iGEM 2023 — Team dnhs-sandiego-ca

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# Lab Notebook

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# Engineering Design Process

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# Protocols Used

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# Introduction

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After running PCR on and purifying several different parts that we planned
on conducting research with, our team ran the first set of experiments to
compare the success of fluorescent signals of mEmerald and mScarlet with the
MucK clone. Samples in the presence of different concentrations of TPA and
different concentrations of IPTG were assayed.

IPTG is a molecular compound used as an inducer of gene expression from a
promoter linked to the operon of certain bacterial strains. In the absence
of IPTG, the repressor protein binds to the operator region, preventing
downstream transcription of the gene. Therefore, IPTG theoretically must
also be present, in addition to TPA, to allow transcription of the reporter
protein and achieve a fluorescent signal. We expected the fluorescence to
initially increase with greater concentrations of IPTG as more binds to the
repressor, then the increase to gradually taper off as the binding
approaches its saturation point (when all available repressor proteins are
bound to IPTG).

In addition, we expected the fluorescent signal to increase with greater
concentrations of TPA, as the presence of TPA should result in more cellular
processes that theoretically enhance the transcription of the reporter
protein, until the binding reaches a saturation point, and the increasing
fluorescence tapers off.

**Fluorescent Signal of Reporter Proteins**

![](https://static.igem.wiki/teams/4651/wiki/results/results1.png)

*Figure 1. Fluorescent signal of MucK with mScarlet in varying TPA and
IPTG concentrations*

![](https://static.igem.wiki/teams/4651/wiki/results/results2.png)

*Figure 2. Fluorescent signal of MucK with mEmerald in varying TPA and
IPTG concentrations*

The measurements of fluorescence demonstrated the noticeable background
signal in the readings as the signal reaches around 200 in mScarlet with
MucK and around 500 in mEmerald with MucK in the control group with a 0 µM
TPA concentration. This group was predicted to have no fluorescence signal.
Furthermore, theoretically there should be no increase in signal when the
IPTG concentration is 0 µM as IPTG (represented by blue on the graphs) is
required to facilitate TPA production. However, the signal increases from
199.106 to 341.493 with mScarlet and from 537.617 to 846.374 with mEmerald
as TPA concentration increases.

The largest fold increase produced in MucK with mScarlet was around 2.25,
whereas the largest fold increase for MucK with mEmerald was roughly 1.77.
While these were both observable changes, they were not significant enough
increases to achieve our goals in developing a sensitive biosensor for TPA.
However, the results were compared with past literature and the use of the
MucK importer was found to be more successful than previously-tested TpaK
importer.

Building on this experiment, our team decided to use the MucK clone with the
mEmerald reporter protein, but also explored other modifications to improve
the sensitivity of the plasmid. mEmerald was chosen over mScarlet as they
have a negligible difference in increased sensitivity, but mEmerald has a
higher signal production in general, indicating potential for a larger
ceiling value and ease to work with.

# Fluorescent Signals of Promoter Modifications

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Instead of focusing on the reporter protein and importers used, the next set
of experiments focused on modifications to the promoter to increase
sensitivity. Three independent modifications were made to the promoter and
compared: removing a potential second restriction binding site, replacing
the critical -10 and -35 sigma factors of the promoter with the
corresponding sigma factors on pTPA3 (a promoter used in previous papers),
and replacing the entire promoter with pTPA3.

![](https://static.igem.wiki/teams/4651/wiki/results/results3.png)

*Figure 3. Fluorescent signal of each promoter modification with
increasing concentrations of TPA and steady IPTG levels*

The first modification (in blue) of removing a potential second restriction
binding site did not impact the fluorescence signal with increasing levels
of TPA concentration. The third modification (in yellow) of replacing the
promoter with pTPA3 had an inverse relationship with TPA concentration.
Neither of these emit a higher fluorescence signal in the presence of more
TPA, and therefore would not make effective biosensors. However, the second
modification (in red) of exchanging has a positive increasing relationship
with the increasing concentration of TPA, making it the best candidate to
improve sensitivity of the plasmid.

# Proof of Concept

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Theoretically, the lack of IPTG should result in a low fluorescence signal,
even in the presence of TPA, as transcription of the reporter protein is
blocked. To test this, the signal of the promoter with the second
modification was compared between an IPTG and no IPTG environment in
increasing concentrations of TPA.

![](https://static.igem.wiki/teams/4651/wiki/results/results4.png)

*Figure 4. Fluorescent signal of the sigma factor promoter modification in
the presence or not in the presence of IPTG with increasing concentrations
of TPA*

For every concentration of TPA, there is a slightly higher fluorescence
signal in the presence of IPTG than not in the presence of IPTG, indicating
the ability of the biosensor to detect TPA to at least some extent. This
method ensures that the signal detected comes from the presence of TPA
rather than some other substance. Slight increases in the signal in the
no-IPTG environment can be explained by errors in the functionality of the
repressor protein or variability in expression.

![](https://static.igem.wiki/teams/4651/wiki/results/results5.png)

*Figure 5. Fluorescent signal of the sigma factor promoter modification or
mEmerald reporter in the presence or not in the presence of IPTG with
increasing concentrations of TPA*

The data from the first fluorescence assay, regarding the sensitivity of
MucK with the mEmerald reporter, and second assay, regarding the increased
sensitivity of the second modification with sigma factors, were compared
with and without TPA to determine the optimal results for maximum
sensitivity of the sensor. There is a noticeable presence of background
signal as the signal is around 500 even without IPTG. The increase in signal
in groups without IPTG (in blue and yellow) can be explained by errors in
the functionality of the repressor protein or expression of the genes.

There is an increase in sensitivity with both the mEmerald reporter and the
sigma factor promoter modifications previously described, although the
promoter modification seems to output a greater signal overall and has a
greater increase in higher concentrations of TPA. Through these results, we
can see the success and applicability of the parts developed in this
research to detect the presence of TPA and use it as a quantifying indicator
of PET degradation.

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