iGEM 2025 — Team whu-china

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# NOTEBOOK

* [Title](#page-header)
* [Introduction](#introduction)
* [High-pass Notebook](#high-pass-connector)
* [Band-pass Notebook](#band-pass)
* [AHL & Protein Degradation](#ahl-protein-degradation)

# Notebook

WHU-China maintains rigorous experimental records as a fundamental part of our year-round practice.
We consider this documentation essential, both for its utility as a reference for future team
members and the wider community, and as a demonstration of our commitment to academic integrity.
These records are compiled in our wet lab notebooks, which contain detailed PDF accounts of every
iGEM experiment, including protocols, results, and the critical insights that have shaped our
project.

## Calculating: High-pass and Connector Notebook

## Calculating: Band-pass Notebook

## Refreshing: AHL Degradation and Protein Degradation

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# ENGINEERING

* [Title](#page-header)
* [Calculating ▼](#calculating)
  + [Biosensors and high-pass bacteria](#biosensors-high-pass)
  + [Band-pass engineered bacteria](#band-pass-engineered)
  + [Connector bacteria](#connector-bacteria)
* [Refreshing ▼](#refreshing)
  + [Light-inducible AHL degradation enzyme](#light-inducible-ahl)
  + [Light-inducible protein degradation](#light-inducible-protein)
* [References](#references)

# Engineering

The Engineering Design Cycle

![Disc Button]({{ cdn_url('engineering/disc-button-new.png') }})

click here

## Calculating

### Biosensors and high-pass bacteria

Cycles in this section:

* [Cycle 1: Ensure the feasibility of
  diffusion experiments](#biosensors-cycle1-title)
* [Cycle 2: Replace T7 promoter with
  constitutive promoter J23106](#biosensors-cycle2-title)
* [Cycle 3: Use medium-copy-number
  backbone](#biosensors-cycle3-title)
* [Cycle 4: Optimize Tra biosensor
  bacteria](#biosensors-cycle4-title)

#### Cycle 1 Ensure the feasibility of diffusion experiments

##### Design

In order to ensure the feasibility of our AHL diffusion experiments, we used the plasmids LasR-deGFP
and LuxR-deGFP from 2024 WHU\_China to construct high-pass engineered bacteria with fluorescence
induced by 3-oxo-C12 HSL and 3-oxo-C6 HSL.

In this cycle, the expression of the downstream corresponding AHL receptor proteins (LuxR and LasR)
are
activated by the T7
promoter and lac operator. The lac operator ensures that LuxR and LasR are only expressed when
needed, thereby reducing the metabolic burden on the engineered bacteria. The deGFP protein is
induced by the promoters Plux and Plas. (Figure 1-1) When adding different concentrations of AHL,
the
bacteria will express different level of GFP.

![Figure 1-1]({{ cdn_url('engineering/1-1.png') }})

Figure 1-1. The genetic circuits of plasmids LuxR-deGFP and
LasR-deGFP from 2024 WHU-China

##### Build

We transformed the plasmids LasR-deGFP and LuxR-deGFP from 2024 WHU\_China into *E. coli* BL21
(DE3) strain. (Figure 1-2)

![Figure 1-2]({{ cdn_url('engineering/1-2-1.png') }})
![Figure 1-2]({{ cdn_url('engineering/1-2-2.png') }})

Figure 1-2. The
plasmid maps of LuxR-deGFP and LasR-deGFP (From 2024 WHU-China).

##### Test

To verify the response of *E. coli* to the two AHL molecules, 3-oxo-C12 HSL and 3-oxo-C6 HSL,
we
implemented liquid induction and solid diffusion experiments.

In liquid induction experiments, we mixed IPTG-induced bacterial cultures with serially diluted AHLs
in
96-well
plates. OD600 and fluorescence intensity were measured using a microplate reader once an hour
(Figure
1-3).

![Figure 1-3]({{ cdn_url('engineering/1-3.png') }})

Figure 1-3. Response curves of Las-deGFP during 12 hours
incubation
in liquid culture.

Through liquid culture and fluorescence detection, we observed that in the first 2 hours, the
biosensors
could detect different concentrations of AHL. However, as time went on, the bacterial proliferation
became excessive, and the detection effect declined (Figure 1-3).

In agar plate colony experiments, we dispensed the culture and different concentration 3-oxo-C12-HSL
at
the positions shown in Figure 1-4A. After 12h incubation, we detected the fluorescence of the
colonies
with inverted fluorescent microscope (Figure 1-4).

![Figure 1-4]({{ cdn_url('engineering/1-4.png') }})

Figure 1-4. Response curves of 3-oxo-C12 HSL and 3-oxo-C6 HSL
after
12 hours of incubation in agar plate colony experiments. **(A)** The spotting
pattern.
The center of the plate is the place where AHL solution is added. **(B)** Overview
of
the plate. **(C)** The dose response curve of LasR-deGFP engineered strain to
3-oxo-C12
HSL. **(D)** The dose response curve of LuxR-deGFP engineered strain to 3-oxo-C6
HSL.

##### Learn

Biosensor of 3-oxo-C6 HSL does not show a good decline curve, which may be due to the high
concentration of AHL in the short diffusion distance, which disturbs the metabolism of engineered
bacteria or inhibits the growth of colonies, while the fluorescence intensity in the far distance
may be caused by leakage expression.

#### Cycle 2 Replace T7 promoter with constitutive promoter J23106

##### Design

To eliminate the induction step required before using the biosensor and to avoid the excessive
expression caused by the T7 strong promoter, we replaced the T7 promoter and lac operator with the
constitutive promoter PJ23106. (Figure. 1-5)

![Figure 1-5]({{ cdn_url('engineering/1-5.png') }})

**Figure 1-5.** The genetic circuit of
PJ23106-LasR-deGFP, which is modified from LasR-deGFP.

##### Build

We performed PCR and Gibson Assembly to build plasmid PJ23106-LasR-deGFP. (Figure 1-6)

![Figure 1-6]({{ cdn_url('engineering/1-6.png') }})

**Figure 1-6.** The plasmid map of
PJ23106-LasR-deGFP.

##### Test

Sequencing confirmed transformation success.

Referencing the work of Fedorec et al. (2024), we employed agar plate colony experiment to validate
the
“highpass”. We dispensed the culture and different concentration 3-oxo-C12-HSL at the positions
shown in
the map (Figure 1-7A) onto the surface of the agar plate, detecting the fluorescence with inverted
fluorescence microscope.

Other concrete details can be found on the [experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

![Figure 1-7]({{ cdn_url('engineering/1-7.png') }})

**Figure 1-7.** **(A)** The spotting
pattern. **(B)** Medium photos. It can be clearly seen that the growth of the
colonies
near the center point (where the AHL solution is added) is worse. **(C)** The
schematic
diagram of the fluorescence results. It is obtained by splicing the photos taken by the
fluorescence
microscope.

##### Learn

In the agar plate colony experiment, colonies closer to the central point, which had access to higher
concentrations of AHL, did not express more GFP. Instead, colonies slightly farther from the centre
point exhibited the highest fluorescence. (Figure. 6A) Observing the colony morphology revealed that
the
colonies located farther from the centre grew better. (Figure. 6B) We speculated that overmuch
3-oxo-C12
HSL may affect the colony growth and thereby influence the fluorescence intensity.

#### Cycle 3 Use medium-copy-number backbone

##### Design

We hypothesized that the primary reasons for poor bacterial growth may include two aspects: First,
the constitutive expression of the receptor protein on the high-copy plasmid (ori is pColE1)
significantly increases the metabolic burden on the engineering bacteria. Second, a high
concentration of the AHL ligand strongly induces the promoter to drive excessive expression of GFP,
further exacerbating metabolic stress. The combined effect of these two factors may lead to plasmid
loss under sustained high metabolic load. To mitigate these issues, we propose the following
strategy: replacing the ori with the medium-copy p15A origin and adopting the Rhl quorum sensing
system that is orthogonal[3] to more systems than Las to construct a more stable biosensor bacteria.
The
following circuit is designed accordingly. (Figure.1-8)

![Figure 1-8]({{ cdn_url('engineering/1-8.png') }})

**Figure 1-8.** The genetic circuit of Rhl biosensor
bacteria.

##### Build

We obtained RhlR and PRhl fragments from Shi-shen Du's Lab. Using Gibson assembly, we inserted these
fragments into the iGEM JUMP collection plasmid pJUMP26-1A (sfGFP) to construct the RhlR-sfGFP
plasmid,
which was then transformed into *E. coli* DH5α. (Figure 1-9, 1-10)

![Figure 1-9]({{ cdn_url('engineering/1-9.png') }})

**Figure 1-9.** The history of RhlR-sfGFP plasmid
construction.figcaption>

![Figure 1-10]({{ cdn_url('engineering/1-10.png') }})

**Figure 1-10.** The map of RhlR-sfGFP plasmid.

##### Test

We performed colony PCR and sequencing to verify successful plasmid construction. Sequencing
confirmed transformation success. (Figure. 1-11)

![Figure 1-11]({{ cdn_url('engineering/1-11.png') }})

**Figure 1-11.** The test of RhlR-sfGFP plasmid
construction. **(A)** The plate shows colonies post-transformation.
**(B)** The result of colony PCR. Obvious bands are seen in the region of
corresponding
size. **(C)** The result of sequencing verified the success of plasmid
construction.

We conducted the agar plate colony experiment on the engineered bacteria to determine whether they
could emit green fluorescence, thereby achieving biosensor functionality, and plotted a series of
dose response curves. (Figure. 1-12)

![Figure 1-12]({{ cdn_url('engineering/1-12.png') }})

**Figure 1-12.** The result of agar plate colony
experiment. **(A)** The map, the medium photo, and the schematic diagram of the
fluorescence results shown at 24 hours. **(B)** The dose response curves of C4-HSL
inducible biosensor bacteria in solid culture.

##### Learn

Through spiral dispensing in agar plate colony experiment, we confirmed that this design generated
consistent biosensor response curves, demonstrating that the ability to detect C4-HSL concentrations
in
solid media. The obtained data display well-defined trends, making them suitable for subsequent
fitting and computational mathematical modeling by the Model Group. Furthermore, Rhl biosensor
bacteria carrying the RhlR-sfGFP plasmid exhibits a highpass filtering behavior, which supports
their use as independent logic gate modules. The relevant verification can be viewed on the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

Building upon this methodology, we subsequently succeeded in constructing the LasR-sfGFP plasmid and
CinR-sfGFP plasmid, whose experimental result meet our expectation. However, we encountered
difficulties during the construction of the TraR-sfGFP plasmid (corresponding to Cycle 4).

##### Build & test of LasR-sfGFP and CinR-sfGFP

Based on the successfully verified RhlR-sfGFP plasmid, we obtained the LasR and Plas fragments from
the T7-LasR-deGFP plasmid using PCR. We then replaced the Rhl system in the RhlR-sfGFP plasmid and
constructed the LasR-sfGFP plasmid using Gibson assembly. (Figure. 1-13A)

Concurrently, the CinR\_AM and Pcin\_AM sequences, derived from the iGEM Distribution Kit, were
utilised to substitute the Rhl system in the RhlR-sfGFP plasmid. The CinR-sfGFP plasmid was then
constructed through Gibson assembly. (Figure. 1-13B)

![Figure 1-13]({{ cdn_url('engineering/1-13.png') }})

**Figure 1-13.** The genetic circuit of biosensor
bacteria and the map of plasmids. **(A)** Las system. **(B)** Cin
system.

We conducted the agar plate colony experiment on the engineering bacteria to determine whether they
could emit green fluorescence, thereby achieving biosensor functionality, and plotted a series of
dose response curves. (Figure. 1-14A, Figure. 1-14B)

![Figure 1-14]({{ cdn_url('engineering/1-14.png') }})

**Figure 1-14.** The results of agar plate colony
experiment. **(A)** The dose response curves of 3-oxo-C12 HSL inducible Las
biosensor
bacteria in solid culture. **(B)** The dose response curves of 3-OH-C14:1-HSL
inducible
Cin biosensor bacteria in solid culture.

#### Cycle 4 Optimize Tra biosensor bacteria

##### Design

Based on the successfully validated RhlR-sfGFP plasmid construct, we utilized the RhlR-sfGFP plasmid
as backbone and incorporated the synthesized sequences of TraR and Ptra from GenScript to construct
the TraR-sfGFP plasmid, thereby establishing the biosensor bacteria for the Tra system. The
following circuit was designed accordingly. (Figure. 1-15)

##### Build

To construct the TraR-sfGFP plasmid, we used Gibson assembly to replace the RhlR and Prhl parts in
the RhlR-sfGFP plasmid backbone with the synthesised TraR and Ptra sequences. After that, we
transformed it into *E. coli* DH5α. (Figure. 1-16, Figure. 1-17)

![Figure 1-16]({{ cdn_url('engineering/1-16.png') }})

**Figure 1-16.** The history of TraR-sfGFP plasmid
construction.

![Figure 1-17]({{ cdn_url('engineering/1-17.png') }})

**Figure 1-17.** The map of TraR-sfGFP plasmid.

##### Test

We performed colony PCR and sequencing. The plasmid was successfully constructed. But in the
subsequent agar plate colony experiment, we did not observe significant fluorescence.

##### Learn

Consult the extant literature on the subject found that, wild type Ptra promoter is nonfunctional in
*E. coli*. To solve the problem of the Tra system's poor functionality, researchers optimized
and
modified the Ptra promoter and TraR protein sequences.[1] (Figure. 1-18)

![Figure 1-18]({{ cdn_url('engineering/1-18.png') }})

**Figure 1-18.** Engineering new QS systems in
*E.
coli*. Though wild type Ptra promoter is nonfunctional, an engineered Plux-tra promoter
(Ptra\*) and a TraR point mutation (to tryptophan) [TraR(W)] significantly increase the Tra
system's
fold-change in the presence of its cognate ligand. Mean values are normalized by lowest
expression
in each panel. Error bars represent SEM (n = 3) [1].

Therefore, we referenced the literature and changed the promoter to Ptra\* and performed point
mutation to the TraR.

##### Re-design & Build

We synthesized Ptra\* using DNA oligo annealing and constructed Ptra\*-TraR-sfGFP plasmid based on
TraR-sfGFP plasmid (Figure 1-19A). Simultaneously, we substituted the amino acid at position 192 of
TraR
with Trp using site-directed mutagenesis , constructing Ptra\*-TraR(W)-sfGFP plasmid (Figure 1-19B).

![Figure 1-19]({{ cdn_url('engineering/1-19.png') }})

**Figure 1-19.** The genetic circuit of biosensor
bacteria and the map of plasmids. **(A)** Ptra\*-TraR-sfGFP. **(B)**
Ptra\*-TraR(W)-sfGFP.

##### Test

We performed colony PCR and sequencing. The plasmids were successfully constructed. However, in the
ensuing agar plate colony experiments, no significant fluorescent signal was detected,
and increasing the concentration of 3-oxo-C8-HSL did not yield any noticeable improvement.

##### Learn

Upon consulting the literature, we discovered that the overall fluorescence response of Tra system
was lower than that of the Las one. [1] (Figure 1-20) Therefore, we hypothesize that enhancing GFP
expression can be achieved by increasing the plasmid copy number or replacing with a stronger
constitutive promoter.

![Figure 1-20]({{ cdn_url('engineering/1-20.png') }})

**Figure 1-20.** Heat map showing GFP abundance for
Tra
& Las system and AHL concentrations. Each column denotes a unique combination of signal (AHL),
receptor (R- protein), and reporter (QS promoter), with rows denoting the concentration of
ligand.
Each value corresponds to the mean fluorescence value measured from a cell population normalized
by
the population’s celldensity; all combinations and concentrations were done in triplicate.
Adapted
from [1].

##### Redesign & build

On one hand, we replaced PJ23106 in the Ptra\*-TraR-sfGFP plasmid with PJ23119, a stronger
constitutive promoter, to construct J23119-Ptra\*-TraR-sfGFP plasmid. (Figure 1-21A) On the other
hand, we replaced the medium-copy p15A origin with the high-copy ColE1 origin to construct
Ptra\*-TraR-ColE1-sfGFP plasmid. (Figure 1-21B)

![Figure 1-21]({{ cdn_url('engineering/1-21.png') }})

**Figure 1-21.** The genetic circuit of biosensor
bacteria and the map of plasmids. **(A)** J23119-Ptra\*-TraR-sfGFP.
**(B)**
Ptra\*-TraR-ColE1-sfGFP.

##### Test

We performed colony PCR and sequencing. The plasmids were successfully constructed.

Subsequently, we conducted the agar plate colony experiment, and found that Tra biosensor bacteria
with Ptra\*-TraR-ColE1-sfGFP plasmid could successfully respond to 3-oxo-C₈-HSL, and emit green
fluorescence. Construction of the Tra biosensor bacteria was successful. (Figure 1-22)

![Figure 1-22]({{ cdn_url('engineering/1-22.png') }})

**Figure 1-22.** The results of agar plate colony
experiment. The dose response curves of 3-oxo-C8-HSL inducible Tra biosensor bacteria in solid
culture.

##### Learn

Following increasing the plasmid copy number, green fluorescence was observed from the Tra biosensor
bacteria, indicating that the functionality of both TraR and the Ptra\* part was successfully
confirmed.

### Band-pass engineered bacteria

Cycles in this section:

* [Cycle 1: Conduct a proof-of-concept
  using the IPTG system](#bandpass-cycle1-title)
* [Cycle 2: Las band-pass circuit](#bandpass-cycle2-title)
* [Cycle 3: Las band-pass circuit
  improvement](#bandpass-cycle3-title)
* [Cycle 4: Las band-pass circuit
  improvement based on IPTG band-pass circuit](#bandpass-cycle4-title)
* [Cycle 5: Rhl band-pass circuit](#bandpass-cycle5-title)
* [Cycle 6: Rhl band-pass circuit
  improvement based on IPTG band-pass circuit](#bandpass-cycle6-title)

#### Cycle 1 Conduct a proof-of-concept using the IPTG system

##### Design

Similar to the “high-pass” circuit, we refered to the work of Fedorec et al. (2024) [2] to design a
“band-pass” genetic circuit. The character of a band-pass filter is that the downstream circuit
cannot
be
activated at too high and too low input signals, only the appropriate concentration of signal
molecules can activate downstream gene expression. Bacteria with a band-pass circuit can be used as
an XOR gate in our project, and can be combined with the “high-pass” bacteria to form a half-adder
or
full-adder
under the condition of three inputs.

Refer to the work of Fedorec et al. (2024) [2] , we designed a band-pass genetic circuit induced by
IPTG
and
the final effect is presented by the expression of sfGFP. The expression of sfGFP is promoted by T7
RNA polymerase(T7 RNAP) and inhibited by PhlF at the same time, only when the expression of both is
at an appropriate level can sfGFP be expressed. This requires the input of IPTG to
be at an appropriate intermediate concentration. When the input IPTG concentration is too low, sfGFP
cannot be effectively expressed due to the lack of T7 RNAP ; and when the concentration of IPTG is
too high, the expression of GFP will be inhibited by PhlF.

In our final design, sfGFP will be replaced by enzymes that can produce other inducible molecules
like N-acyl homoserine lactones (AHLs).

We have designed the following circuit (Figure 2-1) to achieve the effect in the literature. We
constructed two plasmids and transformed them into BL21(DE3) to have all the components in our
bacteria. To regulate the relative expression levels of different components, we used vectors with
different copy numbers.

![Figure 2-1]({{ cdn_url('engineering/2-1.png') }})

**Figure 2-1.** Circuit design of IPTG band-pass
bacteria.

##### Build

Based on the vector pJUMP26-1A(sfGFP) and pJUMP27-1A(sfGFP), we conducted PCR to linearize vectors
and inserted promoter sequences through overlapping ends. Then we conduct Gibson Assembly to
construct the two plasmids. (Figure 2-2) We conduct co-transformation on BL21(DE3), which already
has T7
RNAP gene initiated by lac UV5 promoter on its genome.

![Figure 2-2]({{ cdn_url('engineering/2-2.png') }})

**Figure 2-2.** Plasmids of IPTG band-pass bacteria.

##### Test

To test the IPTG band-pass circuit, we used two different patterns to inoculate colonies and added a
series of concentrations of IPTG solution as input.

We expected colonies closest to and farthest from the input point both display low fluorescence
expression, and other colonies display high fluorescence expression. We used two different patterns
to inoculate colonies and give a series of concentrations of IPTG as input (Figure 2-3). The spiral
pattern is used to verify the character of band-pass bacteria, and the logic gate pattern is used to
find an appropriate combination of input distance and concentration to make the colony work as an
XOR
gate.

![Figure 2-3]({{ cdn_url('engineering/2-3.png') }})

**Figure 2-3.** Two patterns in functional test.
**(A)** Spiral verification. **(B)** Logic gate verification

The result was very consistent with expectation: The colonies closest and farther away from the input
point hardly emit fluorescence, and the colonies in the middle distance emit obvious fluorescence
(Figure 2-4).

![Figure 2-4]({{ cdn_url('engineering/2-4.png') }})

**Figure 2-4.** . Result of spiral verification at
18h
after inoculation and induction (IPTG band-pass circuit).

Further, we found some groups already possessing the properties of XOR logic gates(fig.27).

![Figure 2-5]({{ cdn_url('engineering/2-5.png') }})

**Figure 2-5.** Result of XOR logic gate test (IPTG
band-pass circuit).

##### Learn

The successful validation of the IPTG band-pass circuit demonstrates that our approach to building a
band-pass circuit is feasible. We will imitate the construction method to build a quorum sensing
band-pass circuit.

#### Cycle2: Las band-pass circuit

##### Design

We first tried to construct the Las band-pass circuit firstly (Figure 2-6). The operating principle
of
Las
circuit is similar to that in IPTG circuit in Cycle 1: when the 3-oxo-C12 HSL concentration
is too low, GFP cannot be effectively expressed due to the lack of T7 RNAP; when the concentration
of 3-oxo-C12 HSL is too high, the expression of GFP will be inhibited by PhlF. Only when the
expression of both is at an appropriate level can GFP be expressed.

![Figure 2-6]({{ cdn_url('engineering/2-6.png') }})

**Figure 2-6.** Genetic circuit of Las “band-pass”
bacteria.

##### Build

To build the full circuit, we construct two intermediate plasmids first. Based on the Las half adder
1 in high-pass circuit, iGEM distribution kit and plasmids from 2024 WHU-China, we conducted PCR to
linearize vectors and get fragments, then we conduct Gibson Assembly to construct the two
intermediate plasmids. After that, we conduct the whole Las band-pass plasmid also with PCR and
Gibson Assembly (Figure 2-7).

![Figure 2-7]({{ cdn_url('engineering/2-7.png') }})

**Figure 2-7.** Full plasmid of “band-pass”
bacteria.

##### Test

We successfully obtain the "band-pass" bacteria. Similar to "high-pass" engineered bacteria, we first
performed simplified Agar plate colony experiments. (Figure 2-8) Unfortunately, in the Agar plate
colony
experiments, our "band-pass" bacteria did not show any fluorescence signal.

![Figure 2-8]({{ cdn_url('engineering/2-8.png') }})

**Figure 2-8.** Functional verification pattern of
Las
band-pass circuit.

##### Learn

We encountered many difficulties during the construction process of Las band-pass circuit, especially
the issue of fragment and plasmid loss. We considered the plasmid may be too large and there may be
too many duplicated fragments. Due to the success of cycle1, next step was to try the construction
of IPTG band-pass circuit to build Las band-pass circuit.

#### Cycle 3: Las band-pass circuit improvement

##### Design

Due to the difficulty of the construction of Las system, we decided to reduce the engineering
difficulty by constructing a circuit without T7 RNAP. The new circuit uses Plas to initiate
expression of sfGFP directly (Figure 2-9).

![Figure 2-9]({{ cdn_url('engineering/2-9.png') }})

**Figure 2-9.** Genetic circuit of improved Las
“band-pass” bacteria.

##### Build

To build the full circuit, we conducted overlap PCR to get fragments, then we conduct restriction
cloning
to construct the whole plasmid. (Figure 2-10).

![Figure 2-10]({{ cdn_url('engineering/2-10.png') }})

**Figure 2-10.** Full plasmid of “band-pass”
bacteria.

##### Test

We successfully obtain the "band-pass" bacteria. Similar to IPTG band-pass band-pass circuit, we used
two different patterns to inoculate colonies and give a series of concentrations of IPTG and
3-oxo-C12 HSL at the same time in different proportions as input(Figure 2-3). Unfortunately it still
did
not show any fluorescence signal.

##### Learn

The reason for the lack of fluorescence signal might be excessive expression of phlF. T7
RNAP is a strong factor in initiating downstream gene expression and it is necessary in our circuit.
So next we planned to add T7 RNAP back into our band-pass circuit.

#### Cycle 4 Las band-pass circuit improvement based on IPTG band-pass circuit

##### Design

Based on the experience we have gained from the previous two cycles, we decided to to gradually
replace each component with which in Las band-pass circuit based on the IPTG band-pass circuit in
cycle 1. We replaced lacI with lasR and lac UV5 promoter with Plas.

Theoretically, when both 3-oxo-C12 HSL and IPTG are used for induction, the engineered bacteria will
exhibit a band-pass effect.

##### Build

Based on the plasmid IPTG-band-pass-p15A and IPTG-band-pass-pSC101 in cycle 2-1, we conducted Las
dual plasmid system. Through high fidelity PCR and Gibson Assembly, we get fragment needed and
engineered plasmid IPTG-band-pass-p15A to plasmid Cm-p15A-LasR-PhlF. The final dual plasmid system
is shown below (Figure 2-11).

![Figure 2-11]({{ cdn_url('engineering/2-11.png') }})

**Figure 2-11.** Plasmids of Las-IPTG band-pass
bacteria.

##### Test

Similar to IPTG band-pass band-pass circuit, we used two different patterns to inoculate colonies and
give a series of concentrations of IPTG and 3-oxo-C12 HSL the same time in different proportions as
input(Figure 2-3). The result is very consistent with expectation: The colonies closest and farther
away
from the input point hardly emit fluorescence, and the colonies in the middle distance emit obvious
fluorescence(Figure 2-12).

![Figure 2-12]({{ cdn_url('engineering/2-12.png') }})

**Figure 2-12.** Result of spiral verification at
16h
after inoculation and induction (Las band-pass circuit).The negative value of fluorescence
intensity
is because the value here is smaller than the negative control without AHL induction.

However, due to the limited number of experiment groups, we did not obtain particularly ideal results
in logic gate test(Figure 2-13).

![Figure 2-13]({{ cdn_url('engineering/2-13.png') }})

**Figure 2-13.** Result of XOR logic gate test
(Las-IPTG
system).

##### Learn

The most difficult point is how to adjust the ratio of IPTG and 3-oxo-C12 HSL to achieve band-pass
effect. We originally planned to fully replace the system, but due to time constraints we did not
implement it.

Although we did not obtain obvious XOR logic gate due to time limitations, the band-pass effect
showed by Las-IPTG double-plasmid bacteria has already indicated that it is likely to be an
appropriate concentration ratio of 3-oxo-C12 HSL and IPTG to make it a XOR gate.

#### Cycle 5 Rhl band-pass circuit

As our ultimate goal is to use AHL as the "wire", we have further designed Rhl band-pass circuit
based on Rhl quorum sensing system.

##### Design

The circuit of Rhl band-pass circuit is very similar to which in IPTG band-pass circuit. We use RhlR
as a receptor to receive C4-HSL signals and activate T7 RNAP expression, which can further promote
the expression of sfGFP; and we use phlF as a repressor to inhibit the expression of sfGFP.
Maintaining an appropriate ratio between phlF and T7 RNAP can create a band-pass effect(Figure
2-14).

![Figure 2-14]({{ cdn_url('engineering/2-14.png') }})

**Figure 2-14.** Genetic circuit of Rhl “band-pass”
bacteria.

##### Build

Based on the Rhl high-pass plasmid and iGEM distribution kit, we conducted PCR to linearize vectors
and get fragments, then we conduct Gibson Assembly to construct Rhl band-pass plasmid (Figure 2-15).

![Figure 2-15]({{ cdn_url('engineering/2-15.png') }})

**Figure 2-15.** Full plasmid of “band-pass”
bacteria.

##### Test

Similar to IPTG band-pass circuit, we used two different patterns to inoculate colonies and added a
series of concentrations of C4-HSL as input.

We expect that colonies near the input point show lower fluorescence expression, however the results
did not fully meet our expectation.

##### Learn

The implementation of XOR gate effect strongly depends on the mutual coordination of PhlF, T7 RNAP
and
their relative components. A slight imbalance in the effects of the two elements can lead to
disappointing
results. Additionally, we found that the fluorescence intensity of colonies in all experimental
groups was relatively low, we think it is an issue with the C4-HSL response threshold. In the next
cycle, we will make improvements to address these two issues.

#### Cycle 6 Rhl band-pass circuit improvement based on IPTG band-pass circuit

##### Design

For the two issues in the previous cycle, we have come up with a solution for each. For the weak
overall fluorescence brightness, we found a Rhl promoter(Part:BBa C0071 - parts.igem.org) from Team
Tokyo Tech 2016 whose promoter activity was stronger than that of wild type (previously used)(Figure
2-16).

![Figure 2-16]({{ cdn_url('engineering/2-16.png') }})

**Figure 2-16.** Sequence of Prhl(NM) (BBa\_K1949060)
,
showing the base different from wild type in red.

At the same time, another group of us tried a different way to improve the Rhl band-pass circuit. We
transformed two plasmids into BL21(DE3) strain just like what we did in IPTG band-pass circuit.
Actually, we replaced one of the plasmids in IPTG band-pass circuit to gradually replace IPTG with
C4-HSL (the left one in Figure 2-18).

##### Build

Based on the plasmid Rhl-band-pass in the previous cycle, we conduct PCR and changed the promoter
sequences through overlapping ends. Both Prhl were replaced by Prhl(NM)(Figure 2-17).

![Figure 2-17]({{ cdn_url('engineering/2-17.png') }})

**Figure 2-17.** Plasmid of improved Rhl band-pass
bacteria.

For the dual plasmid: based on the vector pJUMP26-1A(sfGFP), we conducted PCR to linearize vectors
and inserted sequences of Rhl band-pass circuit through overlapping ends. Then we conduct Gibson
Assembly to construct the plasmid. Using this plasmid and plasmid IPTG-band-pass-pSC101 in cycle
2-1, we conduct co-transformation on BL21(DE3), which already has T7 RNAP gene initiated by lac UV5
promoter on its genome. The final dual plasmid system is shown below (Figure 2-18).

![Figure 2-18]({{ cdn_url('engineering/2-18.png') }})

**Figure 2-18.** Plasmids of Rhl-IPTG band-pass
bacteria.

##### Test

Similar to IPTG band-pass circuit, we used two different patterns to inoculate colonies and added a
series of concentrations of IPTG and C4-HSL the same time in different proportions as input(Figure
2-3).

The result is very consistent with expectation: The colonies closest and farther from the input
point hardly emit fluorescence, and the colonies in the middle distance emit obvious fluorescence
(Figure 2-19).

![Figure 2-19]({{ cdn_url('engineering/2-19.png') }})

**Figure 2-19.** Result of spiral verification at
16h
after inoculation and induction (Rhl band-pass circuit).

However, due to the limited number of experiment groups, we did not obtain particularly ideal results
in logic gate test (Figure 2-20).

![Figure 2-20]({{ cdn_url('engineering/2-20.png') }})

**Figure 2-20.** Result of XOR logic gate test
(Rhl-IPTG
band-pass circuit).

##### Learn

The most difficult point is how to adjust the ratio of IPTG and C4-HSL to achieve band-pass effect.
We originally planned to fully replace the system, but due to time constraints we did not implement
it.

Although we did not obtain obvious XOR logic gate activated by AHL due to time limitations, the
band-pass
effect
showed by Rhl-IPTG double-plasmid bacteria has already indicated that it is likely to be an
appropriate concentration ratio of C4-HSL and IPTG to make it a XOR gate.

### Connector bacteria

Cycles in this section:

* [Cycle 1: Use the same AHL for
  transmission (amplifier)](#connector-cycle1-title)
* [Cycle 2: Use different AHL for
  transmission to avoid excessive positive feedback (connector)](#connector-cycle2-title)
* [Cycle 3: Build connectors for
  different
  AHL systems](#connector-cycle3-title)

#### Cycle 1 Use the same AHL for transmission (amplifier)

##### Design

As the signal of one bit may spread the previous digit to cause crosstalk, we use amplifying
bacteria to pull two digit apart from each other. To construct an amplifying bacteria of Las signal,
we designed the LasR-LasI plasmid using the T7-LasR-deGFP plasmid as the vector. The LasR gene is
initiated by the T7 promoter and regulated by the lac operon, thus requiring IPTG to induce
expression. The LasI gene is regulated by the las promoter. When the externally imported
3-oxo-C12AHL
molecules bind to the LasR protein, they can activate the las promoter, express the LasI gene, and
export the Las AHL molecules, thereby amplifying the Las signal, lengthening the distance a signal
can transmit.(Figure 3-1)

![Figure 3-1]({{ cdn_url('engineering/3-1.png') }})

**Figure 3-1.** **(A)** The functional
diagram of the amplifier. **(B)** The genetic circuit of Las amplifier.

##### Build

We obtained the LasR-vector and LasI fragments from LasR-deGFP and LuxR-LasI (from 2024 WHU-China) by
PCR respectively, and then constructed the LasR-LasI plasmid through Gibson assembly.(Figure 3-2)

![Figure 3-2]({{ cdn_url('engineering/3-2.png') }})

**Figure 3-2.** The plasmid map of LasR-LasI.

##### Test

Plasmids were transformed into *E. coli* BL21(DE3). Sequencing verified transformation
success.(Figure 3-3)

![Figure 3-3]({{ cdn_url('engineering/3-3.png') }})

**Figure 3-3.** Sequencing result of LasR-LasI
transformants.

We dispense samples as shown in Figure 3-4. to verify the function of amplifying bacteria. Biosensors
can
detect the Las AHL signal output from the amplifying bacteria through green fluorescence. The
position of eight biosensors are arranged carefully to avoid having the front block the back. Figure
3-5
shows the result.

![Figure 3-4]({{ cdn_url('engineering/3-4.png') }})

**Figure 3-4.** The spotting figure to verify
amplifying
bacteria. The top is the input Las AHL signal(triangle).The amplifying bacteria in the middle
(1cm
from AHL).The bottom eight points are biosensors.

![Figure 3-5]({{ cdn_url('engineering/3-5.png') }})

**Figure 3-5.** Fluorescent photos of amplifiers in
agar
plate colony experiments. The amplifier is inoculated at the square position and the inducer AHL
solution is dripped at the star position.

##### Learn

We found that amplifying bacteria can achieve the amplification effect of receiving input 3-oxo-C12
HSL
signals and output 3-oxo-C12 HSL signals. However, in the absence of 3-oxo-C12 HSL input, downstream
biosensor could still detect 3-oxo-C12 HSL signal and produce green fluorescence due to
amplification by
the amplifying bacteria.

These abnormal phenomena indicate that the amplifying bacteria have certain leakage expression
problems,
that is, they generate 3-oxo-C12 HSL output signals when there is no external 3-oxo-C12 HSL input.We
speculate that positive feedback effect of amplifying bacteria and leaky expression of the T7
promoter
result in the expression of the LasI gene in the absence of 3-oxo-C12 HSL. On the other hand,the
positive feedback between lasR and lasI may also play a role. To further prevent the leakage, we
change
amplifier to connector bacteria in later experiment, which can receive one AHL and turn this signal
to
another.

#### Cycle 2 Use different AHL for transmission to avoid excessive positive feedback (connector)

##### Design

In serial computation, the carry signal generated from a lower-bit operation must propagate to the
next higher-bit position to perform multi-bit computations. We designed the connector bacteria to
transfer the carry output (Ci).

The carry output (Ci) phenotype in half-adder and full-adder bacteria can be enable by the highpass
genetic circuits, which form the basis of our connector bacteria. Within the same connector
bacterium, the two QS systems employed are mutually orthogonal[3] to prevent crosstalk between the
upstream and downstream colonies. (Figure 3-6)

![Figure 3-6]({{ cdn_url('engineering/3-6.png') }})

**Figure 3-6.**The genetic circuit of connector
bacteria. **(A)** LasR-RhlI connector bacteria. **(B)** RhlR-LasI
connector bacteria.

##### Build

In this build, we employed mutually orthogonal QS system, Rhl and Las systems, and each built upon
the plasmid of its own respective biosensor bacteria.

For instance, Gibson assembly was employed to replace the sfGFP gene in the LasR-sfGFP plasmid from
the
Las biosensor bacteria with the RhlI sequence (Figure 3-7A), which has the capacity to produce
C4-HSL.
This allowed us to create the LasR-RhlI connector bacteria (Figure 3-7B), which transmit signals to
downstream colonies. Similarly, we constructed the RhlR-LasI connector bacteria (Figure 3-6C) using
the
RhlR-sfGFP plasmid as the backbone.

![Figure 3-7]({{ cdn_url('engineering/3-7.png') }})

**Figure 3-7.**The construction of two distinct
types
connector bateria. **(A)** The history of connector plasmid construction (Using the
LasR-RhlI plasmid as an example). **(B)** The map of the LasR-RhlI plasmid.
**(C)** The map of the RhlR-LasI plasmid.

##### Test

We performed colony PCR and sequencing. The plasmids were successfully constructed.

Subsequently, we conducted a agar plate colony experiment and found that the AHL ligand
concentration expressed by connector bacteria followed the diffusion curve, but there was certain
leaky expressions.(Figure 3-8)

![Figure 3-8]({{ cdn_url('engineering/3-8.png') }})

**Figure 3-8.**The agar plate colony experiment for
verifying the functionality of connector bacteria. **(A)** The spotting pattern.
**(B)** The dose response curves of C4-HSL inducible RhlR-LasI connector bacteria
in
solid culture. Data is shown at 24 hours. **(C)** The time-stratified response
curves
of 3-oxo-C12-HSL inducible LasR-RhlI connector bacteria in solid culture. The input is 0.5 mM
3-oxo-C12-HSL. **(D)** The time-stratified response curves of C4-HSL inducible
RhlR-LasI connector bacteria in solid culture. The input is 5 mM C4-HSL.

##### Learn

The AHL ligands produced by the connector bacteria were detected by the biosensor bacteria, which
emitted fluorescence in accordance with a diffusion curve. The functional validation of the
connector bacteria was successful in the test.

#### Cycle 3 Build connectors for different AHL systems

##### Design

Similarly, we utilize three pairwise orthogonal QS systems[3]—Rhl, Tra, and Cin system—to construct a
greater variety of connector bacteria which is capable of transmitting QS signals
from different molecules. (Figure 3-9)

![Figure 3-9]({{ cdn_url('engineering/3-9.png') }})

**Figure 3-9.** The genetic circuit of two distinct
types connector bacteria. **(A)** RhlR-TraI connector bacteria.
**(B)**
RhlR-CinI connector bacteria.

##### Build

We utilized Gibson Assembly to replace the sfGFP gene in the RhlR-sfGFP plasmid from the Rhl
biosensor bacteria with TraI and CinI sequences, thereby constructing RhlR-TraI and RhlR-CinI
connector bacteria. (Figure 3-10)

![Figure 3-10]({{ cdn_url('engineering/3-10.png') }})

**Figure 3-10.** The map of two distinct types
connector
bacteria plasmids. **(A)** RhlR-TraI. **(B)** RhlR-CinI.

##### Test

We performed colony PCR and sequencing. The plasmids were successfully constructed.

However, in the ensuing agar plate colony experiments, no significant fluorescent signal
emitted by downstream biosensor bacteria was detected.

##### Learn

The functional validation of the two constructed connectors bacteria was unsuccessful. This may be
because the inducer proteins express an insufficient concentration of AHL ligands, or because the
function of the heterologous proteins is impaired in the *E. coli* chassis. Further
investigation
is
required.

## Refreshing

### Light-inducible AHL degradation enzyme

Cycles in this section:

* [Cycle 1: Verify the functions of
  AiiA
  and explore the experimental conditions](#refreshing-cycle1-title)
* [Cycle 2: AiiA computational split
  sites selection and rational design](#refreshing-cycle2-title)
* [Cycle 3: AiiA light-inducible
  split
  and functional validation](#refreshing-cycle3-title)
* [Cycle 4: LOV light-induced
  Engineering
  and Functional Characterization of the VVD Domain](#refreshing-cycle4-title)

#### Cycle1 Verify the functions of AiiA and explore the experimental conditions

##### Design

In this cycle, we aim to test the function of AiiA to degrade acyl-homoserine lactone (AHL)
molecules and to identify an appropriate method to quantify the degradation efficiency of AiiA. From
a
theoretical standpoint, measuring the enzymatic activity of purified AiiA would be an excellent
approach; however, because screening split sites is required for the light-induced engineering of
AiiA, protein purification at this stage would impose a substantial workload. Therefore, we plan to
first employ a simpler assay to assess AiiA degradation efficiency, and proceed to purification
thereafter. After drawing on the work of Koch et al.(2014)[4] we
will express AiiA in *E. coli* under IPTG induction and incubate the cells in medium
containing
AHLs
to degrade the AHLs. After a period of incubation and degradation, the residual AHL concentration in
the culture supernatant will be detected using the biosensor bacteria. Detailed
experimental procedures are provided in the Measurement section.

##### Build

To evaluate whether AiiA is prone to generating inclusion bodies, we constructed two plasmids,
pGEX-AiiA and pGEX-GST-AiiA, which enable IPTG-inducible expression of AiiA and the GST–AiiA fusion
protein, respectively.(Figure 4-1)

![Figure 1-2]({{ cdn_url('engineering/4-1-1.png') }})
![Figure 1-2]({{ cdn_url('engineering/4-1-2.png') }})

Figure 4-1. The
plasmid map of pGEX-AiiA and pGEX-GST-AiiA

We amplified the DNA fragments used for plasmid construction by PCR, with the AiiA sequence
synthesized by GenScript. The fragments were assembled with the linearized vector via Gibson
Assembly to generate the final plasmids. The assembled plasmids were first transformed into
*E.coli* DH5α strains; single colonies were picked and validated by colony PCR and Sanger
sequencing. (Figure 4-2) After verification, plasmids were isolated and transformed into *E.
coli*
BL21(DE3) strains.

![Figure 4-2]({{ cdn_url('engineering/4-2.png') }})

**Figure 4-2.** **(A)** Colony PCR
results.
White arrows indicate the colony PCR amplication results corresponding to the strains ultimately
used. **(B)** Sanger sequencing result for pGEX-AiiA (DH5α). All other sequencing
results were verified as correct and are not shown.

Finally, BL21 strains containing pGEX-AiiA or pGEX-GST-AiiA plasmids were induced with IPTG, and
Coomassie Brilliant Blue staining was performed to confirm proper protein expression.(Figure 4-3)

![Figure 4-3]({{ cdn_url('engineering/4-3.png') }})

**Figure 4-3.** Coomassie Brilliant Blue staining
results. The AiiA protein has a molecular mass of 28.3 kDa, and the GST–AiiA fusion protein has
a
molecular mass of 55.3 kDa.

##### Test

The experiment for measuring degradation efficiency can be simplified to
induction–degradation–detection. Induction was carried out at 25°C with 0.5 mM IPTG for
approximately 12 h. Cells were then incubated with AHL in a shaker at 37°C for about 2 h,
after which the cells were removed by centrifugation. Fresh medium and the biosensor bacteria were
added, and the mixture was incubated at 37°C in a plate reader with OD and fluorescence recorded
every 4 min. Detailed experimental procedures and data processing are provided in the Measurement
section.

![Figure 4-4]({{ cdn_url('engineering/4-4.png') }})

**Figure 4-4.** The results of AHL molecule
degradation
by AiiA. The annotations in the figure indicate the degradation conditions. “Background” group
denotes the detection condition containing only fresh medium and the reporter strain. The AHL
substrate used was C4-HSL; in the “none” (no-degradation) control, the final AHL concentration
was 1
mM. For all groups other than the background, the AHL concentration prior to degradation was
identical to that of the none group.

Figure 4-4 displays the most representative result. We also evaluated degradation efficiency at
terminal AHL concentrations of 0.5 mM and 0.05 mM, which yielded results consistent with those
observed at 1 mM. The outcomes for the GST–AiiA–expressing strain were likewise consistent with
those for the AiiA-expressing strain.

##### Learn

The results indicate that, even without IPTG induction, leaky expression of AiiA is sufficient to
completely degrade C4 in the medium within 2 h. Under expression conditions of 25°C with 0.5 mM
IPTG, enzymatically active AiiA was produced; consequently, the GST tag will not be used in
subsequent experiments to avoid inclusion body formation. AiiA exhibits strong degradation
efficiency, and our assay was progressively refined during this process. As a next step, we plan to
proceed directly with the light-induced engineering of AiiA.

#### Cycle 2 AiiA computational split sites selection and rational design

##### Design

To gain in-depth structural insights into the AiiA protein and identify suitable split sites, we
employed molecular dynamics simulations and molecular docking to facilitate a more rational
selection of split loci. Given that our AiiA sequence differs slightly from the AiiA structures
available in the PDB, we plan to use AlphaFold3 to obtain the structural model of our AiiA
protein.[5] Subsequently, molecular dynamics simulations and associated analytical methods will be
applied to characterize the flexible regions, active sites, and principal components of AiiA, using
the CHARMM36 force field. The SPELL algorithm will also be utilized to determine split sites based
on split energy and solvent accessible surface area information.[6]

After selecting the split sites, we constructed fusion proteins of split AiiA with VVD[VVD is a
light-oxygen-voltage (LOV) sensory domain that dimerizes in response to blue light, thereby
facilitating the self-assembly of split proteins. Please refer to the introduction in Cycle3
for detailed information]. The
structural models of these fusion proteins were also generated using AlphaFold3, and molecular
dynamics simulations were performed to assess the stability of the fusion constructs. We then
employed protein–protein docking methods, including AlphaFold3, Z-Dock, and Rosetta, to predict the
conformation of the VVD dimer. Using the dimeric protein structure, molecular docking experiments
were conducted with the AHL molecule, followed by further molecular dynamics simulations to evaluate
the enzymatic activity upon dimerization.

Through the above computational approaches, we aim to assist in the screening of optimal split sites
in AiiA, while also refining the in silico methodology based on wet-lab experimental results,
thereby contributing to the improvement of algorithms for identifying protein split sites.

##### Build

We began by determining the three-dimensional (3D) structure of the AiiA protein using
AlphaFold3.(Figure 4-5)

![Figure 4-5]({{ cdn_url('engineering/4-5.png') }})

**Figure 4-5.** Predicted structure of the AiiA
protein.
The three-dimensional model was generated using AlphaFold3.

The necessary scripts for molecular dynamics (MD) simulation and analysis were developed. MD
simulations were subsequently conducted employing the GROMACS for CPU-2021 package.

Simulation Scripts
Copy

```
echo -e "1\n1" |gmx_mpi pdb2gmx -f A1.pdb -o A1.gro
gmx_mpi editconf -f A1.gro -o A1-box.gro -c -d 1.2 -bt dodecahedron
gmx_mpi solvate -cp A1-box.gro -cs spc216.gro -o A1-sol.gro -p topol.top
gmx_mpi grompp -f ions.mdp -c A1-sol.gro -p topol.top -o ions.tpr
echo "13" |gmx_mpi genion -s ions.tpr -o A1-sol-ions.gro -p topol.top -pname NA -nname CL -neutral

gmx_mpi grompp -f em.mdp -c A1-sol-ions.gro -p topol.top -o em.tpr
gmx_mpi mdrun -v -deffnm em
echo "10 0" |gmx_mpi energy -f em.edr -o potential.xvg

gmx_mpi grompp -f nvt.mdp -c em.gro -r em.gro -p topol.top -o nvt.tpr
gmx_mpi mdrun -v -deffnm nvt
echo "16 0" |gmx_mpi energy -f nvt.edr -o temperature.xvg

gmx_mpi grompp -f npt.mdp -c nvt.gro -r nvt.gro -t nvt.cpt -p topol.top -o npt.tpr
gmx_mpi mdrun -v -deffnm npt
echo "18 0" |gmx_mpi energy -f npt.edr -o pressure.xvg
echo "24 0" |gmx_mpi energy -f npt.edr -o density.xvg

gmx_mpi grompp -f md.mdp -c npt.gro -t npt.cpt -p topol.top -o md_0_1.tpr
gmx_mpi mdrun -v -deffnm md_0_1

# Analysis Scripts
echo -e "1 \n 0" | gmx_mpi trjconv -s md_0_1.tpr -f md_0_1.xtc -o md_0_1_noPBC.xtc -center -pbc mol -ur compact

echo -e "4 \n 4" | gmx_mpi rms -s md_0_1.tpr -f md_0_1_noPBC.xtc -o rmsd.xvg -tu ns
echo -e "4 \n 4" | gmx_mpi rms -s em.tpr -f md_0_1_noPBC.xtc -o rmsd_xtal.xvg -tu ns

echo "1" | gmx_mpi gyrate -s md_0_1.tpr -f md_0_1_noPBC.xtc -o gyrate.xvg

echo -e "4 \n 4" | gmx_mpi trjconv -f md_0_1_noPBC.xtc -s md_0_1.tpr -o avg.pdb -dump 0
echo -e "4 \n 4" | gmx_mpi rmsf -f md_0_1_noPBC.xtc -s avg.pdb -o rmsf.xvg -res

echo -e "1 \n 1" | gmx_mpi sasa -f md_0_1_noPBC.xtc -s md_0_1.tpr -o sasa.xvg -tu ns

echo -e "1 \n 1" | gmx_mpi hbond -f md_0_1_noPBC.xtc -s md_0_1.tpr -num hbnum.xvg

echo "1" | gmx_mpi do_dssp -f md_0_1_noPBC.xtc -s md_0_1.tpr -o ss.xpm -sc ss_comp.xvg -tu ns
gmx_mpi xpm2ps -f ss.xpm -o ss.eps

echo -e "4 \n 4" | gmx_mpi covar -f md_0_1_noPBC.xtc -s md_0_1.tpr -o eigenvalues.xvg -v eigenvectors.trr -xpma covara.xpm

echo -e "4 \n 4" | gmx_mpi anaeig -f md_0_1_noPBC.xtc -s md_0_1.tpr -v eigenvectors.trr -proj proj.xvg -first 1 -last 2

echo -e "4 \n 4" | gmx_mpi mdmat -f md_0_1_noPBC.xtc -s md_0_1.tpr -mean dm.xpm -frames frames.xpm -no -n
gmx_mpi xpm2ps -f dm.xpm -o dccm.eps
```

Based on the subsequent use of AlphaFold3 for the folding and docking of the fusion protein, the
folding results were satisfactory. However, the docking results provided by AlphaFold3 exhibited low
confidence scores and lacked consistency across multiple runs. Consequently, we employed an
alternative strategy utilizing the Z-Dock rigid-body docking method followed by Rosetta for flexible
refinement to predict the dimeric conformation of the fusion protein. Unfortunately, the final model
showed a significant discrepancy compared to the VVD dimer conformation reported in the literature.
We conducted some preliminary analysis and discussion within the Learn part. Ultimately, however, as
these experiments did not yield conclusive or meaningful results, this section was omitted from the
final manuscript for brevity.

##### Test

We performed 60 ns molecular dynamics simulations using GROMACS 2021.1. The system was constructed
based on the AiiA structure provided by AlphaFold 3, employing the CHARMM36 force field and the
TIP3P water model. The protein was placed in a dodecahedral water box with a 1.2 nm clearance from
the protein surface. The system underwent energy minimization, followed by 100 ps each of NVT and
NPT equilibration. Production simulations were conducted under the NPT ensemble, with temperature
maintained at 300 K (V-rescale) and pressure at 1 bar (Parrinello–Rahman). A 2 fs integration time
step was used, along with the LINCS constraint algorithm. Trajectory analyses included RMSD, RMSF,
radius of gyration, hydrogen bonding, and principal component analysis to evaluate the structural
stability and dynamic properties of the protein. The PDB file of AiiA used in our study was
submitted to the SPELL web server (https://dokhlab.med.psu.edu/spell/login.php) to obtain the
corresponding split protein output. Additional relevant parameters are provided in the Supplementary
Materials.

![Figure 4-6]({{ cdn_url('engineering/4-6.png') }})

**Figure 4-6.** Root mean square fluctuation (RMSF)
analysis of AiiA from molecular dynamics simulations. The red markers indicate the amino acids
ultimately selected as the split sites. The blue markers highlight residues exhibiting high
flexibility that are located near the active site of AiiA. The green markers denote other highly
flexible residues considered as alternative candidate split sites.

![Figure 4-7]({{ cdn_url('engineering/4-7.png') }})

**Figure 4-7.** Results from the SPELL algorithm.
The
definition of "Split Energy" can be found inin the work of Dagliyan et al. (2018b) [6]. This
parameter is primarily used to evaluate the likelihood of spontaneous reassembly of the split
protein fragments in the absence of other influencing factors.

The selection of split sites was primarily based on RMSF profiles and SPELL results(Figure 4-6,
Figure
4-7), the
selection was also partially informed by the results of Principal Component Analysis (PCA). (see
Supplementary Materials) and visual inspection of the molecular dynamics trajectories. Some
trade-offs were made during the selection process. For instance, while residue 154 exhibits
relatively low flexibility, it was still chosen as a split site due to its favorable Split Energy
and solvent accessible area (SAA) characteristics, and was subsequently validated in experimental
assays. These wet-lab experiments also provide insights into the relationship between local
flexibility and splitting efficiency. Based on the initial round of experimental results, our
strategy will be adaptively adjusted to explore alternative split sites. Following the finalization
of split sites, the structures of the resulting split fusion proteins were predicted using AlphaFold
3 and subjected to molecular dynamics simulations.

![Figure 4-8]({{ cdn_url('engineering/4-8.png') }})

**Figure 4-8.** Molecular dynamics simulation
results of
the split fusion proteins.

A detailed analysis of the simulations revealed several insightful findings. For instance, the choice
of split site appeared to have a minimal impact on the Solvent Accessible Surface (SAS) of the
VVD-cAiiA fusion protein but a more pronounced effect on the SAS of the nAiiA-VVD fusion,
particularly around residues 154-181. Furthermore, splitting at 154-181 significantly altered the
RMSD fluctuations of nAiiA-VVD, leading to a more compact and stable protein conformation.
Consequently, the nAiiA-VVD construct split at site 181-182 exhibited very low RMSD fluctuations
during the simulation, indicating high structural stability. Additionally, the RMSD fluctuations in
VVD-cAiiA were primarily traced to the region encompassing residues 181-208aa.(Figure 4-8)

Integrating these observations, we hypothesize that the region 154-208 constitutes a crucial
hydrophobic core in AiiA (consistent with the SAS profile from SPELL analysis), which interacts with
other hydrophobic patches. From this perspective, residue 208 represents a more favorable split
site.

##### Learn

The molecular dynamics simulations provided valuable insights that aided in our screening of
potential split sites. The wet-lab experiments further demonstrated that the self-assembly
activities of the proteins split at the three selected sites were nearly identical, supporting the
reliability of the SPELL predictions. However, the VVD domain did not exhibit its expected function
in the wet-lab experiments, which prevented us from leveraging these experimental results to refine
our computational methodology.

For protein-protein and molecular docking studies, the dimerization of VVD is known to require
a ligand and involves light-induced conformational changes, which falls beyond the scope of our
current technical capabilities. Conversely, docking simulations conducted solely with AiiA would
provide insights predominantly into the self-assembly of the split fragments, rather than informing
on the functional interaction within the full fusion protein system.

#### Cycle 3 AiiA light-inducible split and functional validation

##### Design

Our strategy for constructing light-induced AiiA is to split AiiA and fuse the fragments to
photosensory proteins; upon light induction, the photosensors dimerize and thereby promote
reconstitution of the split fragments into full-length, functional AiiA. In the absence of light,
the photosensors gradually dissociate, driving disassembly of the reconstituted AiiA and consequent
loss of degradative activity.

First, for the choice of the light-controlled module, we selected VVD. Among LOV-domain proteins, VVD
has the smallest molecular mass and is sensitive to blue light, undergoing rapid dimerization upon
blue-light illumination. In addition, numerous VVD variants have been developed that enhance its
oligomerization propensity in both intracellular and extracellular contexts.

Second, in plasmid design, drawing on the work of Sheets et al. (2020) [7] we fused VVD to the N- and
C-terminus of the split AiiA fragments,
respectively, with flexible linkers between domains. For convenience, the two proteins are expressed
from a single promoter (Figure 4-9).

![Figure 4-9]({{ cdn_url('engineering/4-9.png') }})

**Figure 4-9.** The genetic circuit of the
split-protein
expression.

Finally, for split-site selection, our basic principles were: (1) the site should reside in a
relatively flexible region so as not to disrupt the native secondary structure; (2) the split
fragments should not expose hydrophobic surfaces; and (3) each fragment should preserve its
structural integrity. Moreover, to achieve light-induced activity, the intrinsic
self-complementation of the split fragments must be tuned to an intermediate level—namely, they
should exhibit minimal spontaneous reassembly into the full-length protein without VVD, yet
efficient reconstitution under VVD dimerization. To select the split sites and tune reconstitution
propensity, we primarily referred to the SPELL algorithm described by Dagliyan et al.(2018) [6] and
employed
molecular dynamics simulations and molecular docking to evaluate local flexibility,
hydrophobic/hydrophilic regions, and predicted degradative activity after reconstitution as
reference metrics for candidate sites.

##### Build

In this section, guided by the SPELL predictions, we constructed plasmids encoding split AiiA
variants with split sites at amino acid positions 154, 181, and 208.(Figure 4-10)

![Figure 4-10]({{ cdn_url('engineering/4-10.png') }})

**Figure 4-10.** The expression plasmid map of AiiA
with
a split site at residue 208.

Similar to Cycle 1, the fragments used for plasmid construction were obtained by PCR, with the
AiiA and VVD coding sequences synthesized by GenScript. The fragments were assembled with the
linearized vector using Gibson Assembly to generate the final plasmids. The assembled plasmids were
first transformed into Escherichia coli DH5α; single colonies were picked and verified by colony PCR
and Sanger sequencing. (Figure 4-11) After confirmation, plasmids were extracted and the
corresponding
plasmids were transformed into *E. coli* BL21(DE3).

![Figure 4-11]({{ cdn_url('engineering/4-11.png') }})

**Figure 4-11.** **(A)** Representative
colony PCR results; white arrows indicate the strains which successful amplified and carried
forward
to subsequent experiments. **(B)** Sanger sequencing result for the nAiiA(1–208
aa)–VVD
VVD–cAiiA(209–250 aa) construct in DH5α. All other sequencing results were confirmed to be
correct
and are not shown.

Finally, we also performed Coomassie Brilliant Blue staining to confirm that the proteins were
expressed
correctly.(Figure 4-12)

![Figure 4-12]({{ cdn_url('engineering/4-12.png') }})

**Figure 4-12.** Coomassie Brilliant Blue staining
results. For the construct split at residue 154, the two split fragments have apparent molecular
masses of 35.5 kDa and 29.1 kDa; for the construct split at residue 181, 38.5 kDa and 26.1 kDa;
and
for the construct split at residue 208, 41.5 kDa and 23.1 kDa, respectively.

##### Test

During this cycle, we first optimized our experimental methodology and the biosensor bacteria
(details
can be found in the Measurement section). In the new round of experiments, the relative fluorescence
unit (RFU) of the background group remained stable, unlike the decline observed over time in Cycle
1.

Under the new experimental conditions, we also used BL21 strains containing the
pGEX-nmCherry-VVD-VVD-cmCherry plasmid for degradation incubation as a control. (Initially, we used
BL21 without any plasmid as the control. However, we constructed the aforementioned engineered
strain in Cycle 4 and considered it a more rigorous control. Therefore, we included this additional
experiment during Cycle 4. The plasmid map for this engineered strain is provided in the Cycle 4
section.)

Although the response curve of the biosensor bacteria exhibited some changes—such as an extended
overall
response duration and the response to the AHL started earlier—the final expression intensity
remained unchanged. Furthermore, across multiple experiments, the
response curve of the "None" degradation group demonstrated strong consistency, indicating that the
optimized experimental method offers enhanced reproducibility and yields reliable results.(Figure
4-13)

![Figure 4-13]({{ cdn_url('engineering/4-13.png') }})

**Figure 4-13.** The result of optimal AHL
degradation
experiments.

We plan to conduct degradation experiments at both 37°C and 25°C. The higher temperature of 37°C
promotes AiiA enzyme activity, while 25°C is more conducive to VVD polymerization and the assembly
of the split AiiA fragments. Due to the lack of specialized optogenetic equipment, we cannot
precisely control the intensity of blue light illumination. For the light-exposed group, samples
were incubated in a shaker equipped with blue light sources. The dark control group was incubated in
a separate shaker, with EP tubes wrapped in aluminum foil to block ambient light.

![Figure 4-14]({{ cdn_url('engineering/4-14-n.avif') }})

**Figure 4-14.** Results for the group with the
cleavage
site at 208 aa under degradation conditions at 37°C and 25°C. (A, B) "Background" refers to the
control group containing only fresh medium and the reporter strain. The AHL molecule used was
C4-HSL, and the final concentration of AHL in the non-degradation ("None") group was 0.05 mM.
Except
for the background group, the initial AHL concentration in all other groups before degradation
was
consistent with that of the "None" group.

Results for other cleavage sites were similar to those for site 208 and are not shown here.
As can be observed, detectable activity was present at both 37°C and 25°C, which we attribute to the
self-assembly
of the split protein fragments. However, blue light illumination did not enhance the degradation
efficiency.
Furthermore, BL21 strains containing the pGEX-AiiA plasmid showed effective degradation under
identical
light
conditions, confirming that blue light exposure does not impair AiiA activity. Experimental results
for
other
cleavage sites consistently supported these findings.

![Figure 4-15]({{ cdn_url('engineering/4-15-n.avif') }})

**Figure 4-15.** Consolidated results of the dark
control groups under degradation conditions at 37°C and 25°C. (A, B) "Background" refers to the
control group containing only fresh medium and the reporter strain. The AHL molecule used was
C4-HSL, with a final concentration of 0.05 mM in the non-degradation ("None") group. Except for
the
background group, the initial AHL concentration in all other groups before degradation was
consistent with the "None" group.

The three cleavage sites tested showed similar degradation efficiency. We hypothesize this result
stems
from their comparable splitting energies (as referenced in the SPELL), leading to similar
self-assembly
activities and consequently similar degradation efficiencies.

##### Learn

Unfortunately, despite multiple repetitions of the blue light-induced degradation experiments
under various blue light illumination conditions, the results consistently indicate that our design
was unsuccessful. Based on the collective experimental evidence, we conclude that the failure to
achieve
light control is primarily due to either the inability of VVD to form dimers or the lack of a
promoting
effect of VVD dimerization on the reassembly of the split protein fragments.

In this cycle, our approach was somewhat rushed. For the next steps, we plan to first utilize the
split
mCherry to validate VVD function and identify suitable blue light illumination conditions.

#### Cycle 4 Light-induced Engineering and Functional Characterization of the VVD Domain

##### Design

Given the complexity and non-intuitive nature of the VVD-AiiA experimental procedures in Cycle 3,
which
hindered efficient troubleshooting, we designed Cycle 4 to independently validate the light-induced
function of VVD.The overall design is similar to that of Cycle 3: we split mCherry into N-terminal
(n
mCherry) and C-terminal (c mCherry) fragments, with VVD domain attached to the internal end of each
fragment , whereby blue light irradiation induces VVD dimerization, facilitating the reassembly of
the
two mCherry fragments into a functional protein that emits red fluorescence.

With the exception of the mCherry component, all other elements in the design remain consistent with
those in
Cycle 3. The split sites for mCherry were selected primarily based on the design described in
Fan et al. (2008)[8]. Furthermore, we extended this design by constructing additional plasmids to
investigate
whether the failure in Cycle 3 could be attributed to elements such as the linker or RBS. These
include
variants
with altered linkers, replacement of the RBS with a linker[9], and constructs where both VVD domains
are
fused to the N-terminus of
split mCherry.

##### Build

For this cycle, we constructed the following four plasmids:

![Figure 4-16]({{ cdn_url('engineering/4-16-n.avif') }})

**Figure 4-16.**Plasmid maps constructed for Cycle
4.
**(A)** Design derived from Cycle 3, with split AiiA replaced by split mCherry.
**(B)** Plasmid based on (A) but with the RBS replaced by a flexible protein
linker.
**(C)** Plasmid modified from (A) by altering the fusion configuration of VVD;
here,
both VVD units are placed at the N-terminus of the split mCherry fragments.
**(D)** Plasmid based on (A) but incorporating an alternative protein linker.

Details of the linkers used are provided below:

**linker1: -GSGSGSGSGS- linker2: -GSAGSAAGSGEF- linker3: -GSGSGSGSGSGSGSGSGSGS- linker4:
-GGGGSGGGGS**

Following a strategy similar to that in Cycle 1, the DNA fragments required for plasmid construction
were
obtained by PCR. The mCherry gene fragment was sourced from the 2024 iGEM contribution kit, while
the
VVD gene fragment was synthesized by GenScript. The resulting fragments were assembled with a
linearized
vector using Gibson Assembly to generate the final plasmid constructs.

The assembled plasmids were first transformed into E. coli DH5α cells. Single colonies were selected
and validated by colony PCR and DNA sequencing. After confirmation of the correct sequence, the
plasmids
were extracted and subsequently transformed into E. coli BL21(DE3) cells for downstream
applications.

##### Test

The functional validation of split mCherry was carried out in three main steps: protein expression,
blue light induction, and fluorescence measurement. Experiments were conducted in both liquid and
solid media.

In liquid culture, protein expression was induced overnight at 25°C, followed by illumination with
a blue LED array. Fluorescence intensity was measured using a microplate reader before
and after light exposure.

On solid media, a method similar to diffusion assay was employed: IPTG was spotted at the center of
the
plate,
and bacterial suspension was applied in a spiral gradient around it. After about 12 hours of
incubation,
the plates
were exposed to blue LED light, and fluorescence was visualized before and after illumination using
an
inverted
fluorescence microscope. All procedures were performed under light-shielding conditions, with blue
light
intensity
gradually increased to identify optimal activation conditions.

However, under neither condition—liquid or solid media—did split mCherry produce detectable
fluorescence
before
or after blue light illumination.

##### Learn

A series of Split mCherry assays revealed that our experimental design and conditions failed to
effectively activate
the VVD domain. This issue, which could stem from several factors, is currently under further
investigation.
Previous results have demonstrated that AiiA possesses intrinsic self-assembly activity. We believe
that
achieving
light-induced degradation of AHL by AiiA is feasible, provided that the light-induced dimerization
function of VVD
is properly restored. However, due to time constraints, this particular challenge remains
unresolved.

### Light-inducible protein degradation

Cycles in this section:

* [Cycle 1: Explore degradation
  conditions
  and effects of LOVdeg](#lovdeg-cycle1-title)

#### Cycle 1 Explore degradation conditions and effects of LOVdeg

##### Design

To test the blue-light-inducible protein degradation effect of LOVdeg [6], we fused LOVdeg tag to
mCherry and induced the fused protein expression with IPTG.(Figure 5-1)

Because of the blue light induction, to avoid the quenching effect of blue light, we use the red
fluorescent protein mCherry instead of GFP. Throughout this part, we performed experiments using low
copy number plasmids pSC101 that result in moderate levels of a given protein of interest, for the
reason that degradation as the sole mode of gene expression control may be limiting when proteins
are at very high levels.

![Figure 5-1]({{ cdn_url('engineering/5-1new.png') }})

**Figure 5-1.** The genetic circuits of
mCherry-LOVdeg
and the control group.

##### Build

We utilized the pJUMP27-1A (sfGFP) vector from the iGEM distribution kit to insert the LacI fragment
and mCherry-LOVdeg expression circuit into it via PCR and Gibson assembly, constructing the
pSC101-LacI-mCherry-LOVdeg plasmid. Concurrently, we constructed the control pSC101-LacI-mCherry
plasmid. After construction, we transformed these plasmids into *E. coli*. (Figure 5-2, 5-3)

![Figure 5-2]({{ cdn_url('engineering/5-2-1.png') }})
![Figure 5-2]({{ cdn_url('engineering/5-2-2.png') }})

**Figure
5-2.** The genetic circuits and the corresponding maps of plasmids.
**(A)** pSC101-LacI-mCherry plasmid. **(B)**
pSC101-LacI-mCherry-LOVdeg plasmid.

← Swipe left/right to view full content →

![Figure 5-3]({{ cdn_url('engineering/5-3new.png') }})

**Figure 5-3.** The history of
pSC101-LacI-mCherry-LOVdeg plasmid construction.

##### Test

We performed colony PCR and sequencing to verify successful plasmid construction. Sequencing
confirmed transformation success.

With these bacteria and 1M IPTG induction, we conducted the agar plate colony experiment to
determine whether they could emit red fluorescence, and to validate the LOVdeg tag-mediated
degradation of the fluorescent protein.

At first, the plates in both the light and dark groups were cultured covered with aluminum foil to
block light. At 14 hours, the light group was exposed to blue light for 3 hours, while the dark
group continued light-blocked incubation. Imaging was performed at 18 hours to generate
fluorescence-input distance curves. (Figure 5-4)

![Figure 5-4]({{ cdn_url('engineering/5-4.png') }})

**Figure 5-4.** The result of agar plate colony
experiment 18 hours after inoculation, which showed the dose response curves of mCherry
fluorescence.

##### Learn

We observed a significant brightness difference between the LOVdeg-dark and LOVdeg-light group.
However, no such difference was detected between the control-light and LOVdeg-light groups. One
possibility is that the blue-light illumination time was insufficient. Alternatively, blue light
might have arrested bacterial growth [2], so that 3 h of irradiation was not enough to let the
fluorescence of the control and LOVdeg light groups diverge.

Therefore, we plan to extend the lighting time in order to further observe the degradation effect.

##### Re-test

Based on the previous cycle, we find an appropriate input distance and subsequently conducted agar
plate colony experiments with blue light induction for 10 hours.(Figure 5-5)

![Figure 5-5]({{ cdn_url('engineering/5-5.png') }})

**Figure 5-5.** In agar plate colony experiments,
the
fluorescence of colony showed mCherry protein levels in response to blue light. After 12h
cultured
in dark, the light group was exposed to blue light for 10 hours.

##### Learn

It was successfully verified that LOVdeg tag has a degradation effect under the 10h induction of blue
light.

We plan to replace the plasmid promoter with the QS system promoter to verify whether it can accept
AHL ligands and emit fluorescence, and whether the fluorescent protein can be degraded upon exposure
to blue light.

## References

1. Scott SR, Hasty J. Quorum Sensing Communication Modules for Microbial Consortia. *ACS
   Synth Biol*. 2016 Sep 16;5(9):969-77. doi: 10.1021/acssynbio.5b00286. Epub 2016 May
   19.
2. Fedorec AJH, Treloar NJ, Wen KY, Dekker L, Ong QH, Jurkeviciute G, Lyu E, Rutter JW, Zhang
   KJY, Rosa L, Zaikin A, Barnes CP. Emergent digital bio-computation through spatial diffusion
   and engineered bacteria. *Nat Commun*. 2024 Jun 8;15(1):4896. doi:
   10.1038/s41467-024-49264-3.
3. Jonkergouw C, Savola P, Osmekhina E, van Strien J, Batys P, Linder MB. Exploration of
   Chemical Diversity in Intercellular Quorum Sensing Signalling Systems in Prokaryotes.
   *Angew Chem Int Ed Engl*. 2024 Jan 8;63(2):e202314469. doi: 10.1002/anie.202314469.
   Epub 2023 Nov 10.
4. Abramson, J., Adler, J., Dunger, J. et al. Accurate structure prediction of biomolecular
   interactions with AlphaFold 3. *Nature* 630, 493–500 (2024). <https://doi.org/10.1038/s41586-024-07487-w>
5. Dagliyan, Onur, Andrey Krokhotin, Irem Ozkan-Dagliyan, Alexander Deiters, Channing J. Der,
   Klaus M. Hahn, and Nikolay V. Dokholyan. "Computational design of chemogenetic and
   optogenetic split proteins." *Nature Communications* 9, no. 1 (2018): 4042.
6. Nathan Tague, Cristian Coriano-Ortiz, Michael B Sheets, Mary J Dunlop (2024) Light-inducible
   protein degradation in *E. coli* with the LOVdeg tag *eLife* 12:RP87303. <https://doi.org/10.7554/eLife.87303.3>
7. Baloban, M., Manoilov, K.Y., Karasev, M.M. et al. Photoswitchable intein for light control of
   covalent protein
   binding and cleavage. *Nat Commun* 16, 8263 (2025). <https://doi.org/10.1038/s41467-025-63595-9>
8. Fan, J.Y., Cui, Z.Q., Wei, H.P., Zhang, Z.P., Zhou, Y.F., Wang, Y.P., Zhang, X.E. Split mCherry
   as a new red
   bimolecular fluorescence complementation system for visualizing protein-protein interactions in
   living cells.
   *Biochem Biophys Res Commun* 367(1), 47-53 (2008). <https://doi.org/10.1016/j.bbrc.2007.12.101>

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# EXPERIMENTS

* [DNA Cloning Methods▼](#nucleic-acid-experiments)
  + [PCR](#pcr)
  + [Transformation](#transformation)
  + [Plasmid Extraction](#plasmid-extraction)
  + [Gel Extraction](#gel-extraction)
  + [PCR Purification](#pcr-purification)
  + [Restriction Cloning](#restriction-cloning)
  + [Seamless Cloning](#seamless-cloning)
  + [Casting an Agarose Gel](#casting-agarose-gel)
  + [Gel Electrophoresis](#gel-electrophoresis)
* [Protein Analysis ▼](#protein-experiments)
  + [Coomassie Blue Staining](#culture-characterization)
  + [GST Affinity Column](#gst-affinity-column)
* [Functional Verification Assays ▼](#measurement)
  + [Liquid Culture Characterization](#liquid-culture-characterization)
  + [Agar Plate Colony Experiments](#agar-plate-colony-experiments)

# Experiments

## DNA Cloning Methods

### PCR

* 1. Set up assembly reactions in PCR-tubes for a total volume of 50 µL as follows:

| Component | Volume |
| --- | --- |
| ddH2O | up to 50µl |
| 2×Phanta Max Buffer | 25µl |
| dNTP Mix (10mM each) | 1µl |
| Forward Primer (10 µM) | 2µl |
| Reverse Primer (10 µM) | 2µl |
| Phanta Max Super-Fidelity DNA Polymerase | 1µl |
| Template DNA | xµl |

* 2. Mix gently and spin down.
* 3. Place PCR-tubes in thermocycler and run the following program:

| Step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 95°C | 3 min | 25~35 cycles |
| Denaturation | 95°C | 15 sec |
| Annealing | 56 ~ 72 °C | 15 sec |
| Extension | 72°C | 30 -60 sec/kb |
| Final Extension | 72°C | 5 min |

### Transformation

* 1. Thaw chemically competent cells on ice.
* 2. Add DNA (30ng), and pipette gently to mix.
* 3. Incubate the suspension for 25 minutes on ice.
* 4. Heat shock is done by incubating the cells for 45 seconds at 42℃.
* 5. Chill on ice for 2 min.
* 6. Add 700μl of LB medium and incubate for 1 hour at 37℃, 200rpm in order to obtain antibiotic
  resistance.
* 7. Centrifuge at 5000rpm for 1min, discard the supernatant to 100 μl and spread onto a plate with
  appropriate antibiotics.
* 8. Incubate overnight at proper temperature.

### Plasmid Extraction

* 1. Transfer 1 - 5 ml of overnight (12 - 16 h) culture to a centrifuge tube, and centrifuge at 10,000
  rpm (11,500 × g) for 1 min. Discard the culture medium and place the tube inverted on a blotting
  paper to drain the liquid.
* 2. Add 250 μl of Buffer P1 (make sure that RNase A has been added) to the centrifuge tube containing
  the precipitated bacterial cells, and mix thoroughly by pipetting or vortexing.
* 3. Add 250 μl of Buffer P2 to the mixture from Step 2. Mix by gently inverting the tube 8 - 10 times
  to completely lyse the cells.
* 4. Add 350 μl of Buffer P3 to the mixture from Step 3. Immediately invert the tube gently 8 - 10
  times to fully neutralize Buffer P2. At this time, white flocculent precipitates should form.
  Centrifuge at 12,000 rpm (13,400 × g) for 10 min.
* 5. Place FastPure DNA Mini Columns into a Collection Tube 2 ml. Carefully transfer the supernatant
  from Step 4 to the FastPure DNA Mini Columns with a pipette, taking care not to disturb the
  precipitates. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place
  the FastPure DNA Mini Columns back into the Collection Tube.
* 6. Add 600 μl of Buffer PW2 (make sure that absolute ethanol has been added) to the FastPure DNA
  Mini Columns. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place
  the FastPure DNA Mini Columns back into the Collection Tube.
* 7. Repeat Step 6.
* 8. Place the FastPure DNA Mini Columns back into the Collection Tube. Centrifuge the empty column at
  12,000 rpm (13,400 × g) for 1 min to completely remove the residual wash buffer.
* 9. Place the FastPure DNA Mini Columns in a new sterile 1.5 ml centrifuge tube. Add 30 -
  100 μl of Elution Buffer to the center of the spin column membrane. Leave the system at room
  temperature for 2 min, and centrifuge at 12,000 rpm (13,400 × g) for 1 min to elute the DNA.
* 10. Discard the FastPure DNA Mini Columns and store the extracted DNA at -20°C to prevent
  degradation.

### Gel Extraction

* 1. Column equilibration: add 500 µl Buffer BL to the Spin Column CA2 (put Spin Column CA2 into a
  collection tube). Centrifuge for 1 min at 12,000 rpm in a table-top microcentrifuge. Discard the
  flowthrough, and put Spin Column CA2 back into the collection tube.
* 2. Cut the DNA fragment from agarose gel with a clean, sharp scalpel. Weigh the gel slice in a clean
  1.5 ml EP tube.
* 3. Add equivalent volume of Buffer PN to the gel (If the gel is 0.1 g, it is defaulted to be 100μl,
  then add 100μl Buffer PN). Incubate at 50°C by inverting up and down the tube until the agarose gel
  dissolves completely. (If DNA fragment is <300 bp, it is recommended to add isopropanol which is 1/2
  volume of Buffer PN to the agarose gel sample after the gel is completely dissolved.)
* 4. When the gel dissolves completely and the solution temperature turns to room temperature,
  transfer the mixture to the Spin Column CA2 (put Spin Column CA2 into a collection tube). Let the
  column stand for 2 min at room temperature, then centrifuge for 30-60 sec at 12,000 rpm in a
  table-top microcentrifuge. Discard the flow-through; place the Spin Column CA2 back into the
  collection tube again.
* 5. Wash the Spin Column CA2 with 600 µl Buffer PW (ensure that ethanol has been added) and
  centrifuge for 30-60 sec at 12,000 rpm. Discard the flow-through and place the Spin Column CA2 back
  into the collection tube.
* 6. Repeat Step 5.
* 7. Place the Spin Column CA2 back to the collection tube and centrifuge at 12,000 rpm for 2 min to
  remove residual Buffer PW. Discard the flow-through, and place column with the cap open for several
  minutes to air dry the membrane.
* 8. Transfer the Spin Column CA2 to a clean 1.5ml centrifuge tube. Add appropriate volume of Buffer
  EB to the center of the membrane, incubate at room temperature for 2 min, then centrifuge at 12,000
  rpm for 2 min.

### Purification of PCR Products

* 1. Column equilibration: add 500 μl Buffer BL to the Spin Column CB2 (put Spin Column CB2 into a
  collection tube). Centrifuge for 1 min at 12,000 rpm. Discard the flow-through, and then place Spin
  Column CB2 back into the collection tube.
* 2. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix.
* 3. Transfer the mixture to the Spin Column CB2, incubate at room temperature for 2 min. Centrifuge
  for 30-60 sec at 12,000 rpm in a table-top microcentrifuge. Discard the flow-through, and then place
  Spin Column CB2 back into the same collection tube.
* 4. Add 600 µl Buffer PW (ensure that ethanol (96-100%) has been added) to the Spin Column CB2 and
  centrifuge for 30-60 sec at 12,000 rpm. Discard the flow-through, and place Spin Column CB2 back in
  the same collection tube.
* 5. Repeat step 4.
* 6. Centrifuge at 12,000 rpm (~13,400×g) for 2 min to remove residual Buffer PW. Discard the
  flow-through, and allow the column to air dry with the cap open for several minutes to dry the
  membrane.
* 7. Place the Spin Column CB2 in a clean 1.5 ml microcentrifuge tube. Add 30-50 μl Buffer EB to the
  center of membrane, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm.

### Restriction Cloning

#### 1.Digestion

* (1) Reaction system:

| Component | Volume |
| --- | --- |
| ddH2O | up to 50µl |
| Restriction Enzyme 1 | 1μl |
| Restriction Enzyme 2 | 1μl |
| DNA | 1μg |
| 10X NEBuffer | 5μl (1X) |

* (2) Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction
  tube.
* (3) Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
* (4) Incubate for 1 hour at the enzyme-specific appropriate temperature.

#### 2. Ligation

* (1) Reaction system:

| Component | Volume |
| --- | --- |
| ddH2O | up to 20μl |
| 10X Ligation Buffer | 2μl |
| Vector DNA (4 kb) | 50 ng (0.020 pmol) |
| Insert DNA (1 kb) | 37.5ng(0.060 pmol) |
| T4 DNA Ligase (350U/μl) | 1μl |

* (2) Program: 16℃ for 1-5h

### Seamless Cloning

#### 1. Calculation of the amount of vectors and fragments

The total insertion volume of the optimal fragment and carrier in the recombinant reaction system is
0.02-1 pmol, 1-3 fragments is 0.02-0.5 pmol, and 4-6 fragments is 0.5-1 pmol. The optimum molar
ratio
of vector to insert fragment was 1:3. The corresponding DNA quality can be calculated by the
following
formula:

* (1) Linearized vector usage (ng) =(the number of base pairs × 0.65 × vectors and fragments pmol)
  / (1+3n)
* (2) Insert fragment usage (ng) =(the number of base pairs × 0.65 vectors and fragments pmol × 3)
  / (1+3n)
* (3) n represents the number of inserted fragments

#### 2. Recombination reaction system

It is recommended to prepare on ice. All components should be mixed well before use.

| Component | 1-3 fragments | 4-6 fragments |
| --- | --- | --- |
| ddH2O | Up to 20µL | Up to 20µL |
| Hieff Clone™ Universal One Step Cloning Kit | 10µL | 10µL |
| Total fragments | 0.02-0.5 pmol | 0.5-1 pmol |

#### 3. Recombination reaction conditions

* (1) After the preparation of the system, mix them gently and collect the reaction solution to
  the bottom of the tube by brief
  centrifugation.
* (2) When one fragment is inserted and the total amount of DNA is less than 300 ng, the
  recommended reaction condition is 50 ℃ for 5 min; When the number of inserted fragments is 2-4,
  the recommended reaction condition is 50 ℃, 15 min; When the number of inserted fragments is
  5-6, the recommended reaction condition is 50℃ for 30 min. lt is recommended that the reaction
  be carried out on instruments with accurate temperature control such as PCR instrument or water
  bath.
* (3) The reaction products can be transformed directly or stored at - 20℃ and thawed and
  transformed when necessary.

### Agarose Gel Preparation

* 1. Mix the agarose powder with TAE Buffer.
* 2. Boil the mixture in a microwave until agarose is completely dissolved (1-2 min).
* 3. Add the DNA Dye.
* 4. Camp the casting tray so it will not leak and put in the correct comb. Pour the liquid gel
  slowly into the chamber to avoid bubbles, and wait for it to get solidified.

### Agarose Gel Electrophoresis

* 1. Carefully remove the comb. Put the gel in a running chamber with the wells facing the
  cathode. Fill the chamber with 1X TAE buffer until the gel is completely submerged.
* 2. Load the samples.
* 3. Put the cover on the chamber and attach the plugs to the power supply. Run the gel at 120 V
  for 45 minutes.
* 4. Take a picture of the gel using the gel imager.

## Protein Analysis

### Coomassie Blue Staining

* 1. Wash the gel (the purpose of this step is to wash away interfering substances such as SDS
  from the gel to reduce the staining background and increase the staining sensitivity):

+ (1) Place the gel in a suitable container, add 50ml of deionized water and microwave on high
  for 3min.
+ (2) (Optional) Pour off the deionized water, add 50ml of deionized water and microwave on
  high for 3min. Adding this step can improve the detection sensitivity about 3-10 times.
+ (3) Shake for 5 min on a orbital shaker or horizontal shaker.

* 2. Staining: pour off the liquid carefully, aspirate the residual liquid, add about 20 ml of
  rapid staining solution of Coomassie Brilliant Blue and stain for 10-30 min. Add an appropriate
  amount of rapid staining solution (about 20ml per 8-cm gel), so that the staining solution can
  cover the gel, and the surface of the
  liquid is at least three gels above the thickness of the gel is appropriate, room temperature
  (20-25℃) in the orbital shaker for staining. The
  staining time at room temperature is 10-30 min, and the actual staining time can be adjusted
  according to the staining effect.
* 3. Decolorization (optional): Add about 100ml of deionized water and shake on a shaker to
  decolorize. Every 5-15 min,carefully pour off the liquid, add 100 ml of deionized water and
  continue to decolorize the gel on a shaker. Usually 30-120 min of decolorization will result in
  a very low background or even perfect gel staining.

### GST Affinity Column Protein Purification

Based on experimental results and observations, the protocol from beyondtime kit has been revised and
optimized as follows:

#### 1. IPTG-Induced Expression of GST-Tagged Protein in E. coli

The following protocol describes the widely used IPTG-induced expression system. For optimization of
induction conditions, please refer to the detailed instructions specific to your expression system.
Other induction systems should follow their respective guidelines.

* a. Pick a single colony expressing the GST-tagged protein and inoculate it into 3-10 ml of LB medium
  containing the appropriate antibiotic. Incubate overnight at 37°C with shaking.
* b. Dilute the overnight culture 1:20 into fresh, pre-warmed (37°C) LB medium containing the
  appropriate antibiotic. For example, inoculate 5 ml of overnight culture into 100 ml of pre-warmed,
  antibiotic-supplemented LB. The culture volume depends on the required protein yield: 3-10 ml for
  initial small-scale verification; 100-200 ml for routine expression and purification; and 1 L or
  more for preparative-scale purification. For improved expression outcomes, a 1:100 dilution of the
  overnight culture is recommended, although this will require a longer incubation time to reach the
  target OD.
* c. Incubate the culture at 37°C with shaking for approximately 30-60 minutes, or until the OD₆₀₀
  reaches 0.6-1.0.
* d. Add IPTG to a final concentration of 1 mM and continue incubation at 37°C for 2-4 hours to induce
  protein expression.

***Note:** Prior to IPTG addition, remove a small aliquot of the culture to serve as an
uninduced control. This control can be incubated separately for the same duration or sampled
directly. Optimal IPTG concentration, induction temperature, and induction time should be determined
experimentally for each specific protein.*

* e. Transfer the culture to centrifuge tubes. Harvest the cells by centrifugation at 4,000 × g for 20
  minutes or 15,000 × g for 1 minute at 4°C. Discard the supernatant. The cell pellet can be processed
  immediately for lysis or stored at -20°C or -80°C for future use. Frozen pellets should be thawed on
  ice for 15 minutes before proceeding.

#### 2. Reagent Preparation

* a. **10X GSH Solution:** Dissolve the 184 mg of GSH provided in the kit in 6 ml of the
  supplied Elution Buffer (requires GSH addition). Mix thoroughly to obtain the 10X GSH solution. The
  prepared solution can be stored at -20°C and remains stable for at least one year.
* b. **Elution Buffer:** Mix the provided Elution Buffer (requires GSH addition) with the
  10X GSH solution at a 9:1 ratio (e.g., combine 9 ml of Elution Buffer with 1 ml of 10X GSH
  solution). The resulting mixture is the working Elution Buffer. Due to the susceptibility of GSH to
  oxidation, the Elution Buffer should be prepared fresh and can be stored at 4°C for up to two weeks.
  The final composition of the Elution Buffer is 50 mM Tris, 150 mM NaCl, 10 mM GSH, pH 8.0.

#### 3. Large-Scale Purification of GST-Tagged Protein

This method is suitable for purifying larger quantities of protein (e.g., from a starting culture volume
of 150 ml or more).

* a. Following step 1(e), for fresh or thawed bacterial pellets, add Lysis Buffer at a ratio of 4 ml
  (a range of 2-5 ml is acceptable) per gram of wet cell pellet. Resuspend the cells thoroughly. If
  necessary, add an appropriate protease inhibitor cocktail (e.g., Beyotime's P1025/P1026 for
  bacterial extracts or P1005/P1006 general-use protease inhibitor cocktail) to the lysis buffer
  before cell disruption.
* b. Add lysozyme to a final concentration of 1 mg/ml and mix thoroughly. Incubate the suspension on
  ice or in an ice-water bath for 30 minutes.

***Note:** Lysozyme can be prepared as a 100 mg/ml stock solution in Lysis Buffer,
aliquoted, and stored at -20°C. It should be added to the sample just before use.*

* c. Lyse the cells using sonication on ice. Apply sonication at a power of 200-300 W, using pulses of
  10 seconds followed by 10-second intervals, for a total of 6 cycles.

***Note:** The specific sonication protocol should be optimized for the particular
ultrasonic instrument used.*

* d.(Optional) If the lysate remains highly viscous after sonication, add RNase A to a final
  concentration of 10 µg/ml and DNase I to 5 µg/ml, and incubate on ice for 10-15 minutes.
  Alternatively, shear the genomic DNA by repeatedly passing the lysate several times through a
  syringe fitted with a narrow-gauge needle.
* e. Centrifuge the lysate at 10,000 × g for 20-30 minutes at 4°C. Collect the clarified supernatant
  and keep it on ice or in an ice-water bath. Save 20 µl of the supernatant for subsequent analysis.

***Note:** The supernatant must be clear and free of insoluble material before proceeding
to the purification step. The presence of insoluble impurities will significantly compromise the
purity of the final protein product.*

* f. Take 1 ml of well-resuspended BeyoGold™ GST-tag Purification Resin. Centrifuge at 1,000 × g for
  10 seconds at 4°C and discard the storage solution. Add 0.5 ml of Lysis Buffer to resuspend and
  equilibrate the resin. Centrifuge again and discard the liquid. Repeat this equilibration step 1-2
  more times. After the final equilibration, add approximately 4 ml of the clarified bacterial lysate
  supernatant to the resin. Incubate at 4°C with gentle shaking on a orbital shaker for 60 minutes.
* g. Load the mixture of lysate and BeyoGold™ GST-tag Purification Resin into the provided affinity
  chromatography empty column (3 ml).

***Note:** An alternative method is to first prepare a column with 1 ml of the 50%
BeyoGold™ GST-tag Purification Resin slurry, equilibrate it with 0.5 ml of Lysis Buffer (2-3 times),
then load the ~4 ml of clarified lysate supernatant. The flow-through can be collected and reloaded
onto the column 3-5 times to enhance binding efficiency. While the batch binding method (mixing
before loading) is slightly more cumbersome, it often promotes better binding of the GST-tagged
protein to the resin.*

* h. Open the bottom cap of the purification column and allow the liquid to drain out by gravity.
  Collect approximately 20 µl of the flow-through for subsequent analysis.
* i. Wash the column 5 times, each time with 0.5-1 ml of Lysis Buffer. Collect about 20 µl of the wash
  flow-through after each wash for subsequent analysis. During the washing and subsequent elution
  steps, the protein content in the washes and eluates can be monitored quickly using the Bradford
  method (P0006) to determine if the number of washes or elutions should be adjusted.

***Note:** If the purity of the final protein is insufficient, the number of washes can
be increased by 2-3 times. If required, the GST tag can be cleaved from the fusion protein using
enzymes such as PreScission Protease (P2302/P2303), TEV Protease (P2307/P2308), or Thrombin to
release the target protein. Refer to step 5 for detailed methods on GST tag cleavage and
removal.*

* j. Elute the target protein 6-10 times, each time with 0.5 ml of Elution Buffer. Collect each
  elution fraction into separate centrifuge tubes. The collected eluates contain the purified
  GST-tagged protein samples.

## Functional Verification Assays

### Liquid Culture Characterization

* 1. The optical density of overnight cultures is measured at 600 nm and then diluted at a specific
  ratio.
* 2. The diluted culture is incubated at 37 °C with shaking for 3 h. (If induction of AHL receptor
  protein expression is required, 0.5 mM IPTG is added at a final concentration when the OD600 reaches
  0.6, followed by a 1-hour induction period.)
* 3. Bacterial culture and the appropriate signal molecule solution are mixed in the wells. The total
  volume in each well is adjusted to 200 µL using medium.
* 4. The plate is placed in the plate reader and incubated at 37 °C with continuous shaking for 3 h.
  Absorbance (600 nm) and fluorescence (excitation 484 nm, emission 510 nm) are measured every 6 min.

### Agar Plate Colony Experiments

This protocol is adapted from Fedorec et al. (2024).

* 1. The plates, which are filled with about 12 mL of solid medium containing the appropriate
  antibiotic, are allowed to dry at 55 °C for ~2.5 min in a laboratory oven to prevent interference
  from condensed water.
* 2. The optical density of overnight cultures is measured at 600 nm, and the cultures are diluted to
  an OD600 of 0.6.
* 3. 0.3 μL of the diluted culture and 1.0 μL of the appropriate signal molecule solution are
  dispensed onto the surface of the agar at the positions shown in the corresponding spotting patterns
  (placed beneath the culture dish).
* 4. The plates are left to dry until the droplets are no longer visible (approximately 15 min).
* 5. The plates are then placed in an incubator at 37 °C and imaged with a fluorescence microscope at
  12 h, 15 h, 18 h, 21 h, and 24 h to monitor growth and fluorescence.

{% include 'footer2.html' %}
{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

# RESULTS

* [Part 1: Orthogonal Quorum Sensing Systems
  ▼](#part1)
  + [Preliminary Experiment](#preliminary-experiment)
  + [High-pass Engineered Bacteria](#high-pass-engineered)
  + [Band-pass Engineered Bacteria](#band-pass-engineered)
  + [Orthogonality](#orthogonality)
  + [Connector Bacteria](#connector-bacteria)
* [Part 2: Construction of Logic Gates
  ▼](#part2)
  + [High-pass Logic Gates](#high-pass-logic)
  + [Band-pass Logic Gates](#band-pass-logic)
  + [Carry Input Simulation](#carry-input-simulation)
* [Part 3: Light-inducible Degradation
  ▼](#part3)
  + [AHL Degradation Enzyme](#ahl-degradation)
  + [Protein Degradation Tag](#protein-degradation)

# Results

## Part 1: Orthogonal Quorum Sensing Systems with Biosensors

As the foundation of our project, we constructed a variety of AHL-induced biosensors. A series of
experiments was conducted to test diffusion response curves and to identify the orthogonal groups
between AHL-biosensors pairs.

### Preliminary Experiment

To validate the feasibility of our diffusion and serial experiments, we used the plasmids LasR-deGFP
and
LuxR-deGFP from [2024
WHU\_China](https://2024.igem.wiki/whu-china/) with *E. coli* BL21(DE3) strain, constructing high-pass engineered bacteria
with fluorescence induced by 3-oxo-C12 HSL (LasR-deGFP strain) and 3-oxo-C6 HSL (LuxR-deGFP strain).

After confirming the sequences of the engineered bacteria, we established the solid diffusion
response
curves for the LasR-GFP and LuxR-GFP reporter bacteria using agar plate colony experiments (Figure
1A)
and
fluorescence microscopy (Figure 1B). The fluorescence intensity decreases with the increasing
diffusion
distance. At higher AHL concentrations, more colonies exhibit detectable fluorescence (Figure 1C).

![Preliminary experiment]({{ cdn_url('results/fig1.png') }})

**Figure. 1** Fluorescence photos and response
curves of
colonies on solid plate. **(A)** The spotting pattern. The center of the plate is
the
place where AHL solution is added. **(B)** Overview of the plate. It can be seen
that
the
fluorescence response intensity decreases with the increase of the distance from the AHL added
point. **(C)** Fluorescent photos of colonies at different diffusion distances
under
four
3-oxo-C12 HSL concentrations, each concentration repeated twice. **(D)** The dose
response curve corresponding to distance of four concentrations of 3-oxo-C12 HSL after 12 hours
of
incubation.

![Preliminary experiment]({{ cdn_url('results/fig2.png') }})

**Figure. 2** The dose response curve of lasR-deGFP
engineered strain to 3-oxo-C12 HSL after 12 hours of incubation in agar plate colony
experiments.
**(A)** The dose response curve of lasR-deGFP engineered strain to 3-oxo-C12 HSL.
**(B)**
The dose response curve of LuxR-deGFP engineered strain to 3-oxo-C6 HSL.

In the agar plate colony experiments, the fluorescence intensity of LasR-GFP reporter decreases
significantly with the increase of distance (Figure 1D, Figure 2). Biosensor of 3-oxo-C6 HSL does
not
show a good decline curve, presumably because proximal high AHL concentrations perturbed
engineered-bacterium metabolism or colony growth, whereas distal fluorescence arose from leakage
expression.

### High-pass Engineered Bacteria

Based on the preliminary experiments, we constructed the biosensor bacteria to detect C4-HSL,
3-oxo-C8
HSL, 3-OH-C14:1 HSL molecules, and further established solid diffusion response curves for these
strains, including 3-oxo-C12 HSL biosensor bacteria for extending our selection of orthogonal AHL
systems. (Figure 3)

![Preliminary experiment]({{ cdn_url('results/fig3.png') }})

**Figure 3** Agar plate colony experiments of
high-pass
engineered bacteria and results. **(A)** The spotting pattern. The center of the
plate
is
the place where AHL solution is added. **(B)** The dose response curve
corresponding to
distance of six concentrations of C4-HSL. **(C)** The dose response curve
corresponding
to distance of six concentrations of 3-oxo-C8 HSL. **(D)** The dose response curve
corresponding to distance of six concentrations of 3-oxo-C12 HSL. **(E)** The dose
response curve corresponding to distance of six concentrations of 3-OH-C14:1 HSL. Although the
culture time increased, the maximum fluorescence intensity remained unchanged. As time went on,
the
colonies located at greater input distance showed fluorescence, indicating the diffusion of AHL
in
solid agar plate.

Although the
culture time increased, the maximum fluorescence intensity remained unchanged. As time went on, the
colonies located at greater input distance showed fluorescence, indicating the diffusion of AHL in
solid agar plate.

### Band-pass Engineered Bacteria

To implement the half adder, we engineered a band-pass strain expressing sfGFP under IPTG induction,
since the IPTG induced strain is more likely to exhibit band-pass characteristics. Following
induction
with specific concentrations of IPTG and AHL, only the colonies with medium diffusion distance
showed
high fluorescence intensity, indicating that band-pass engineered bacteria were successfully
constructed.

![Preliminary experiment]({{ cdn_url('results/fig4.png') }})

**Figure 4** Response curves of band-pass engineered
bacteria. The horizontal axis is the diffusion distance. The negative value of fluorescence
intensity is because the value here is smaller than the negative control without AHL induction.
**(A)** The response curves induced by three concentrations of IPTG solution.
**(B)** Response curves induced by 5m IPTG and 0.1mM 3-oxo-C12 HSL solutions.
**(C)** Response curves induced by 1m IPTG and 10mM C4-HSL solutions.

### Orthogonality

We evaluated the orthogonality of the five AHL signaling systems by quantifying their crosstalk
indices.
The crosstalk index was defined as the ratio of the activation strength caused by a non-cognate
molecule
to that induced by the cognate molecule. As shown in the heatmap (Figure 5), most systems exhibited
limited responses to non-cognate signals (off-diagonal elements, low values). However, certain
degrees
of crosstalk were observed between specific pairs, indicating partial overlap in recognition.

![Preliminary experiment]({{ cdn_url('results/fig5.png') }})

**Figure 5** Crosstalk index of five AHL signaling
systems. The horizontal axis is the AHL system. The vertical axis is the AHL system. The color
is
the crosstalk index.

Our biocomputer design requires three orthogonal AHL systems. Based on the experimental results, we
identified two candidate triplets: (1) 3-oxo-C6/LuxR, C4/RhlR, and C14:1/CinR; (2) C4/RhlR,
3-oxo-C8/TraR, and C14:1/CinR. Because LuxR-based biosensors suffer from strong crosstalk[1], we rejected the first set and adopted the second, whose
orthogonality had been validated previously in the study.

Considering the widespread use and experimental convenience of 3-oxo-C12/LasR in synthetic biology,
we
also included it when needed.

Our project design includes an amplifier, which synthesizes AHL after receiving the inducer to extend
the
diffusion distance. Through agar plate colony experiments, the engineered bacteria as amplifiers
have
the ability to synthesize AHL molecules, but there is leakage expression. (Figure 6)

![Preliminary experiment]({{ cdn_url('results/fig6.png') }})

**Figure 6** Fluorescent photos of amplifiers in
agar
plate colony experiments. The amplifier is inoculated at the square position and the inducer AHL
solution is dripped at the star position.

### Connector Bacteria

In the operation of our biological calculator, the interaction of different QS systems and their
sensors
is involved. In order to verify the feasibility of series connection, we constructed RhlR-LasI and
LasR-RhlI engineered bacteria, and measured the fluorescence intensity of the corresponding
downstream
biosensors.

When the upstream connector received the inducer, the fluorescence intensity of the biosensor was
significantly higher than that without inducer. However, from Figure 7B we can see that there is a
leakage
expression in the connector within 1-2 cm input distance.

![Preliminary experiment]({{ cdn_url('results/fig7.png') }})

**Figure 7** Response curves of biosensors located
downstream
of the connector bacteria. **(A)** Fluorescence intensity of Las biosensor when C4-HSL
(binds
to RhlR) is added upstream the RhlR-LasI connector or not. **(B)** Fluorescence
intensity
of
Rhl biosensor when 3-oxo-C12 HSL (binds to LasR) is added upstream the LasR-RhlI connector or not.

## Part 2: Construction of Logic Gates

After constructing high-pass and band-pass engineered strain, we explored suitable conditions for
them to
be used as logic gates. By changing the concentration, diffusion distance and diffusion time of the
inducer, we constructed **AND** gates, **OR** gates, **XOR**
gates and other logic gates
successfully.

### High-pass Engineered Bacteria

We applied our high-pass bacterial cultures onto the agar plates following the droplet guidance map
shown
below for four different biosensors (Figure 9A). To simulate logic gate inputs, AHL droplets were
deposited on the left (01), right (10) or both sides (11) of the bacterial droplets.. In this way, a
single plate can generate multiple logic gate setups with varying input distances, allowing us to
examine their corresponding outputs. Figure 8 shows the ideal fluorescence intensity of colonies
programmed to perform AND, OR and XOR logic gates.

![Preliminary experiment]({{ cdn_url('results/fig8.png') }})

**Figure 8** Ideal fold change fluorescence of
colonies
when work as different logic gates. Fold change of fluorescence intensity: the fluorescence
intensity of a given input state normalized to the basal state (input “00”, no AHL).
**(A)** Ideal fold change fluorescence of an AND gate. Only when inducer droplets
are
added on both sides, it would show high fluorescence intensity. **(B)** Ideal fold
change
fluorescence of an OR gate. The colony would show high fluorescence intensity as long as the
inducer
is added. **(C)** Ideal fold change fluorescence of an XOR gate. Only when inducer
droplets are added on one side of the colony, it would show high fluorescence intensity.

As shown below, The 3D plots effectively help us visualize the distribution patterns of AND and OR
gates
across different systems, providing a basis for determining the formation range of each gate.

![Preliminary experiment]({{ cdn_url('results/fig9.png') }})

**Figure 9** The 3D plots illustrate our
high-throughput
screening of engineered strains carrying four different AHL systems, evaluated across three
dimensions: AHL concentration, incubation time, and distance from the AHL source.
**(A)**
The droplet guidance map. **(B)** High-throughput screening of the corresponding
logic
gates for High-pass C4-HSL biosensor. **(C)** High-throughput screening of the
corresponding logic gates for High-pass 3-oxo-C8 HSL biosensor. The one on the left comes from a
wider concentration range, in order to obtain better results, we repeated the experiment at
concentrations below 25mM and obtained the result on the right. **(D)**
High-throughput
screening of the corresponding logic gates for High-pass 3-OH-C14:1 HSL biosensor.
**(E)** High-throughput screening of the corresponding logic gates for High-pass
3-oxo-C12 HSL biosensor.

Screening revealed reproducible AND and OR gate responses, albeit with variable signal strengths. The
following figures highlight the representative conditions under which we observed the relatively
pronounced AND and OR gate behaviors in each system.

RhlR
TraR
CinR
LasR

![RhlR Representative conditions]({{ cdn_url('results/fig10-1.png') }})

![TraR Representative conditions]({{ cdn_url('results/fig10-2.png') }})

![CinR Representative conditions]({{ cdn_url('results/fig10-3.png') }})

![LasR Representative conditions]({{ cdn_url('results/fig10-4.png') }})

**Figure 10** The representative conditions under
which
the AND and OR gate behaviors can be observed in each system.

From the results, we observed that OR gates generally produced stronger outputs with clearer signals,
whereas AND gates often showed slightly weaker responses, with partial activation at the 01 and 10
input
states.

To address this limitation, we plan to introduce a T7 RNA polymerase under the control of
AHL-responsive
promoters to drive EGFP expression. This design is expected to sharpen the response curve and
enhance
the overall signal strength.

### Band-pass Engineered Bacteria

The band-pass logic gate **XOR** was inoculated following the same schematic protocol as
the
high-pass logic gate.

During the construction of the band-pass engineered strain, we initially chose IPTG as the inducer,
based
on references [1].

The band-pass response was more evident to the naked eye than in quantitative measurements. Due to
time
constraints, we did not perform high-throughput screening, but we speculate that an IPTG induction
concentration between 0.1-5 M could potentially achieve an even stronger band-pass response.

Nevertheless, these results demonstrate the feasibility of forming a band-pass circuit and provide
strong
support for its future application in the AHL system.

Next, we attempted to partially replace the IPTG circuit with the Las circuit and Rhl circuit.

![Preliminary experiment]({{ cdn_url('results/fig11.png') }})

**Figure 11** The representative conditions under
which
the XOR gate behavior can be observed induced by IPTG and AHL solutions. **(A)**
XOR
gated condition of IPTG band-pass engineered bacteria. **(B)** XOR gated condition
of
Las-IPTG and Rhl-IPTG band-pass engineered bacteria.

Despite time constraints, we have not yet identified a combinatorial scheme capable of implementing
an
XOR logic gate. However, we have already obtained some results as evidence that these systems can
achieve XOR
effect (Figure 4B, Figure 4C).

### Carry Input Simulation

We experimentally simulated a critical step in the computational process: the calculation and
handling of
carry inputs.

Taking the “110” input in the first panel of Figure B as an example, we added 1 mM C4-HSL (as
indicated
in the
figure legend) to the two triangles at the top of the spotting pattern to activate the RhlR-LasI
connector located
at the square below. The connector then produced C12-oxo-HSL, which diffused to the LasR-sfGFP
engineered bacteria located
0.75 cm away from the center (as indicated in the figure legend), representing the first “1” of the
110
input. Simultaneously,
1 mM C12-oxo-HSL (as indicated in the figure legend) was added to one of the two triangles located 2
cm
below the center,
representing the second “1” of the 110 input. In this way, the fluorescence intensity of the central
LasR-sfGFP colony reflects
its response to this input pattern.

The schematic below illustrates our design (Figure 12A). Four parameters were varied in the
experiment:
(i)
C4-HSL concentration activating the RhlR-LasI carry connector (square symbol in Figure 12A), (ii)
the
spacing between
connector Ci-1 and the current bit, and (iii-iv) the concentrations and positions of primary inputs
Ai
and Bi.

Due to experimental constraints, the current bit's unit was implemented using only the high-pass
engineered strain LasR-EGFP (central circle). By measuring its fluorescence intensity, we were able
to
determine its response to the three inputs and thus infer the computed output.

The input combination 000 indicates that the connector is present on the plate but not activated with
AHL. 100 corresponds to activating only the connector, while 001 indicates only one of the primary
inputs (Ai or Bi) is present. 011 means both A and B are present, and 110 corresponds to the
connector
plus one of the primary inputs (Ai or Bi).

Each output was named using hexadecimal notation, which can be converted to an eight-bit binary
format
representing the corresponding input combination.

To reduce the workload of initial screening, the inputs A and B were considered equivalent, and the
combinations 010 and 101 were omitted from both the experiments and the truth table.

![Preliminary experiment]({{ cdn_url('results/fig12.png') }})

**Figure 12** Carry input simulation.
**(A)**
The droplet guidance map of carry input simulation on agar plate. The two small triangles at the
top
represent the primary inputs from the previous bit, while the square beneath them represents the
previous bit's computational unit, responsible for generating the carry signal (Ci-1). In our
experiment, this unit is implemented as an RhlR-LasI AHL connector, which receives C4-HSL and
produces C12-oxo-HSL. The central circle represents the current bit's computational unit, which
receives the carry signal along with the primary inputs (Ai and Bi) from the left and right
triangles below, where the C12-oxo-HSL for LasR were added. **(B)** Experimental
results
of carry input simulation under different spatial and temporal configurations. Each panel shows
the
fold change of fluorescence output under various input states (000-111), with the corresponding
truth table and hexadecimal notation listed below. The input combination "000" indicates that
the
connector (RhlR-LasI) is present on the plate but not activated by AHL. "100" corresponds to
activating only the connector, while "001" indicates the presence of only one primary input (Ai
or
Bi). "011" represents both primary inputs (A and B), and "110" corresponds to the connector plus
one
primary input. To simplify screening, the symmetric combinations "010" and "101" were omitted.
The
bar plots show fold change fluorescence at each state, and representative images of colonies are
displayed above each plot.The specific activation conditions of the connector (RhlI) and the
input
signals A and B (LasR)—including concentration and distance—are indicated in the schematic
legends
on the right side of each panel.

The experimental results demonstrate that the current bit's computation can produce outputs
corresponding
to multiple logic gates depending on the input conditions. However, the carry input (Ci-1) does not
yet
exert the same weight as the primary inputs A and B, preventing the output from perfectly matching
the
expected computational behavior.

We plan to further optimize factors such as distance, timing, and inducer concentration, aiming to
balance the influence of the previous carry input and the two primary inputs. Achieving
approximately
equal weighting would allow all three inputs to contribute equally to the current bit's computation.

## Part 3: Light-inducible Degradation

### AHL Degradation Enzyme

In this part, we used the constructed biosensors to quantify AHL depletion. Microplate assays
confirmed
that AiiA degrades all tested AHL variants efficiently. AiiA expression was induced in the degrading
bacteria, which were then incubated with AHL in 2YT medium After incubation, the degrading bacteria
were
removed by centrifugation. Then the detection bacteria and fresh 2YT medium were added to detect the
remaining AHL concentration in the 96-well plate.

![Preliminary experiment]({{ cdn_url('results/fig13.png') }})

**Figure 13** Degradation conditions are indicated
in
the
figure. "None" represents the sample without any degradation process. "Background" is the
control
group containing solely fresh 2YT medium and the engineered biosensor *E.coli* strain.
**(A)** Degradation of C4-HSL at a final concentration of 0.05 mM.
**(B)**
Degradation of 3-oxo-C6 HSL at a final concentration of 0.1 μM. **(C)** Degradation
of
3-oxo-C12 HSL at a final concentration of 1 μM. **D** Degradation of 3OC-14:1 HSL
at a
final concentration of 0.1 μM.

For detailed methods, please refer to the [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) and [Measurement](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D) sections. AiiA's broad substrate spectrum and high degradation
efficiency met the requirements of our project. Subsequently, we purified AiiA using a GST-affinity
tag
and preliminarily validated its activity on solid media (for details, see Supplementary Materials),
facilitating further experiments.

In order to further understand AiiA's structural characteristics, we performed molecular dynamics
(MD)
simulations. The computational methods and parameters are described in the [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) section and
Supplementary Materials.

![Preliminary experiment]({{ cdn_url('results/fig14.png') }})

**Figure 14** Molecular dynamics simulations of
AiiA.
**(A)** RMSF analysis of AiiA molecular dynamics simulations. The amino acid
ultimately
selected as the split site is highlighted in red line. Residues with high flexibility located
near
the AiiA active site are shown in blue line. Potential alternative split sites identified within
flexible regions are marked in green line. **(B)** Results of the SPELL algorithm.
The
Split Energy (for details, see [Reference SPELL]) is a parameter primarily used to evaluate the
intrinsic propensity of the split protein fragments to reassemble spontaneously in the absence
of
other driving factors. **C** Molecular dynamics simulation of the split fusion
protein.
This panel shows the simulation results for the fusion protein (incorporated with the VVD
domain)
after splitting.

To identify flexible regions, we employed Root Mean Square Fluctuation (RMSF) analysis. However, not
all
flexible regions are suitable candidates, as active sites can also exhibit flexibility. Therefore,
we
examined the AiiA protein structure and its ligand-binding sites to exclude potential functional
regions, such as residues 66, 135, and 168.

The SPELL algorithm provided Split Energy and Solvent Accessible Area (SAA) data for each residue.
This
information helps us to understand the self-assembly activity and hydrophilicity of the split site.
Molecular dynamics (MD) simulations were used to assess the structural stability of the split fusion
proteins and to provide supplementary evidence for the SPELL results. For instance, the region
spanning
residues 150-200 was identified as a critical hydrophobic core that contributes to the compactness
and
stability of the AiiA structure.

Based on these in silico findings, we initially selected three split sites for subsequent
experimental
validation.

Following the identification of these candidate sites, we constructed the corresponding plasmids and
proceeded with functional assays. Subsequently, the degradation efficiency experiment was carried
out
using the same experimental method as mentioned above.

![Preliminary experiment]({{ cdn_url('results/fig15.png') }})

**Figure 15** Functional validation of split-AiiA
constructs under different conditions. Legends in the figure indicate the degradation
conditions.
"None" represents the sample without any degradation. "Background" is the control group
containing
only fresh 2YT medium and the engineered biosensor E.coli strain. The AHL molecule used was
C4-HSL
at a final concentration of 0.05 mM. **(A)** Validation of the experimental method.
Using
BL21(DE3) strains without AiiA expression does not affect the final response intensity.
**(B)** Results for the AiiA split at 208aa in the Dark or Light(blue LED light)
environment under degradation conditions at 37°C . **(C)**, **(D)**
Results
for three chosen split sites in the Dark environment under degradation conditions at 37°C and
25°C.

Initial observations using the split mCherry system indicated that the split AiiA constructs retained
partial degradation activity at both 37°C and 25°C. We attribute this activity to the self-assembly
of
the protein fragments. However, no enhancement in degradation efficiency was observed upon blue
light
illumination.

As a positive control, we conducted parallel experiments under identical light conditions using
BL21(DE3)
strain containing the pGEX-AiiA plasmid (This strain expresses the full length AiiA without
splitting) .
The positive control demonstrated effective degradation, confirming that the blue light illumination
itself does not cause significant damage to AiiA activity within the experiment.

The three different split sites yielded similar degradation efficiencies. We hypothesize that this
result
is due to their similar Split Energy values, which likely led to comparable self-assembly
propensities
and, consequently, similar functional outputs. This consistency confirms the effectiveness of our
initial in silico screening methodology for selecting split sites.

We are currently investigating the underlying reasons for the lack of light-induced enhancement in
degradation efficiency.

### Protein Degradation Tag

For the light-inducible degradation module of result display proteins, we chose the modular
degradation
tag LOVdeg and fused it with mCherry. We used solid plate colony experiments to test its efficiency
(Figure 12). Ten hours of blue-light exposure reduced mCherry-LOVdeg fluorescence by > 50 %. This
indicates
that our LOVdeg tag can effectively accelerate the degradation rate of target protein under blue
light.

![Preliminary experiment]({{ cdn_url('results/fig16.png') }})

**Figure 16** In solid plate colony experiments, the
fluorescence of colonies showed mCherry protein levels in response to blue light. After 12h
cultured
in dark, the light group was exposed to blue light for 10 hours.

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