iGEM 2021 — Team NTNU-Trondheim

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# Team:NTNU-Trondheim/Notebook

SulFind 2021

[![](https://static.igem.org/mediawiki/2021/0/02/T--NTNU-Trondheim--Logo_ensfarget.png)](https://2021.igem.org/Team%3ANTNU-Trondheim)

PROJECT

[Project Description](https://2021.igem.org/Team%3ANTNU-Trondheim/Description)
[Implementation](https://2021.igem.org/Team%3ANTNU-Trondheim/Implementation)
[Contribution](https://2021.igem.org/Team%3ANTNU-Trondheim/Contribution)
[Safety](https://2021.igem.org/Team%3ANTNU-Trondheim/Safety)
[Engineering](https://2021.igem.org/Team%3ANTNU-Trondheim/Engineering)

LAB

[Experiments](https://2021.igem.org/Team%3ANTNU-Trondheim/Experiments)
[Results](https://2021.igem.org/Team%3ANTNU-Trondheim/Results)
[Notebook](https://2021.igem.org/Team%3ANTNU-Trondheim/Notebook)

PARTS

[Improvement](https://2021.igem.org/Team%3ANTNU-Trondheim/Improve)
[Parts](https://2021.igem.org/Team%3ANTNU-Trondheim/Parts)

HUMAN PRACTICES

[Human Practices](https://2021.igem.org/Team%3ANTNU-Trondheim/Human_Practices)
[Communication](https://2021.igem.org/Team%3ANTNU-Trondheim/Communication)
[Education](https://2021.igem.org/Team%3ANTNU-Trondheim/Education)

TEAM

[Team members and project sponsors](https://2021.igem.org/Team%3ANTNU-Trondheim/Team)
[Attributions](https://2021.igem.org/Team%3ANTNU-Trondheim/Attributions)
[Collaboration](https://2021.igem.org/Team%3ANTNU-Trondheim/Collaborations)
[Partnership](https://2021.igem.org/Team%3ANTNU-Trondheim/Partnership)

![](https://static.igem.org/mediawiki/2021/1/17/T--NTNU-Trondheim--Banner_Notebook.jpeg)

This page contains our day-to-day work in the laboratory. As we worked on two approaches for the main part
of the
summer, the documentation of the laboratory work is divided into the *Heme protein approach* and the
*Sulfide-responsive transcriptional repressor approach.* In the fall of 2021 we performed work
related to
*Improvement of an existing part.*

## Week 27

**07/07/21**

* Prepared and autoclaved laboratory equipment* Prepared LB medium

**08/07/21**

*Heme protein approach:*

* Prepared stock solution with Myoglobin and made aliquots with concentration 0.1 g/L.

## Week 28

**12/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Diluted primers for cloning to a final concentration of 10 μM.

**13/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the efficiency of the supercompetent DH5-alpha *E. coli* cells, and incubated
  them overnight.* PCR-amplified the pENZ004 plasmid for Gibson assembly.* Analyzed the samples from PCR on an agarose gel.* Removed the WT plasmid from the PCR samples with Dpn1 enzyme digest.

*Heme protein approach:*

* Prepared stock solution with Cy3 NHS ester.* Prepared stock solution of Potassium Phosphate buffer.* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**14/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Suspended DNA sequences from IDT.* Performed PCR purification, Gibson assembly and heat shock transformation.* Counted the number of colonies from the competent cell test from yesterday.

*Heme protein approach:*

* Purified labeled myoglobin from 13/07/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution and (H2S) dilutions.

**15/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Transferred cells to LB amp medium and incubated overnight.

**16/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Isolated plasmid DNA from the transformed DNA. *NB!* At step 10 only 30 μL EB buffer
  should be added,
  instead of 30 μL as the protocol states.* Prepared Glycerol stock.

## Week 29

**19/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Performed Gibson assembly and transformation for design 2 fragments.* Performed restriction digest and gel analysis.* Tested the whole cell sensor with (H2S) and design 1 (A, B & C) - part 1. Frozen
      transformed
      bacteria were thawed
      on ice and added to a 5 ml solution of LB(Amp). Incubated overnight.

**20/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Performed Gibson assembly and transformation for design 2 fragments.* Sent to Eurofins for Sanger sequencing (Miniprepped DNA was dried out overnight, to make it
    concentrated enough
    for sequencing. Template and sequencing primers were mixed in accordance with Eurofins
    protocol and sent for
    sequencing).* Transformation with design 2A (gibson assembly product) - part 1.* Tested the whole cell sensor with (H2S) and design 1 (A, B & C) - part 2.
        Bacterial solutions
        from 19/07/21 were
        transferred to LB solution and incubated overnight.

*Heme protein approach:*

* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**21/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Performed transformation with design 2A* Sent 2BS-C1, 2BS-C2, 2BL-C1, 2BL-C2 and 2AL to Eurofins for Sanger sequencing.* Tested the whole cell sensor with (H2S) [2000,85 M] and design 1 (A, B & C) -
      part 3. Diluted
      bacterial solution
      and added analytes. Incubated it for 17 hours

*Heme protein approach:*

* Purified labeled myoglobin from 20/07/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution [5 mg/L] and (H2S) dilutions [0.05-0.3 umol/L]. Measured
  fluorescence with
  Tecan.

**22/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the whole cell sensor with (H2S) [2000,85 M] and design 1 (A, B & C),
  measured
  fluorescence - part 3;* Froze design 2 cells, mini prepped and sent to Eurofins for sanger sequencing.

**23/07/21**

*Heme protein approach:*

* Performed emission/excitation scan measurements on the Tecan to identify emission/excitation
  peaks of the Cy3
  fluorophore used in the heme approach. Used the labeled myoglobin from 21/07/21.

**25/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the whole cell sensor with (H2S) (GSSH). Frozen transformed bacteria were
  thawed on ice
  and added to a 5 ml
  solution of LB(Amp). Incubated overnight.

## Week 30

**26/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the whole cell sensor with (H2S) (GSSH). Bacterial solutions from the
  previous day were
  transferred to LB
  solution and incubated overnight.

  *Heme protein approach:*

  + Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**27/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the whole cell sensor with (H2S) (GSSH) and design 1 (A, B & C). Diluted
  bacterial
  solution and added
  analytes. Incubated it for 17 hours.

*Heme protein approach:*

* Purified labeled myoglobin from 26/07/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution [5 mg/L] and (H2S) dilutions [0, 1, 5, 10, and 25 umol/L].
  Measured
  fluorescence with Tecan.

**28/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the whole cell sensor with (H2S) (GSSH) and design 1 (A, B & C), measured
  fluorescence.

*Heme protein approach*:

* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**29/07/21**

*Sulfide-responsive transcriptional repressor approach:*

* Froze bacteria, mini prepped and sent design 2A to Eurofins for Sanger sequencing.

*Heme protein approach*:

* Purified labeled myoglobin from 28/07/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution [25 mg/L] and (H2S) dilutions [0.25-1.5 umol/L]. Measured
  fluorescence with
  Tecan.

**01/08/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the whole cell sensor with (H2S) (GSSH) and design 1A,1B,1C, 2AS, 2AL,
  2BS, 2BL. Frozen
  transformed
  bacteria were thawed on ice and added to a 5 ml solution of LB(Amp). Incubated overnight.

## Week 31

**02/08/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the whole cell sensor with (H2S) (GSSH) and design 1A,1B,1C, 2AS, 2AL,
  2BS, 2BL.
  Bacterial solutions from
  the previous day were transferred to LB solution and incubated overnight.* Colony PCR of 2AL and 2AS

*Heme protein approach:*

* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**03/08/21**

*Sulfide-responsive transcriptional repressor approach:*

* Colony PCR - the results indicated that 1A, 1B, 1C and 2A do not work.* Tested the whole cell sensor with (H2S) (GSSH) and design 2BS. Diluted bacterial
    solution and
    added analytes.
    Incubated it for 17 hours.

*Heme protein approach*:

* Purified labeled myoglobin from 02/08/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution [5 mg/L] and (H2S) dilutions [0-1.5 umol/L]. Measured
  fluorescence with
  Tecan.

**04/08/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested whole cell sensor with (H2S) (GSSH) and design 2BS, measured fluorescence.

*Heme protein approach*:

* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**05/08/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the whole cell sensor with (H2S) (GSSH) and design 2BS, measured
  fluorescence again.

  *Heme protein approach:*

  + Purified labeled myoglobin from 04/08/21, measured myoglobin concentration and
    labeling ratio, prepared
    labeled protein solution [5 mg/L] and (H2S) dilutions [0-1.5 umol/L].
    Measured
    fluorescence with Tecan.

**06/08/21**

*Sulfide-responsive transcriptional repressor approach:*

* Tested the whole cell sensor with (H2S) and design 2BS, measured fluorescence.

**08/08/21**

*Heme protein approach*:

* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

## Week 32

**09/08/21**

*Heme protein approach:*

* Purified labeled myoglobin from 08/08/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution [5 mg/L] and (H2S) dilutions [0, 0.001, 0.01, 0.1, 1, 10, uM].
  Measured
  fluorescence with Tecan
  in addition to a kinetic study.* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**10/08/21**

*Heme protein approach:*

* Purified labeled myoglobin from 09/08/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution [5 and 25 mg/L] and (H2S) dilutions [0, 0.001, 0.01, 0.1, 1, 10,
  uM].
  Measured fluorescence with
  Tecan.* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**11/08/21**

*Heme protein approach:*

* Purified labeled myoglobin from 10/08/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution [1, 5, 10, 20, 40, mg/L] and (H2S) dilutions [0.1 uM]. Measured
  fluorescence
  with Tecan in
  addition to a kinetic study.

**15/08/21**

*Heme protein approach:*

* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

## Week 33

**16/08/21**

*Heme protein approach:*

* Purified labeled myoglobin from 15/08/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution [1, 5, 10, 20, 40 mg/L] and (H2S) dilutions [1 uM]. Measured
  fluorescence
  with Tecan in addition
  to a kinetic study.* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**17/08/21**

*Heme protein approach:*

* Purified labeled myoglobin from 16/08/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein solution [1, 5, 10, 20, 40 mg/L] and (H2S) dilutions [0.1 uM].
  Measured
  fluorescence with Tecan.* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**18/08/21**

*Heme protein approach:*

* Purified labeled myoglobin from 17/08/21, measured myoglobin concentration and labeling
  ratio, prepared labeled
  protein dilutions and (H2S) dilutions. Tested two different kinetic studies.
  The first
  with labeled protein [1, 5,
  10 and 20 mg/L] and (H2S) [0.1 uM], and another with labeled protein [5 mg/L]
  and (H2S)
  [0.001, 0.01, 1.0 and 2.0
  uM].

## Week 36

**10/09/21**

*Heme protein approach:*

* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**11/09/21**

*Heme protein approach:*

* Purified labeled myoglobin from 10/09/21, measured myoglobin concentration and labeling ratio,
  prepared labeled
  protein solution [10 mg/L] and (H2S) dilutions [1 uM].

  Performed a kinetic study where different time intervals ( 3 sec, 30 sec, 1 minute and 5
  minutes).

  *Improvement of an existing part:*

  + Started the cloning process (PCR, PCR gel analysis and Dpn1 enzyme digest).

## Week 37

**13/07/21**

*Improvement of an existing part:*

* Picked two colonies of each sample from 11/07/21, and inoculated in LB/amp overnight.

**14/07/21**

*Improvement of an existing part:*

* Transferred bacteria cell culture into LB/amp, incubated from 08:45 to 12:45. Diluted IPTG to 40
  and 400 uM in
  bacteria culture, and incubated 3-5 hours in 37 degrees. Tested bacteria with IPTG and a control
  (bacteria
  without IPTG) with Tecan.* Miniprep and sequencing.

**15/07/21**

*Improvement of an existing part:*

* Made negative control culture for UV and fluorescence measurements.

**16/07/21**

*Improvement of an existing part:*

* Measured fluorescence intensity and recorded UV images of grown cultures.

## Week 40

**05/10/21**

*Heme protein approach:*

* Labeled myoglobin with Cy3 NHS ester, incubated overnight.

**06/10/21**

*Heme protein approach, implemented in Lab on a Chip:*

* Purified labeled myoglobin from 05/10/21, measured myoglobin concentration and labeling ratio,
  prepared labeled
  protein solution [10 mg/L] and (H2S) dilutions [0.1, 0.5 and 1 uM]. Lab on a Chip was
  used to
  measure the
  fluorescence.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NTNU-Trondheim/Engineering

SulFind 2021

[![](https://static.igem.org/mediawiki/2021/0/02/T--NTNU-Trondheim--Logo_ensfarget.png)](https://2021.igem.org/Team%3ANTNU-Trondheim)

PROJECT

[Project Description](https://2021.igem.org/Team%3ANTNU-Trondheim/Description)
[Implementation](https://2021.igem.org/Team%3ANTNU-Trondheim/Implementation)
[Contribution](https://2021.igem.org/Team%3ANTNU-Trondheim/Contribution)
[Safety](https://2021.igem.org/Team%3ANTNU-Trondheim/Safety)
[Engineering](https://2021.igem.org/Team%3ANTNU-Trondheim/Engineering)

LAB

[Experiments](https://2021.igem.org/Team%3ANTNU-Trondheim/Experiments)
[Results](https://2021.igem.org/Team%3ANTNU-Trondheim/Results)
[Notebook](https://2021.igem.org/Team%3ANTNU-Trondheim/Notebook)

PARTS

[Improvement](https://2021.igem.org/Team%3ANTNU-Trondheim/Improve)
[Parts](https://2021.igem.org/Team%3ANTNU-Trondheim/Parts)

HUMAN PRACTICES

[Human Practices](https://2021.igem.org/Team%3ANTNU-Trondheim/Human_Practices)
[Communication](https://2021.igem.org/Team%3ANTNU-Trondheim/Communication)
[Education](https://2021.igem.org/Team%3ANTNU-Trondheim/Education)

TEAM

[Team members and project sponsors](https://2021.igem.org/Team%3ANTNU-Trondheim/Team)
[Attributions](https://2021.igem.org/Team%3ANTNU-Trondheim/Attributions)
[Collaboration](https://2021.igem.org/Team%3ANTNU-Trondheim/Collaborations)
[Partnership](https://2021.igem.org/Team%3ANTNU-Trondheim/Partnership)

![](https://static.igem.org/mediawiki/2021/e/e9/T--NTNU-Trondheim--Banner_Engineering.jpeg)

Several approaches were considered when developing a biosensor with adequate sensing capability for detecting
hydrogen sulfide H2S. After extensive research the two most promising approaches were brought into
the lab to be tested. One of the ideas was a whole cell sensor, the other was a molecular sensor with heme
proteins implemented in a microfluidic chip. Both projects went through the engineering cycle, illustrated in
Figure 1, which consists of the following steps: design, build, test and learn. On this page we describe the
engineering cycle for both projects.

![alt_text](https://static.igem.org/mediawiki/2021/thumb/3/39/T--NTNU-Trondheim--engineering_cycle.png/603px-T--NTNU-Trondheim--engineering_cycle.png "image_tooltip")

**Figure 1**: The four steps of the engineering cycle.

## Engineering of heme-based sensor

We were interested in designing a molecular sensor because it offers immediate and selective detection. These
are some of the most important sensor qualities because H2S-levels in the water tanks can rise
quickly. In our initial research phase we investigated several approaches to using heme-proteins as a basis for
a molecular H2S sensor. This was largely motivated by the known interactions between many
heme-proteins and H2S, such as with cytochrome c oxidase, hemoglobin and myoglobin [1]. This
interaction is non-covalent and reversible, which offers a possibility for continuous detection. Early on, we
considered the use of cantilevers to detect interaction in a heme-protein with high H2S-affinity, as
well as variations on heme-based oxygen sensors. As we continued our investigation, we discovered a seemingly
more robust way of detecting H2S-heme interactions, that is by using myoglobin conjugated with a
fluorophore [2].

After deciding to use myoglobin as the basis of our sensor, we needed to find a way to create a detectable
signal for when H2S binds to the protein. For myoglobin, this is possible using the principle of
Förster Resonance Energy Transfer, also known as FRET. This is a method commonly used to study protein
interactions based on an energy transfer that occurs between an energy donor and acceptor, usually fluorophores,
in close proximity [3]. This energy transfer produces a fluorescent signal that can be detected. In our sensor
system, we can use FRET to detect H2S-binding because the absorption spectrum of myoglobin shifts
when interacting with H2S [4]. The particular method of energy transfer we implemented is called
absorption into fluorescence, and is achieved by labeling the N-terminus of myoglobin with a fluorophore that
has an emission peak corresponding to an area of interest in the absorption spectra of myoglobin [5]. This area
of interest can be seen in Figure 2 around 550nm. When exciting the fluorophore, energy from the fluorescence
will be absorbed into the myoglobin, and therefore changes in the absorption can be detected by fluorescence.

![alt_text](https://static.igem.org/mediawiki/2021/thumb/b/bc/T--NTNU-Trondheim--metMb_Absorption.png/647px-T--NTNU-Trondheim--metMb_Absorption.png "image_tooltip")

**Figure 2:** Absorption spectrum of ferric myoglobin, before and
after addition and reaction with Na2S. The line in black shows the absorption spectrum before the
reaction, and the blue after the reaction. The area of interest for our heme-based H2S sensor is the
hump around 550 nm, where there is an increase in absorption for the reaction between ferric myoglobin and
H2S. *Figure from (Jensen & Fago, 2018) [6]*

Our main objective in the testing phase for our myoglobin-fluorophore approach was to investigate the optimal
concentrations of fluorophore-bound myoglobin using different H2S concentrations. The first stages of
testing were done outside of the microfludic chip to assess the different sensor parameters. As presented in our
[Results Page](https://2021.igem.org/Team%3ANTNU-Trondheim/Results)higher
fluorophore-myoglobin concentrations seemed to increase the variance in the measurements. Additionally,
we found lower variance when slightly increasing the distance between the excitation and emission measurement
bands used for measuring the fluorescence signal in the myoglobin-fluorophore. Correcting for these factors, we
managed to get a signal at very low H2S concentrations. However, there were still some issues with
variance, lack of a general trend and linearity, potentially caused by different factors which are further
described in the discussion segment of our
[Results Page](https://2021.igem.org/Team%3ANTNU-Trondheim/Results). The main factors
here were the use of non-isolated measurement wells, no degassing and potential oversaturation
of the reaction mixture. From our testing, we learned that the further development of this approach would
therefore require a fluorescence measurement instrument with closed measurement wells or chambers, to prevent
H2S diffusion, and a degasser to deoxygenate the sample before and after to prevent H2S
oxidation. This would however require more resources than currently available to us, but it seems probable that
this would increase the effectiveness of the sensor significantly. On the other hand, these limitations could be
reduced when moving the sensor system to a microfluidic chip.

## Design of Microfluidic Chip

The design of our microfluidic chip was a quite simple one, and it was for a flow focusing device. It consisted
of three fluid inlets; one for the myoglobin solution, one for the H2S solution and one final inlet
for the carrier oil. In the first phase of the microfluidic chip was the droplet generator (Figure 3). There,
the inlets for the myoglobin solution and the H2S solution merged together. Right afterwards this new
mix flowed past the carrier oil inlet, making individual and separated droplets from the flow. The second part
of the chip was the mixing phase, where a mixing channel in a zigzag shape ensured proper mixing of myoglobin
and H2S to make sure fluorescence would occur (Figure 4). Last phase was the collection chamber, a
linearly expanding channel that caught all of the droplets within it. These droplets would then be collected and
the fluorescence measured off-chip (Figure 5).

Here are some of our parameters for the flow setup:

* Flow rate of heme solution = 30 µl/hr* Flow rate of H2S solution = 30 µl/hr* Flow rate oil phase = 250 µl/hr* Droplet generation frequency = 400 Hz* Droplet volume = 41 pl

![alt_text](https://static.igem.org/mediawiki/2021/thumb/b/bf/T--NTNU-Trondheim--Merging_microfluidic.gif/800px-T--NTNU-Trondheim--Merging_microfluidic.gif "image_tooltip")

**Figure 3:** Droplet generator. The two first inlets are for
the myoglobin and H2S solution respectively. These merge and meet the carrier oil, making droplets.

![alt_text](https://static.igem.org/mediawiki/2021/4/4b/T--NTNU-Trondheim--Mixing_microfluidic.gif "image_tooltip")

**Figure 4:** Mixing channel to ensure proper mixing for myoglobin and H2S.

## ![alt_text](https://static.igem.org/mediawiki/2021/3/38/T--NTNU-Trondheim--Collection_microfluidic.gif "image_tooltip")

**Figure 5:** Collection chamber for the droplets.

## The SQR approach

We began our research by looking into sulfide-binding molecules, and came across the transcriptional repressor
protein SqrR, which showed promise as a component in a whole-cell sulfide sensor. This repressor protein and the
way it functions in transcriptional regulation became the foundation for the “SQR” part of our project. Even
though we ultimately ended up ending the SQR approach early to focus on the Heme approach and microfluidic chip
design, we went through one full iteration of the engineering cycle, **design**ing genetic
constructs that would act as sulfide sensors, **build**ing the designs by cloning the constructs
into bacteria, **test**ing our sensors with varying levels of H2S,
**learn**ing from our mistakes, and **design**ing new constructs.

**Design → Build → Test → Learn → Design**

SqrR is a repressor protein that is involved in sulfide-dependent gene expression in the bacterium
*Rhodobacter capsulatus* [7]. When no sulfide is present, SqrR binds to the Psqr promoter, inhibiting the
expression of the gene encoding Sulfide:quinone oxidoreductase (SQR). SQR is an enzyme that oxidises sulfide to
zero-valent sulfur (S0), which can then conjugate with multiple different nucleophiles or low molecular weight
thiols to form reactive sulfur species (RSS) [7]. Upon reaction with a RSS, a di-, tri-, or tetrasulfide bond
forms between two cysteine residues of SqrR, resulting in a conformational change that reduces its DNA binding
affinity. RSS thereby functions as inducers of the genes under control of the Psqr promoter that is repressed by
SqrR [7].

### Design 1.0

We made two different genetic designs that utilize the SqrR protein. The first insert was designed to be modular
and allow for the steps of sulfide oxidation, and RSS sensing to be separated on a microfluidic chip. First
H2S would be reacted with Glutathione disulfide (GSSG), so that glutathione persulfide (GSSH), a RSS,
is formed. The GSSH would then diffuse into engineered cells with the genetic insert, where SqrR is inhibited,
resulting in expression of the reporter gene. This process is illustrated in Figure 6. The insert consisted of a
fluorescent reporter gene placed under control of the Psqr promoter, *sqrR*, the gene that encodes SqrR,
placed under control of a constitutive promoter, as well as biobrick ribosomal binding sites and terminators.
The insert design is illustrated in Figure 7.

![alt_text](https://static.igem.org/mediawiki/2021/thumb/c/c5/T--NTNU-Trondheim--design_1_h2s_detection.png/800px-T--NTNU-Trondheim--design_1_h2s_detection.png "image_tooltip")

**Figure 6**: Schematic representation of hydrogen sulfide
detection by the GSSG-dependent design.

![alt_text](https://static.igem.org/mediawiki/2021/thumb/a/a1/T--NTNU-Trondheim--pigeon_design_1.png/800px-T--NTNU-Trondheim--pigeon_design_1.png "image_tooltip")

**Figure 7**: Components of composite parts for hydrogen sulfide
detection by the GSSG-dependent design. *Figure designed using Pigeon [8].*]

Three different inserts were designed like this. In two of the parts, CreiLov was used as a reporter gene.
CreiLov is a flavin-binding fluorescent protein that does not require oxygen for fluorescence, and can therefore
be used in anaerobic environments [9]. The protein was used so that integration into a microfluidic chip might
be feasible. We also varied the constitutive promoter used for SqrR expression, so that the relative strength of
the promoters were different. This was done because we suspected that the expression level of SqrR would affect
the sensitivity of the sensor.

We also designed inserts that would function as a whole-cell sensor without the need for any additional
components. In this design, the bacterium would also express the SQR enzyme, and therefore oxidise
H2S inside the cell. The process is illustrated schematically in Figure 8, and the part design is
shown in Figure 9. These designs were in part inspired by the Nanjing-China iGEM team in 2017. Link to their
design is
[here.](https://2017.igem.org/Team%3ANanjing-China/Design)

![alt_text](https://static.igem.org/mediawiki/2021/thumb/c/cc/T--NTNU-Trondheim--design_2_h2s_detection.png/800px-T--NTNU-Trondheim--design_2_h2s_detection.png "image_tooltip")

**Figure 8**: Schematic representation of hydrogen sulfide
detection by the whole-cell design.

![alt_text](https://static.igem.org/mediawiki/2021/thumb/8/84/T--NTNU-Trondheim--pigeon_design_2.png/798px-T--NTNU-Trondheim--pigeon_design_2.png "image_tooltip")

**Figure 9**: Components of composite parts for hydrogen sulfide
detection by the whole-cell design. *Figure designed using Pigeon [8]*.]

As with the previous design, we also made variants where the reporter protein was either CreiLov or GFP, and the
strength of the constitutive promoters varied.

Read more about the design on our
[Parts Page.](https://2021.igem.org/Team%3ANTNU-Trondheim/Parts)

### Build

We ordered all the designs fully synthesized as linear fragments from Integrated DNA Technologies (IDT). IDT was
unable to synthesize the construct containing both SQR and GFP encoding genes, likely because of the length of
the construct.

We inserted the constructs into the plasmid vector pENZ004, a plasmid with a mid-range copy-number and Amp
resistance genes, and used Gibson assembly to clone the vector into *E. coli* DH5-alpha cells. We then
confirmed the success of the cloning by sequencing, to confirm the presence of our constructs in the bacterial
plasmids. The entire procedure, as well as troubleshooting steps, is detailed on our
[Lab Notebook Page.](https://2021.igem.org/Team%3ANTNU-Trondheim/Notebook)

We were unable to successfully clone one of the constructs for the whole-cell design. After doing some
troubleshooting, and attempting several Gibson assemblies and transformations we decided to move on with testing
the four constructs that were successfully cloned into our bacteria, so that we would have sufficient time for
testing.

Read more about the cloning procedure on our [Results Page.](https://2021.igem.org/Team%3ANTNU-Trondheim/Results)

### Test

The bacteria transformed with the GSSH-dependant construct were tested by measuring the absorbance and
fluorescence of transformed bacteria on a Tecan plate reader after incubating the bacteria with varying
concentrations of freshly prepared GSSH (a RSS). The bacteria containing the SQR-construct was tested by
measuring the absorbance and fluorescence of transformed bacteria on a Tecan plate reader after incubating the
bacteria with varying concentrations of freshly dissolved Sodium sulfide (Na2S). Fluorescence was
measured at 450 nm for excitation and 495 nm emission for the CreiLov parts, and 475 nm for excitation and 545
nm emission for the GFP parts.

We tested the design with the following concentrations of H2S ranging from 0 to 1000 ug/L, based on
results we received from Carlo Lazado about the H2S levels dangerous to fish. Read more about this on
our
[Human Practises Page.](https://2021.igem.org/Team%3ANTNU-Trondheim/Human_Practices)

We found no significant difference in observed fluorescence between the bacteria that had been incubated with
GSSH or H2S and the control. The fluorescence measurements are presented on our [Results Page.](https://2021.igem.org/Team%3ANTNU-Trondheim/Results)

### Learn

We troubleshooted our experiment design in the lab by varying the medium the bacteria were grown in, increasing
the H2S concentrations, and treating the bacteria with GSSH/H2S earlier in their growth
phase. Time did not allow further troubleshooting in the lab, as we needed to focus on the Heme and microfluidic
chip part of our project, which were showing more promise.

Our advisor Alex raised the topic of whether GSSH would be able to cross the cell membrane. The molecule is too
big to diffuse across the cell membrane. This is a major flaw in our design, and made us reconsider the modular
approach where sulfide is oxidised outside the cell.

Upon further analysis of the genetic sequences we had ordered from IDT we became aware of two mistakes. When
inserting the one of the biobrick RBS (Part:BBa\_B0030), was accidentally copied from the registry as dsDNA
instead of ssDNA, resulting in the sequence being inserted twice and inverted. This could likely have an effect
on the ribosome binding to the transcript. Perhaps more disastrous, we had removed upstream purification and
cleavage sites from the CreiLov gene, and omitted inserting an ATG codon back into the sequence. These mistakes
occurred because we were pressed for time when ordering our constructs, but are unfortunate and could have been
prevented if more time was allocated for double-checking our constructs.

### Design 2.0

Even though we did not have time to order and test new genetic constructs, we made a second design of the
construct where SQR is expressed within the cell, as this approach alleviates the need for GSSH crossing a cell
membrane. We fixed the mistakes with the RBS and the missing start codon for CreiLov in the construct where SQR
is expressed in addition to the sensor sequences. In addition, we also changed a part of DNA at the beginning of
the sequence that could form hairpin structures, and potentially caused problems with the PCR primers.

If time had permitted, we would have ordered this improved design, and tested in the lab. We would order the
part as smaller parts that we could assemble, so that it would be less likely that problems would occur during
synthesis. We would also have tested CreiLov alone with a constitutive promoter, to add another layer of
modularity when troubleshooting.

#### References

1. Pietri, R., E. Román-Morales, and J. López-Garriga, *Hydrogen sulfide and hemeproteins: knowledge and
mysteries.* Antioxid Redox Signal, 2011. **15**(2): p. 393-404.

2. Strianese, M., et al., *Myoglobin as a new fluorescence probe to sense* H2S*.*
Protein Pept Lett, 2011. **18**(3): p. 282-6.

3. Sekar, R.B. and A. Periasamy, *Fluorescence resonance energy transfer (FRET) microscopy imaging of live
cell protein localizations.* J Cell Biol, 2003. **160**(5): p. 629-33.

4. Nicholls, P., *The formation and properties of sulphmyoglobin and sulphcatalase.* Biochem J, 1961.
**81**(2): p. 374-83.

5. Strianese, M., et al., *Absorption into fluorescence. A method to sense biologically relevant gas
molecules.* Nanoscale, 2011. **3**(1): p. 298-302.

6. Jensen, B. and A. Fago, *Reactions of ferric hemoglobin and myoglobin with hydrogen sulfide under
physiological conditions.* Journal of Inorganic Biochemistry, 2018. **182**: p. 133-140.

7. Shimizu, T., et al., *Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of
sulfide-dependent photosynthesis.* Proc Natl Acad Sci U S A, 2017. **114**(9): p.
2355-2360.

8. Bhatia, S. and D. Densmore, *Pigeon: A Design Visualizer for Synthetic Biology.* ACS Synthetic
Biology, 2013. **2**(6): p. 348-350.

9. Zou, W., K. Le, and M.L. Zastrow, *Live-Cell Copper-Induced Fluorescence Quenching of the Flavin-Binding
Fluorescent Protein CreiLOV.* ChemBioChem, 2020. **21**(9): p. 1356-1363.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NTNU-Trondheim/Experiments

SulFind 2021

[![](https://static.igem.org/mediawiki/2021/0/02/T--NTNU-Trondheim--Logo_ensfarget.png)](https://2021.igem.org/Team%3ANTNU-Trondheim)

PROJECT

[Project Description](https://2021.igem.org/Team%3ANTNU-Trondheim/Description)
[Implementation](https://2021.igem.org/Team%3ANTNU-Trondheim/Implementation)
[Contribution](https://2021.igem.org/Team%3ANTNU-Trondheim/Contribution)
[Safety](https://2021.igem.org/Team%3ANTNU-Trondheim/Safety)
[Engineering](https://2021.igem.org/Team%3ANTNU-Trondheim/Engineering)

LAB

[Experiments](https://2021.igem.org/Team%3ANTNU-Trondheim/Experiments)
[Results](https://2021.igem.org/Team%3ANTNU-Trondheim/Results)
[Notebook](https://2021.igem.org/Team%3ANTNU-Trondheim/Notebook)

PARTS

[Improvement](https://2021.igem.org/Team%3ANTNU-Trondheim/Improve)
[Parts](https://2021.igem.org/Team%3ANTNU-Trondheim/Parts)

HUMAN PRACTICES

[Human Practices](https://2021.igem.org/Team%3ANTNU-Trondheim/Human_Practices)
[Communication](https://2021.igem.org/Team%3ANTNU-Trondheim/Communication)
[Education](https://2021.igem.org/Team%3ANTNU-Trondheim/Education)

TEAM

[Team members and project sponsors](https://2021.igem.org/Team%3ANTNU-Trondheim/Team)
[Attributions](https://2021.igem.org/Team%3ANTNU-Trondheim/Attributions)
[Collaboration](https://2021.igem.org/Team%3ANTNU-Trondheim/Collaborations)
[Partnership](https://2021.igem.org/Team%3ANTNU-Trondheim/Partnership)

![](https://static.igem.org/mediawiki/2021/1/1e/T--NTNU-Trondheim--Team.jpg)

Protocols for all the various techniques and procedures are presented in detail in this section. The
protocols
are divided into three parts, one set of protocols for the heme approach, one set for the
sulfide-responsive
transcriptional repressor and protocols used by both approaches.

## Heme Protein Approach

* [Preparing potassium phosphate buffer (PPB) and stock solutions](https://static.igem.org/mediawiki/2021/4/4a/T--NTNU-Trondheim--preparing_potassium.pdf)* [Detecting
    (H2S) with labeled myoglobin](https://static.igem.org/mediawiki/2021/e/e1/T--NTNU-Trondheim--det.pdf)

## Sulfide-Responsive Transcriptional Repressor Approach

* [Sulfide
  and GSSH solution](https://static.igem.org/mediawiki/2021/f/f7/T--NTNU-Trondheim--sulfide_gssh.pdf)* [Sulfide solution for direct contact with bacteria](https://static.igem.org/mediawiki/2021/0/04/T--NTNU-Trondheim--sulfide_bacteria.pdf)

* [Polymerase chain
  reaction](https://static.igem.org/mediawiki/2021/c/c6/T--NTNU-Trondheim--PCR.pdf)* [Gel
    analysis](https://static.igem.org/mediawiki/2021/7/7d/T--NTNU-Trondheim--gel.pdf)* [Dpn1
      enzyme
      digest](https://static.igem.org/mediawiki/2021/c/c1/T--NTNU-Trondheim--dpn1.pdf)* [PCR
        purification](https://static.igem.org/mediawiki/2021/e/e8/T--NTNU-Trondheim--PCR_purification.pdf)* [Gibson
          assembly](https://static.igem.org/mediawiki/2021/b/bf/T--NTNU-Trondheim--gibson.pdf)* [Heat
            shock transformation](https://static.igem.org/mediawiki/2021/d/d2/T--NTNU-Trondheim--heat_shock.pdf)* [Isolation of plasmid DNA](https://static.igem.org/mediawiki/2021/2/2e/T--NTNU-Trondheim--isolation_of_DNA.pdf)* [Enzyme restriction and gel analysis](https://static.igem.org/mediawiki/2021/1/1c/T--NTNU-Trondheim--restriction_digest_analysis.pdf)* [Sanger
                  sequencing](https://static.igem.org/mediawiki/2021/3/38/T--NTNU-Trondheim--sanger.pdf)

## Protocols used by both approaches

- [Methylene
  blue method](https://static.igem.org/mediawiki/2021/8/8c/T--NTNU-Trondheim--Metylen.pdf)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NTNU-Trondheim/Results

SulFind 2021

[![](https://static.igem.org/mediawiki/2021/0/02/T--NTNU-Trondheim--Logo_ensfarget.png)](https://2021.igem.org/Team%3ANTNU-Trondheim)

PROJECT

[Project Description](https://2021.igem.org/Team%3ANTNU-Trondheim/Description)
[Implementation](https://2021.igem.org/Team%3ANTNU-Trondheim/Implementation)
[Contribution](https://2021.igem.org/Team%3ANTNU-Trondheim/Contribution)
[Safety](https://2021.igem.org/Team%3ANTNU-Trondheim/Safety)
[Engineering](https://2021.igem.org/Team%3ANTNU-Trondheim/Engineering)

LAB

[Experiments](https://2021.igem.org/Team%3ANTNU-Trondheim/Experiments)
[Results](https://2021.igem.org/Team%3ANTNU-Trondheim/Results)
[Notebook](https://2021.igem.org/Team%3ANTNU-Trondheim/Notebook)

PARTS

[Improvement](https://2021.igem.org/Team%3ANTNU-Trondheim/Improve)
[Parts](https://2021.igem.org/Team%3ANTNU-Trondheim/Parts)

HUMAN PRACTICES

[Human Practices](https://2021.igem.org/Team%3ANTNU-Trondheim/Human_Practices)
[Communication](https://2021.igem.org/Team%3ANTNU-Trondheim/Communication)
[Education](https://2021.igem.org/Team%3ANTNU-Trondheim/Education)

TEAM

[Team members and project sponsors](https://2021.igem.org/Team%3ANTNU-Trondheim/Team)
[Attributions](https://2021.igem.org/Team%3ANTNU-Trondheim/Attributions)
[Collaboration](https://2021.igem.org/Team%3ANTNU-Trondheim/Collaborations)
[Partnership](https://2021.igem.org/Team%3ANTNU-Trondheim/Partnership)

![](https://static.igem.org/mediawiki/2021/9/9b/T--NTNU-Trondheim--Banner_Results.jpeg)

## Heme approach results

### Experiment overview

We performed 16 experiments, which can be divided into three categories based on what variable was explored.
This includes varying H2S concentrations, varying protein concentrations and kinetic studies
(varying the time)
with 2-3 parallels. All experiments involved the observation of the reaction between H2S and
N-terminus
Cy3-tagged myoglobin, using fluorescence intensity (FI) measurements. Additionally, two emission/excitation
scans were performed to find the optimal excitation and emission bands for these measurements. The
instrument
used for all fluorescence intensity measurements was the Tecan Infinite 200Pro.

### Initial results

In the graph below are the results of experiment 1. Here we measured using 5 mg/L of labeled myoglobin-Cy3
reaction mixture, and varying H2S concentrations around the level which is supposed to be
unhealthy, not deadly,
for fish in RAS. For the measurement we used the optimal excitation/emission wavelengths for our
fluorophore,
Cy3, which is 550nm and 570nm. As you may see, these results are fairly unpromising, as there is high
variance
and no discernible trend. Further, we would expect our 0-value to be the highest, as the presence of
H2S should
lower the fluorescence intensity of the reaction mixture. However, we got odd values for our blank wells
(which
is just water and buffer), at around 50000 FI, much higher than all the values for the measurement points.

![alt_text](https://static.igem.org/mediawiki/2021/c/cd/T--NTNU-Trondheim--new_figure1.png "image_tooltip")

**Figure 1:** Fluorescence intensity measurements for varying H2S
concentrations using 5
mg/L labeled protein reaction mixture. Measurement parameters were 550nm for excitation, 570nm for emission
and
with 60 gain.

### Excitation and emission measurements

Due to the high variance and high FI values for the blank measurements from our initial results, we
suspected
that the excitation/emission parameters might be too close. The instrument we used for fluorescence
measurement
uses a 9nm wide band for emission measurement, and a 20nm band for excitation. Since our chosen
excitation/emission peaks for the first experiment were 550/570, the strange values might be caused by
spectral
overlap or poor calibration of the instrument. To investigate further we performed excitation and emission
scans
for the respective emission and excitation values. The results for these scans can be found in figure 2, for
the
excitation scan, and figure 3, for the emission scan.

![alt_text](https://static.igem.org/mediawiki/2021/d/df/T--NTNU-Trondheim--new_figure2.png "image_tooltip")

**Figure 2:** Excitation scan of 5 mg/L reaction mixture (1) and 25 mg/L reaction mixture (2),
as
well as control solutions containing the corresponding myoglobin concentrations and blank solutions.
Measurement
parameters were 520-560nm in 2nm increments for excitation (only 520-544 is shown in the figure due
overreading), 570nm for emission and with 60 gain.

![alt_text](https://static.igem.org/mediawiki/2021/3/3d/T--NTNU-Trondheim--new_figure3.png "image_tooltip")

**Figure 3:** Emission scan of 5 mg/L reaction mixture (1) and 25 mg/L reaction mixture (2), as
well as control solutions containing the corresponding myoglobin concentrations and blank solutions.
Measurement
parameters were 550nm for excitation, 560-600nm in 2nm increments for emission (only 576-600nm is shown in
the
figure due to overreading), and with 60 gain.

From these results, we can see that there is a large increase in fluorescence intensity when measuring
emission
at 570nm and exciting higher than 545nm, and likewise there is an increase when exciting at 550nm and
measuring
emission lower than 575nm (it is important to note that for the emission values the real area is ±4.5nm, and
for
excitation it’s ±10nm). For the other values we can see that the reaction mixture measurements have the
highest
fluorescence intensity, so ideally we want to measure in this area. Using this, we decided to measure with
excitation/emission pairs such as 540nm/570nm, 540nm/580nm and 550nm/580nm for the next few experiments. We
then
saw that 540nm/580nm had the lowest variance of the three pairs, and similar results, so we then decided to
use
only 540nm/580nm for practical reasons. Due to this, the rest of the results will be from the 540nm/580nm
data.

### Further results

Following our excitation/emission scans, we performed the measurements for experiment 2 using our new
measurement parameters, and got better results. This time, our blank measurements were close to zero, our
zero-measurements were the highest, there was lower overall variance and we got somewhat of a better trend,
though not perfect. We used a wider range of H2S concentrations for this experiment, which at the
higher values
has a large molar excess over the 5 mg/L reaction mixture (around 42 times for 25 micromolar of
H2S)

![alt_text](https://static.igem.org/mediawiki/2021/e/e9/T--NTNU-Trondheim--new_figure4.png "image_tooltip")

**Figure 4:** Fluorescence intensity measurements for varying H2S concentrations
using
5 mg/L bound
protein reaction mixture. Measurement parameters were 540nm for excitation, 580nm for emission and with 120
gain.

Using the three parallels from each measurement, we set up a two tailed T-test table to study the
statistical
significance of the data. This is shown in table 1, and is color coded such that any value below 0.025 is a
deep
green, above 0.050 yellow and red towards 1. Here we can see that we have good statistical significance for
the
difference between the zero-measurements and 1uM measurements, as well as between zero and 5. There is
however
very low significance between 1uM and 5uM, which makes sense since their means are so close. Additionally,
there
is good significance between 5um and 10um, but in the opposite direction from what we’d prefer (increasing
FI by
concentration instead of decreasing).

**Table 1:** Pairwise results of two-tailed
T-test
for each of the
measurements

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| T-test | Blank | 0 uM | 1 uM | 5 uM | 10 uM | 25 uM |
| Blank | x | 0.0000 | 0.0014 | 0.0000 | 0.0003 | 0.0007 |
| 0 uM | 0.0000 | x | 0.0240 | 0.0009 | 0.0565 | 0.0864 |
| 1 uM | 0.0014 | 0.0240 | x | 0.5457 | 0.1568 | 0.1885 |
| 5 uM | 0.0000 | 0.0009 | 0.5457 | x | 0.0160 | 0.0401 |
| 10 uM | 0.0003 | 0.0565 | 0.1568 | 0.0160 | x | 0.6086 |
| 25 uM | 0.0007 | 0.0864 | 0.1885 | 0.0401 | 0.6086 | x |

Due to the unwanted upwards trend towards the higher values, we speculated that the large molar excess could
be
over-saturating the reaction mixture, so in experiment 3 we tried using a higher mixture concentration (25
mg/L)
and a narrower/lower range of H2S concentrations (in the ranges that was deadly for fish in RAS).
As you can see
in figure 5, this did not go well. We got very high variance, no discernible trend and the zero-measurement
wasn’t significantly higher than the measurements with H2S.

![alt_text](https://static.igem.org/mediawiki/2021/7/74/T--NTNU-Trondheim--new_figure5.png "image_tooltip")

**Figure 5:** Fluorescence intensity measurements for varying H2S concentrations
using
25 mg/L
bound protein reaction mixture. Measurement parameters were 540nm for excitation, 580nm for emission and
with
100 gain.

We then lowered the protein concentration back to 5 mg/L, and performed a series of experiments with
different
H2S ranges, trying to determine a more or less linear range for this protein concentration. In
figure 6 and 7 we
see the results of experiment 5 and 6, which both had similar trends as in experiment 2, where there is
first a
dip in fluorescence intensity at lower concentrations then an increase. In both experiment 5 and 6 the
measurements for the first H2S value, 0.1uM and 0.001uM respectively, gave a statistical
significant change in
FI from the zero-measurements.

![alt_text](https://static.igem.org/mediawiki/2021/9/9a/T--NTNU-Trondheim--new_figure6.png "image_tooltip")

**Figure 6:** Fluorescence intensity measurements for varying H2S concentrations
using
5 mg/L bound
protein reaction mixture. Measurement parameters were 540nm for excitation, 580nm for emission and with 120
gain.

![alt_text](https://static.igem.org/mediawiki/2021/c/c6/T--NTNU-Trondheim--new_figure7.png "image_tooltip")

**Figure 7:** Fluorescence intensity measurements for varying H2S concentrations
using
5 mg/L bound
protein reaction mixture. Measurement parameters were 540nm for excitation, 580nm for emission and with 120
gain.

### Investigations:

Following these experiments, we tried investigating different protein concentrations, as well as the effect
of
time. In figure 8 we see the results of experiment 8, where the FI of several protein concentrations are
shown,
measured with three parallels at three time-points. As evident, time doesn’t seem to be a large factor in
this
experiment. Additionally, we can observe that the larger the protein concentration the higher the variance,
particularly past 10 mg/L.

![alt_text](https://static.igem.org/mediawiki/2021/b/b1/T--NTNU-Trondheim--new_figure8.png "image_tooltip")

**Figure 8:** Fluorescence intensity measurements for varying reaction mixture concentrations
at
three separate timepoints. Measurement parameters were 540nm for excitation, 580nm for emission and with 100
gain.

To investigate the reaction with H2S further, we performed a series of kinetic experiments. These
measured the
fluorescence intensity of the reaction mixture with and without H2S present over a period of 30
minutes. The
results for one of these experiments, experiment 14, is presented in figure 9. Here we see that in the very
beginning there is good separation between the H2S FI (blue line) and null FI (orange line), but
this shrinks
over time, and at about 1200s the H2S FI is in fact larger than the null FI. An important thing
to
note was that
it was unexpected for the null FI to decrease at all, since this should only happen if the reaction mixture
comes in contact with H2S. This happened in all the kinetic experiments we performed.

![alt_text](https://static.igem.org/mediawiki/2021/f/fe/T--NTNU-Trondheim--new_figure9.png "image_tooltip")

**Figure 9:** Fluorescence intensity measurements for kinetic study over 30 minutes, using 10
mg/L
bound protein reaction mixture and 0.1 uM H2S. Measurement parameters were 540nm for excitation,
580nm for
emission and with 100 gain.

Due to the strange results of the previous kinetic experiments, we decided to investigate whether the light
from
the fluorescence measurements could be degrading either the fluorophore or myoglobin component in the
reaction
mixture, leading to false readings. In experiment 16, we tried checking for this by performing several 30
minute
kinetic measurements, but with varying measurement intervals; 5, 30, 60 and 300 seconds. The results are
presented in figure 10, and here it is evident that with the most frequent kinetic intervals of 5 seconds,
there
was a significant drop in FI compared to less frequent intervals. This is visible when comparing the 5s
measurements the red lines) to the grouping of the other intervals’ measurements. However, even for the less
frequent intervals the null FI is still dropping over time, and though there is better separation over time
this
also shrinks to close to the same FI value for many of the lines.

![alt_text](https://static.igem.org/mediawiki/2021/b/b4/T--NTNU-Trondheim--new_figure10.png "image_tooltip")

**Figure 10:** Fluorescence intensity measurements for kinetic study over 30 minutes, using 10
mg/L
bound protein reaction mixture and 1 uM H2S. The experiment was run several times using different
measurement
intervals, and these intervals are A for 5s, B for 30s, C for 300s and D for 60s.
Measurement parameters were 540nm for excitation, 580nm for emission and with 100 gain.

## Discussion

We managed to get statistically significant differences between null measurements and certain H2S
measurements
with our reaction mixture early on. Beyond that, however, most experiments in our project involved
investigating
different factors that affect the variance of our measurements, and potentially the trend between different
H2S
concentration measurements. Of these, we are fairly certain that light exposure is a big factor in
determining
FI measurements over time, which makes sense given that the fluorophore we used is light sensitive.
Unfortunately, a lot of other important factors were harder for us to test due to our limited resources and
instruments.

However, inferring from the results, we speculate that a lot of the variance could be explained by the free
diffusion of H2S from the open wells into the air inside the fluorescence measurement instrument
and then back
into the null wells. Specifically, the decrease in FI of the null measurements in the kinetic experiments
seems
to indicate this. This speculation is based on H2S being very volatile, and air diffusion within
a
somewhat
enclosed space can distribute H2S in a higher concentration effectively. The diffusion back into
the solution in
the null well should be less effective, but given the sensor having a low enough limit of detection it would
explain the several lines in experiment 16 stabilizing at the same FI value over time. To check if this was
the
case, we would have needed a fluorometric instrument with closed and separated measurement wells .

There are also some other factors that also could affect the variance. Firstly, H2S can be
oxidized
very easily,
which means that in addition to the H2S lost to diffusion, there might be a substantial decrease
in
H2S while
it’s in solution. To prevent this we would have needed a closed system which we would deoxygenate using a
degasser, bubbling argon or nitrogen through. Secondly, the oversaturation of H2S in the reaction
mixture with
respect to a potentially linear measurement area could explain both variance and the lack of a general trend
for
our measurements. Myoglobin has a higher chance to irreversibly bind to H2S when it’s present in
a
relatively
high concentration, which we suspect would not produce a signal (decrease in FI), and could explain the
increase
in FI generally at higher H2S concentrations as seen in experiment 2 and 5. To investigate this
meaningfully
would require us to lower the variance caused by the prior factors, since as we saw in experiment 3 and 8
the
variance seems to increase substantially with increasing the protein concentration.

In order to make sure that H2S was produced in the prepared solutions of Na2S, we used the methylene blue
method. Using this method, we observed a colour change to blue, verifying the presence of H2S. We could
therefore rule out a lack of H2S production as a source of error in our experiment.

## Conclusions

In summary, although we have some results indicating the potential for an H2S sensor with a very
low level of
detection, the variance and lack of trend in important ranges severely inhibit the sensor performance in
other
areas. We however note that improvement may only require better suited hardware. As our proposed
implementation
would be in an LOC, some of the factors affecting variance might be diminished, such as the H2S
diffusion
between measurement wells.

## SQR approach results

On this page the results from the experiments detailed on our
[experiments page](https://2021.igem.org/Team%3ANTNU-Trondheim/Experiments) are
briefly presented.

### Cloning

The five constructs ordered from IDT were cloned into plasmid vectors by Gibson assembly. Next, the plasmids
were transformed into supercompetent *Escherichia coli* cells. Sanger sequencing was used to verify
if
the cloning had been successful. Four out of five designs came out successful.

The plasmid vector, pENZ004, was PCR amplified with primers containing overhangs corresponding to the ends
of
the constructs. Since some of the constructs were identical on either side, three plasmid amplifications
were
made. The PCR gel analysis, shown in figure 1, showed that all three plasmids had been PCR amplified, to the
desired length (ca 2.1 kb).

![alt_text](https://static.igem.org/mediawiki/2021/f/f5/T--NTNU-Trondheim--Results.jpg "image_tooltip")

**Figure 11:** PCR gel analysis of PCR products after pENZ004 is amplified with primers
containing
overhangs for Gibson assembly. 1kb ruler is used on either side of the plasmids.

### Testing

The next step was to test the whole cell sensors. Design 1 was tested with GSSH produced from GSSG and
specific
concentrations of H2S. Design 2 was tested with H2S directly. Exact concentrations and
procedures are found on our [experiments page](https://2021.igem.org/Team%3ANTNU-Trondheim/Experiments)

When exposing the whole cell sensor to GSSH (design 1) and H2S (design 2) the intensity of the
fluorescence signal was expected to increase with increasing H2S concentration. Unfortunately,
none
of the whole cell sensors showed a fluorescence signal when observed under fluorescent light, see figure 2,
or
when fluorescent was measured on a Tecan plate reader, see figure 3.

![alt_text](https://static.igem.org/mediawiki/2021/thumb/8/8d/T--NTNU-Trondheim--Results2.jpg/800px-T--NTNU-Trondheim--Results2.jpg "image_tooltip")

**Figure 12:**  Our whole cell sensors after treatment with GSSH and H2S under UV
light.
On the
right, a control containing only LB medium. There was no sign of fluorescence in the samples.

![alt_text](https://static.igem.org/mediawiki/2021/3/31/T--NTNU-Trondheim--new_figure13.png "image_tooltip")

**Figure 13:**  Measured fluorescence using a H2S sensing whole cell sensor plotted
against H2S concentration. The system is designed to be sensitive for S2- and includes
the
reporter gene CreiLOV and a weaker promoter for SqrR. No trend is observed in the data.

As detailed on our
[engineering page](https://2021.igem.org/Team%3ANTNU-Trondheim/Engineering)
it was discovered that mistakes made during the ordering of our constructs resulted in the CreiLov gene
being
inserted without a start codon. In addition, the ribosome binding site sequence was inserted twice and
inverted,
likely disturbing translation of the constructs. It is also a possibility that the cell membrane is
impermeable
to GSSH due to the size of the molecule.. To improve the approach, an in vitro kit could be utilized. The
boundary of the cell membrane would then be eliminated. If time had permitted we would have liked to fix
these
mistakes and study the approach further. Read more about this on our [engineering page](https://2021.igem.org/Team%3ANTNU-Trondheim/Engineering)