338003)
sequence.

The basal expression of the CMV-promoterused
part:
[BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004) is very high. This promoter was only used to validate the
successful cloning and was replaced by several other promoters in the further course of the project.
These promoters should respond to LPS stimulation, one toxin of a bacterial biofilm. LPS should lead
to an increased expression of the downstream genes via the NFκB pathway. To make this
possible, the IL6-promotercreated part:
[BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008)
was used first. However, because this promoter is not compatible with the
BioBrick system of the iGEM Foundation, an additional mutated promoter (IL6mutcreated part:
[BBa\_K3338005](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338005))
was synthesized.
Therefore the thymine at position 371 was replaced by an adenine.

Furthermore, two promoters (NF-κB-AP1-minCMV1created part:
[BBa\_K3338007](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338007)
and
NF-κB-AP1-minCMV2created part:
[BBa\_K3338002](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338002))
were designed and developed by ourselves. The minimal CMV-promoterused
part:
[BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004) was
taken as a basis and extended in two different variants with binding sites for the inflammatory
molecules AP-1 and NFκB. These are induced by the bacterial toxin LPS. This should enable a
differentiation of lipopolysaccharide-induced expression of reporter proteins from basal expression.
These self-designed promoters are shown in the figure below.

![](https://static.igem.org/mediawiki/2020/5/50/T--Hannover--File_engeneering_6.png)

**Figure 6:** Schematic representation of the designed promoters. Two different combinations
of binding sites for inflammatory molecules were combined with the minimal CMV-promoter.

The process of engineering was completed with the subsequent successful cloning. The self-designed
promoters and both IL6-promotercreated part:
[BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008)s
were kindly provided by IDT and could be ligated directly into the
pEGFP-C2created part:
[BBA\_K3338020](http://PARTS.IGEM.ORG/WIKI/INDEX.PHP?TITLE=PART:BBA_K3338020)
backbone by clever selection of the connection points. Now the LPS dependence of the
individual promoters could be traced by the expression of the reporter proteins. The data of the
different experiments can be read in the chapter [results](Results).

## Engineering of our measuring chamber

The chamber measuring principle is intuitive. We follow the principle of magnetophoresis, which aims
to sort the particles inside the chamber in two output channels based on their magnetic properties.
For this, a magnet is placed on one side of the chamber, attracting the magnetic particles and thus
sorting magnetic and non-magnetic particles into different channels.

As source for an external magnetic field, we use a neodymium-alloy magnet. Two solutions stream
through the input channels into the chamber. One being the probe solution with the magnetic
particles, the other one being a buffer solution. Because of the low stream velocities and the small
size of the particles, a laminar flow is build up inside the chamber. This prevents unwanted mixing
of the two solutions.

Evaluation of the experiment is done by counting the number of particles at each output for different
strengths of the magnetic field and calculating the ratio of the counts. If the particles in the
probe solution exhibit magnetic properties, the count ratio will change significantly, when changing
the magnetic field strength.

![](https://static.igem.org/mediawiki/2020/7/74/T--Hannover--File_engeneering_7.png)

**Figure 7:** The measuring chamber involves two channels one with a probe, the other with a
buffer solution. A neodymium-alloy magnet is used to produce the magnetic field

In the mixing channel, the magnetic field of the magnet interacts with the magnetic dipole moment of
the particle. This interaction results in a force exerting on the magnetic particles moving inside
the mixing channel. As a result, the trajectory of the particle is altered. If paramagnetic
particles are used, the force is attractive, which means that the particles are accelerated towards
the magnet [[3]](#lit_3). In this case, it is important, that the probe
solution is streamed in the input
channel facing away from the magnet. Only in this case, a significant increase in the number of
paramagnetic particles in the output channel can be observed, when adding an external magnetic
field. Comparing the number of particles at each output channel, more particles will be counted at
the channel faced towards the magnet. If the ratio of the counts is calculated, it will increase or
respectively decrease with a change in the magnetic field strength.

## Bibliography and references

1. Uebe, R., Henn, V., & Schüler, D. (2012). The MagAcreated part:
   [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) protein of Magnetospirilla is not involved in
   bacterial magnetite biomineralization. Journal of bacteriology, 194(5), 1018–1023.
2. Tannous B. A. (2009). Gaussia Luciferasecreated
   part:
   [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) reporter assay for monitoring biological processes in
   culture and in vivo. Nature protocols, 4(4), 582–591.
3. Tarn, Mark D., et al. On-chip diamagnetic repulsion in continuous flow. Science and technology
   of advanced materials, 2009.

* Say Hi:
* [Facebook](https://de-de.facebook.com/iGEMTeamHannover/)
* [Instagram](https://www.instagram.com/igem_teamhannover/)
* [Twitter](https://twitter.com/igem_hannover)
* Email
* [Github](https://github.com/igemsoftwareadmin/Hannover)
* 👋

* We are grateful to all of our
  [Sponsors!](https://2020.igem.org/Team%3AHannover/Sponsors)

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

# Team:Hannover/Experiments

iGEM Hannover 2020

# InToSens

## IGEM Team Hannover

[Home](https://2020.igem.org/Team%3AHannover)

Team

<Team>
<Attributions>
<Collaborations>
<Sponsors>

Project

<Contribution>
<Description>
<Design>
<Engineering>
<Experiments>
<Notebook>
[Proposed Implementation](Implementation)
[Proof of Concept](Proof_Of_Concept)
<Results>

Parts

[Parts Overview](Parts)
[Registry: Our best new basic part](http://parts.igem.org/Part%3ABBa_K3338002)
[Registry: Our best new composite part](http://parts.igem.org/Part%3ABBa_K3338010)[Registry: Improvement of an existing part](http://parts.igem.org/Part%3ABBa_K3338002)

<Safety>

[Human Practices](Human_Practices)

Awards

<Education>
<Model>
<Software>

[Judging Forms](https://igem.org/2020_Judging_Form?id=3338)

☰

# Experiments

We have uploaded our main experimental protocols as PDF file as detailed below. We have focused on protocols which involve our mammallian HeLa cells and our measuring chamber. Please feel free to contact us if you have any further questions.

## [Preparation of growth medium (PDF)](https://static.igem.org/mediawiki/2020/1/15/T--Hannover--experiments_growth_medium_preperation.pdf)

In this protocol, we describe how the growth medium of HeLa cells is prepared.

## [Passaging of HeLa cells (PDF)](https://static.igem.org/mediawiki/2020/9/9f/T--Hannover--Passaging_of_HeLa_cells.pdf)

This protocol explains how we sub-cultivated / passaged the HeLa cells.

## [Counting of HeLa cells (PDF)](https://static.igem.org/mediawiki/2020/e/ef/T--Hannover--experiments_counting_cells.pdf)

To use defined cell numbers in the experiments, it is important to count the cells before seeding during subcultivation. This is described in this protocol.

## [Transfection of HeLa cells (PDF)](https://static.igem.org/mediawiki/2020/7/7b/T--Hannover--Transfection_of_HeLa_cells.pdf)

This protocol describes to possibilties to transfect HeLa cells: lipid-based transfection (lipofection) and electroporation. We used this protocol to transfect all our engineered DNA constructs.

## [Preparation of the electroporation buffer (PDF)](https://static.igem.org/mediawiki/2020/d/d4/T--Hannover--Preparation_of_electroporation_buffer.pdf)

This protocol points to our recipe which we used in HeLa cell electroporation.

## [LPS stimulation of HeLa cells (PDF)](https://static.igem.org/mediawiki/2020/8/88/T--Hannover--LPS-stimulation_of_HeLa_cells.pdf)

In this protocol, the stimulation of HeLa cells with lipopolysaccharides is detailed. We used this protocol in our stimulation experiments.

## [Measurement of luminescence (PDF)](https://static.igem.org/mediawiki/2020/b/bf/T--Hannover--luminescence_assay.pdf)

We describe how we measured the luminescence signal of gaussia luciferase in our experiments.

## [Magnetophoresis protocol (PDF)](https://static.igem.org/mediawiki/2020/f/f9/T--Hannover--magnetophoresis.pdf)

This protocol outlines the main steps to use our magnetophoresis measurement chamber. If you need more details, please do not hestitate to contact us.

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# Team:Hannover/Results

iGEM Hannover 2020

# InToSens

## IGEM Team Hannover

[Home](https://2020.igem.org/Team%3AHannover)

Team

<Team>
<Attributions>
<Collaborations>
<Sponsors>

Project

<Contribution>
<Description>
<Design>
<Engineering>
<Experiments>
<Notebook>
[Proposed Implementation](Implementation)
[Proof of Concept](Proof_Of_Concept)
<Results>

Parts

[Parts Overview](Parts)
[Registry: Our best new basic part](http://parts.igem.org/Part%3ABBa_K3338002)
[Registry: Our best new composite part](http://parts.igem.org/Part%3ABBa_K3338010)[Registry: Improvement of an existing part](http://parts.igem.org/Part%3ABBa_K3338002)

<Safety>

[Human Practices](Human_Practices)

Awards

<Education>
<Model>
<Software>

[Judging Forms](https://igem.org/2020_Judging_Form?id=3338)

☰

# Results

**Summary:**
This page describes the **functional** results of our experiments in detail. To learn
everything about our parts, for example, about their verification, principle and so on - please
follow us
to <Parts>. The presented results enabled us to obtain our [Proof of Concept](Proof_Of_Concept). They are
based on the various aspects, which we considered during <Design> and <Engineering> and follow the
principles of
our [Proposed Implementation](Implementation). Firstly, we will
describe
all **[functional biological results](#biology_results)** and
secondly those obtained with
**[our measuring chamber](#chamber_results). Our results verified the in vitro applicability of our inflammatory toxin sensor [BBa\_K3338010](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338010)**.

# The InflammatoryToxinSensor can detect and report bacterial toxins In order to generate our cell-based sensor for the early and minimally-invasive detection of bacterial infection, we designed and cloned many different genetic constructs. These constructs are composed of different combinations of the single parts we use for our sensor and therefore had to be characterized and validated. As proof of concept experiments, we performed in vitro studies in which we transfected HeLa cells with either genetic construct and subsequently analyzed the cells’ protein expression by appropriate methods. Expression of MagA and Gaussia Luciferase in HeLa cells In a first set of experiments, we aimed to show that the expression of our two reporters – MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) and Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) – was possible in HeLa cells. For this, we designed two genetic constructs with either reporter gene under the control of the constitutively active CMV promoterused part: [BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004). In the case of MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000), we further genetically introduced a fluorescent tag (eGFP) to the protein to make expression analysis via fluorescent microscopy possible. For the generation of both plasmids, NEB HiFi DNA cloning kit was used. The DNA sequence of the respective reporter gene was amplified by PCR using appropriate primers for subsequent introduction into the previously linearized plasmid backbone pEGFP-C2created part: [BBa\_K3338020](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338020). Successful cloning was verified by DNA sequencing. Transfection of HeLa cells with either plasmid, respectively, was performed by lipofection (ViaFect transfection reagent) or electroporation. The constitutive expression of the eGFP-MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) fusion protein was analyzed by fluorescent microscopy 24 hours after transfection. Figure 1 shows that transfected HeLa cells synthesized MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) and that the protein was not localized in the area of the nucleus or the cytoplasm, but rather in membrane regions. This finding is in good agreement with our expectations, as in magnetotactic bacteria, MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) is a transmembrane protein serving as an iron transporter. ![](https://static.igem.org/mediawiki/2020/4/49/T--Hannover--results_1.png) **Figure 1:** Representative microscopy image of eGFP-MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) expressing HeLa cells. Both fluorescence (left) and brightfield channel (middle) as well as a merge (right) are shown. Scale bar: 10 µm. The constitutive expression of Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) was analyzed by performing a luminescence assay. In this, the enzyme’s substrate coelenterazine was added to the cell culture supernatant, which is expected to contain the Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) that was secreted by the transfected HeLa cells. Since luminescence is produced in the reaction of Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) with its substrate, we qualitatively detected the enzyme’s expression via camera. Figure 2 shows that luminescence was only observed when supernatant from previously transfected HeLa cells was utilized, while usage of negative controls such as cell culture media or supernatant from non-transfected HeLa cells did not lead to a generation of light. On the one hand, these results demonstrate the expression of Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) by transfected HeLa cells. On the other hand, it also shows that the protein is secreted by the cells. Furthermore, this study proves the functionality of the Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) by showing that it actually exhibits the expected enzymatic activity. ![](https://static.igem.org/mediawiki/2020/7/79/T--Hannover--results_2.png) **Figure 2:** Luminescence assay showing activity of Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) in supernatant of previously transfected HeLa cells. Negative controls: cell culture media or supernatant from non-transfected HeLa cells. Taken together, the results we achieved by these first experiments suggest that both our chosen reporters can be expressed by human epithelial cells such as HeLa. While we showed the correct intracellular localization of MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000), we further demonstrated the functionality of Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001), making both proteins possibly well suited for our intended sensor concept. Simultaneous expression of MagA and Gaussia Luciferase in HeLa cells – Introduction of P2A In our final sensor, we aim to use both MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) and Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) as reporters which are expressed under control of the same, single promoter. To achieve this goal, it needs a special part in our genetic construct which connects both reporters and enables the simultaneous synthesis of both proteins. For this, we tested two options: IREScreated part: [BBa\_K3338004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338004) and P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003) site. IREScreated part: [BBa\_K3338004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338004) stands for Internal Ribosomal Entry Site and P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003) represents a 2A peptide which encodes for a self-cleaving peptide. The difference between these two “connections” is that they control/regulate the formation of the two reporter proteins at different stages of the expression. The use of IREScreated part: [BBa\_K3338004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338004) enables a second ribosome to bind to the mRNA independently from the 5’-cap, thereby leading to the generation of the two individual reporter proteins: MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) and Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001). In contrast, the presence of a P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003) site first leads to a long, single mRNA transcript containing MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000), P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003) and Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001), which is cleaved into its individual components only during translation. In the end, both IREScreated part: [BBa\_K3338004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338004) and P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003) can lead to the expression of our two reporters, but the stoichiometry of both proteins might be different in either case. Since our goal is to let both reporters be expressed in a stoichiometrically equal manner, we designed two genetic constructs that carry the fluorescent proteins eGFP and mCherry as well as either IREScreated part: [BBa\_K3338004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338004) or P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003) as connecting part. In both cases, we used the constitutively active CMV promoterused part: [BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004) to control the expression. For the generation of both plasmids, NEB HiFi DNA cloning kit was used. The DNA sequence of the single parts was amplified by PCR using appropriate primers for subsequent introduction into the previously linearized plasmid backbone pEGFP-C2created part: [BBa\_K3338020](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338020). Successful cloning was verified by DNA sequencing (please see <Parts>.). To compare the stoichiometry of simultaneous expression of both fluorescent proteins in the case of IREScreated part: [BBa\_K3338004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338004) and P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003), respectively, transfection of HeLa cells with either plasmid was performed by lipofection (ViaFect transfection reagent) or electroporation and fluorescence microscopy was used for subsequent analysis. As demonstrated in Figure 3, we observed a large difference in fluorescence intensity – meaning expression strength – of eGFP and mCherry in the case of the IREScreated part: [BBa\_K3338004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338004) construct. Further, many eGFP positive cells did not show an mCherry signal. In contrast, in the case of the P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003) construct, nearly all cells that expressed eGFP were mCherry positive as well. The mCherry fluorescence intensity is higher than observed for the IREScreated part: [BBa\_K3338004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338004) construct and it is roughly comparable with the respective eGFP signal. ![](https://static.igem.org/mediawiki/2020/b/b5/T--Hannover--results_3.png) **Figure 3:** Representative microscopy images of HeLa cells transfected with CMV-eGFP-IREScreated part: [BBa\_K3338004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338004)-mCherry (top) or CMV-eGFP-P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003)-mCherry (bottom). Images of three channels are shown: green fluorescence (left), red fluorescence (middle) and brightfield (right). Scale bar: 100 µm. As a result, we decided to implement the P2Acreated part: [BBa\_K3338003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338003) site as connecting part for simultaneous expression of both reporters in our sensor concept. How to make our construct “sensing” Until this point, we have constantly utilized the constitutively active CMV promoterused part: [BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004) to analyze the gene expression for characterization of the sensor’s reporters. For our sensor concept though, a promoter with a low basal expression and an activity that is inducible by inflammatory signals is needed to actually make our construct sensing. A broad variety of bacterial components, including for example the bacterial endotoxin lipopolysaccharide (LPS), act as such inflammatory signals and are detected by human cells via binding to extracellular Toll-like receptor domains. In the course of the signal transmission, these receptors often induce the intracellular NF-κB pathway, in which the transcription factor NF-κB is released. After translocation to the nucleus, NF-κB can bind to specific DNA promoter sequences thereby activating gene expression. Hence, for our sensor concept, we aimed to decipher the best conditional promoter design for NF-κB sensing. For this, we tested four different inducible promoters, which are described in more detail below. For every promoter, we designed a genetic construct composed of the promoter and Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) as reporter for quantitative readout of expression strength. For the generation of all plasmids, NEB HiFi DNA cloning kit was used. The DNA sequence of the single parts was amplified by PCR using appropriate primers for subsequent introduction into the previously linearized plasmid backbone pEGFP-C2created part: [BBa\_K3338020](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338020). Successful cloning was verified by DNA sequencing. For analysis, HeLa cells were transfected with either plasmid. The plasmid carrying Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) under control of the CMV promoter served as reference. On the day after transfection, cells were treated with different concentrations of LPS (0, 0.5, 1 or 2 µg/mL) for 3 hours in order to stimulate the NF-κB pathway. Subsequent analysis of expression strength was performed by luminescence assay. Therefore, cell culture supernatant was collected 24 hours as well as 48 hours after LPS treatment and luminescence was quantitatively read out. A schematic representation of the experimental procedure is shown in Figure 4. ![](https://static.igem.org/mediawiki/2020/f/f5/T--Hannover--results_4.png) **Figure 4:** Schematic representation of the experimental procedure. HeLa cells, which were seeded the day before transfection, were transfected with the respective plasmid via lipofection. On the day after transfection, cells were treated with different concentrations of LPS for 3 hours. 24 hours and 48 hours after treatment, the expression strength was quantified by performing a luminescence assay. **CMV promoter** As mentioned before, the CMV promoterused part: [BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004) is characterized by its constitutive activity with rather high basal expression levels. Due to this, we did not expect to observe any LPS induced changes in the promoter’s activity, making it an ideal reference. The results, which are shown in Figure 5, are in good agreement with this and demonstrate the importance of choosing the right promoter to make our sensor concept work. ![](https://static.igem.org/mediawiki/2020/a/af/T--Hannover--results_5.png) **Figure 5:** Relative activity of CMV promoterused part: [BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004) in HeLa cells 24 hours (A) and 48 hours (B) after treatment with different concentrations of LPS for 3 hours, normalized to untreated control (0 µg/mL LPS). Data shown represents mean ± SEM of n=4 biological replicates. Statistical analysis was performed by unpaired t-test in comparison to untreated control, significance level: 10 %, significance is indicated by asterisk. **Interleukin-6 promoter** The Interleukin-6 (IL6-promotercreated part: [BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008)), which plays an important role in (patho)physiological inflammatory processes, features such a desired NF-κB inducible activity. Thus, we chose this promoter for our sensor concept. Figure 6 shows that LPS treatment of cells indeed led to an increased expression of Gaussia luciferase. While the relative enhancement is rather small, it is still significant. ![](https://static.igem.org/mediawiki/2020/5/53/T--Hannover--results_6.png) **Figure 6:** Relative activity of IL6-promotercreated part: [BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008) in HeLa cells 24 hours (A) and 48 hours (B) after treatment with different concentrations of LPS for 3 hours, normalized to untreated control (0 µg/mL LPS). Data shown represents mean ± SEM of n=4 biological replicates. Statistical analysis was performed by unpaired t-test in comparison to untreated control, significance level: 10 %, significance is indicated by asterisk. As the sequence of the IL6-promotercreated part: [BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008) contains a restriction site which is not compatible with the iGEM BioBrick system though, we introduced a point mutation at this site. We named the resulting part IL6mutcreated part: [BBa\_K3338005](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338005) promoter. Figure 7 shows that, in contrast to our expectations and to the behavior of the native IL6-promotercreated part: [BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008), LPS stimulation of HeLa cells carrying the plasmid with Gaussia Luciferasecreated part: [BBa\_K3338001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338001) under control of the IL6mutcreated part: [BBa\_K3338005](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338005) promoter did not lead to an enhanced expression within 24 or 48 hours, respectively, after treatment. While it seems as if LPS treatment negatively influenced the promoter activity, no significant changes could be detected in comparison to untreated control. These results indicate that the introduced point mutation might have led to an unpredicted negative effect on the promoter’s activity in terms of its NF-κB sensing capabilities. Unfortunately, this makes the IL6mut promoter unsuitable for our sensor concept. ![](https://static.igem.org/mediawiki/2020/4/4b/T--Hannover--results_7.png) **Figure 7:** Relative activity of IL6mut promoter in HeLa cells 24 hours (A) and 48 hours (B) after treatment with different concentrations of LPS for 3 hours, normalized to untreated control (0 µg/mL LPS). Data shown represents mean ± SEM of n=4 biological replicates. Statistical analysis was performed by unpaired t-test in comparison to untreated control, significance level: 10 %, significance is indicated by asterisk. **NF-κB-AP1-minimal-CMV promoters** Besides the IL6-promotercreated part: [BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008) and the CMV promoterused part: [BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004), we further tested two self-designed promoters (NF-κB-AP1-minCMV1created part: [BBa\_K3338007](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338007) and NF-κB-AP1-minCMV2created part: [BBa\_K3338002](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338002)). These were based on a minimal CMV promoter with AP1 and NF-κB transcription factor binding sites. Using Matlab, we generated random sequences containing three AP1 and NF-κB binding sites each and the minimal CMV promoter. For generation of the construct, we used synthesized sequences. ![](https://static.igem.org/mediawiki/2020/9/9e/T--Hannover--results_8.png) **Figure 8:** Schematic representation of the generation of the NF-κB-AP1-minCMV promoters. Combination of both transcription factor binding site sequences (3 times each) and the minimal CMV promoter (A) yielded the two synthetic promoter sequences used for our project (B). As seen in Figure 9 and Figure 10, both synthetic NF-κB-AP1-minCMV promoters exhibited a significant upregulation of luciferase expression upon LPS stimulation. This behavior was observed 24 hours as well as 48 hours after stimulation. In the case of NF-κB-AP1-minCMV1 promoter, significance increased after 48 hours compared to 24 hours. These results suggest that both self-designed promoters could serve as potential candidate parts for our final sensor construct. ![](https://static.igem.org/mediawiki/2020/6/63/T--Hannover--results_9.png) **Figure 9:** Relative activity of NF-κB-AP1-minCMV1 promoter in HeLa cells 24 hours (A) and 48 hours (B) after treatment with different concentrations of LPS for 3 hours, normalized to untreated control (0 µg/mL LPS). Data shown represents mean ± SEM of n=4 biological replicates. Statistical analysis was performed by unpaired t-test in comparison to untreated control, significance level: 10 %, significance is indicated by asterisk. ![](https://static.igem.org/mediawiki/2020/4/44/T--Hannover--results_10.png) **Figure 10:** Relative activity of NF-κB-AP1-minCMV2 promoter in HeLa cells 24 hours (A) and 48 hours (B) after treatment with different concentrations of LPS for 3 hours, normalized to untreated control (0 µg/mL LPS). Data shown represents mean ± SEM of n=4 biological replicates. Statistical analysis was performed by unpaired t-test in comparison to untreated control, significance level: 10 %, significance is indicated by asterisk. **Conclusion** Taken together, we showed that the IL6-promotercreated part: [BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008)‘s as well as our two self-designed NF-κB-AP1-minCMV promoters‘ activities can be induced by LPS stimulation. Since this feature is essential for our sensor’s capability to detect a bacterial biofilm, we concluded that these promoters pose potential candidate parts for our final sensor construct. A low basal expression of the reporter proteins is important for our sensor concept Besides the sensing capability, what else do we need to consider? To obtain a high sensitivity of our sensor, it needs a low basal expression level. Moreover, we do not want constitutive expression of both our reporter proteins in the human body. Consequently, we compared all basal expression levels based on luminescence. Our results (Figure 11) show that, indeed, the native IL6-promotercreated part: [BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008) proved to be the best choice as it resulted in a very low basal expression. Only the BioBrick system adapted IL6mut promoter showed an even lower basal expression, but unfortunately, this is possibly due to the fact that the promoter was not really active and inducible anymore in general. Interestingly, both self-designed promoters, which are based on the minimal CMV promoter, exhibited a strong enhancement of basal expression compared to the CMV promoterused part: [BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004). One reason for this observation might be an overall increased transfection efficiency due to the lesser size of the plasmid. Further experiments are needed to clarify. ![](https://static.igem.org/mediawiki/2020/f/f6/T--Hannover--results_11.png) **Figure 11:** Relative basal activity of all tested promoters in HeLa cells 48 hours (A) and 72 hours (B) after transfection. Data was normalized to CMV promoterused part: [BBa\_I712004](http://parts.igem.org/wiki/index.php?title=Part:BBa_I712004) which served as reference. Data shown represents mean ± SEM of n=4 biological replicates. Taken together, we showed that the native IL6-promotercreated part: [BBa\_K3338008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338008)’s properties suit best for our sensor concept. While the synthetic promoters proved to be very good for LPS detection, based on their high basal expression, they could not fully fulfil our purpose though. So we had our Inflammatory Toxin Sensor ready: [BBa\_K3338010](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338010). Can we characterize magnetic cells? Our idea was to build and operate a measuring chamber, which will allow future iGEM Teams to characterize their own magnetic cells or particles in a fast and efficient way. We used magnetic beads to proof our measuring concept and identify possible problems with our chamber. Iron microparticles with a diameter of 10 µm to 100 µm and iron-oxide beads with a diameter of 1 µm were used. The particles were suspended in water in the particle solutions. During our first trials we had problems with the entrapment of air bubbles in the chamber. Air bubbles were in general bigger than our fluid channels, therefore shutting it down completely by clogging the micro fluid channels. To resolve this problem we added soap to our solution, reducing the surface tension of the water and making the formation of bigger bubbles impossible. The magnetic field was measured using a Hall-effect sensor. ![](https://static.igem.org/mediawiki/2020/4/4e/T--Hannover--results_12_2.png) **Figure 12:** Measured magnetic field of the Neodymium-alloy-magnet. The measuring chamber is connected to 200 µl pipette tips. These are used as injector, as well as collection vials. The inputs are fed by two syringe pumps with B Braun Original Perfusor®. We choose 10 mm /s as a valid flow velocity for observation and particle acceleration. Also we determined that at this velocity the impact of diffusion would be minimal. We calculated that at this speed the flow would be laminar using the Reynolds number: \[ R={\mu L \over \nu} \] Therefore the volumetric flow rate of the mixing chamber had to be: \[\dot{V} = 40 ml/h\] To guaranty separated output streams we adjusted the input volumetric flow rate, the rate for the particle solution has to be slightly lower than for the buffer solution. Thus no particles end up in the output if not attracted by the magnet. Thus we used \[\dot{V} = 23 ml/h \] for the buffer solution and \[\dot{V} = 17 ml/h \] for the particle solution. For counting, we used a brightfield microscope. We placed a droplet of our output solution on a glass coverslip and took an image with the microscope. Afterwards we used the Particle Analysis plugin of the software ImageJ [1]. ![](https://static.igem.org/mediawiki/2020/9/98/T--Hannover--results_13.png) **Figure 13:** The measuring and analyzing process of magnetophoresis. We were able to observe an influence of the magnet on the particles within our measuring chamber, which verified our concept of a magnetophoresis-micro-fluidic-system. Figure 14 and figure 15 show the results of our trails with two different kind of magnetic particles. The particles were counted by the ImageJ software and then the ratio of the moved particles was calculated. All values were then displayed in a boxplot diagram. With an increase of the magnetic field, the ration of moved particles is increased, for each the iron micro particles as well as the magnetic beads. ![](https://static.igem.org/mediawiki/2020/4/47/T--Hannover--results_14.png) **Figure 14:** Ratio of iron micro particles moved by the magnetic field in our measuring chamber. ![](https://static.igem.org/mediawiki/2020/2/2a/T--Hannover--results_15.png) **Figure 15:** Ratio of magnetic beads moved by the magnetic field in our measuring chamber. However, some particles were moved into a different channel even without an eternal magnetic field. Diffusion and most likely convection are the reason for this phenomenon, due to design and manufacturing errors in our measuring chamber. For the iron micro particles with a diameter of 100 µm to 10 µm even small magnetic fields with 8.7 mT showed a drastic increase of movement of the particles. This increase in movement compared to the smaller 1 µm magnetic beads can be contributed to their bigger volume and therefore increased magnetic momentum. On the other hand, the drag of the bigger surface is smaller than the magnetic force moving the particles in the surrounding fluid. For the magnetic beads, a defined increase of movement is detectable at 45 mT and above. The measuring chamber is valid for characterizing magnetic particles, for the cellular experiments with our MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) expressing cells some adjustments are needed. The magnetic momentum in the MagAcreated part: [BBa\_K3338000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3338000) cells is located in the magentosomes in nanosized magnetite crystals. Therefore the magnetic field for the measurement has to be at least 50 mT following the experiments with the magnetic beads. Also the drag of the cell will be increased compared to the beads. This is analyzed in ongoing experiments. Bibliography and references 1. Rueden, C. T., Schindelin, J., Hiner, M. C., DeZonia, B. E., Walter, A. E., Arena, E. T., & Eliceiri, K. W. (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC bioinformatics, 18(1), 529.

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