iGEM 2022 — Team xjtu-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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![](https://static.igem.wiki/teams/4182/wiki/chaos/bg1.jpg)

## Notebook

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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![](https://static.igem.wiki/teams/4182/wiki/chaos/bg1.jpg)

Engineering success

* Overview
* Research
* Design
* Build
* Test
* Learn

## Engineering success

### Overview

We offer an environmentally friendly
biofertilizer that attempts to solve the global
ecological security and economic problems caused
by the widespread use of chemical herbicides
through synthetic biology. We constructed an
engineered *E. coli* that produces aspartic acid
and extracellular polysaccharide (EPS), a novel
herbicide, under blue light and can be released
into soil in a controlled manner at high
temperatures, avoiding overuse of herbicides and
possible residues, and promoting water retention
and sand fixation of EPS. Our system consists of
a proplasmid that converts glucose into a key
precursor, GPP, and multiple functional plasmids
that synthesize herbicides and EPS under blue
light control. At the same time, our engineered
cells would release herbicides and EPS
containing lytic genes at a high temperature
above 42℃. About 10% of the bacteria will escape
the lysis process and recover, facilitating a
new round of controlled production and release
of herbicides and EPS. The intelligent synthesis
and release of our biofertilizers will maximize
the effects of herbicides and EPS, contributing
to the environment and society.

### Research

To realize the controllable synthesis and
release of products, after an extensive
literature survey, we found that the arabinose
operon can be engineered to regulated downstream
gene expression by blue light induction instead
of arabinose chemical induction [1].

Chemically induced gene expression systems are
valuable tools to control biological processes
for applications in basic science and
biotechnology. While for the tuned and spatial
control of gene expression, chemically induced
systems have some limitations-they are unable to
achieve complex spatiotemporal regulation [2],
and often lack reversibility or require washing
steps to achieve it [3].

These limitations can be overcome by using
light, rather than small molecules, as external
triggers. Under the light, for example,
pulsating inputs that alternate between dark
(off) and maximum intensity (fully on) can be
produced [4] and have been shown to lead to
effects not achievable with graded intensity
light, such as reduced cell-to-cell variability
in gene expression [5]. Indeed, the amount of
cell-to-cell variation can be adjusted by
adjusting the duty cycle, defined as the
fraction of time that light is fully on,
providing a new mode of control for studying
stochasticity in gene expression[6]. This type
of pulsatile input has also recently been found
to enhance the biosynthesis of products in
engineered cells, enabling a new type of
bioreactor operation that is much easier to
handle than chemical induction [7]. The enzyme
expression was adjusted to increase the
fermentation yield [8]. The above findings are
of great help for the design of our
light-controlled herbicide production.

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/1.png)

FIG. 1 Schematic diagram of blue light
induction regulatory system

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/2.png)

FIG. 2 Schematic diagram of blue
light-induced regulation system

### Design

Based on the above hypothesis and the ideas
provided by the literature, we designed the
upstream regulator- the chimeric VVD-AraC fusion
protein by replacing the arabinose binding and
dimerization domain of arabinose operon with a
blue-light responsive VVD domain, which will
dimerization under light and promote the
downstream PBAD promoter. We selected sfGFP as
the reporter to verify the regulation of the
system. In order to test the effect of VVD-AraC
expression level on the downstream gene
expression, three promoters-native Pc, J23101
and porin promoter was selected in our study
(BBa\_K4182001, BBa\_K4182002, BBa\_K4182003). The
blue-light inducible circuit is shown as
follows.

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/3.png)

FIG. 3 Verification circuit diagram of blue
light-induced regulation system

### Build

According to our design, the VVD gene from
Streptomyces were chemically synthesized, and
the AraC-ParaBAD promoter in arabinose operon
was amplified from *E. coli*, and eSD
from *E. coli* was served as the ribosome binding
site. The three promoters-native Pc, J23101, and
porin was obtained by PCR. All the fragments
were ligated into pBBRMCS1 vector in one step
via Golden Gate Assembly. Figure 4-6 shows the
PCR fragments used for the circuit construction.
The recombinant plasmids were verified by colony
PCR as shown in Figure 7 and 8, which are
further confirmed by sequencing. As a result,
three plasmids PVVDH-Pc, PVVDH-J23101,
PVVDH-porin, were successfully constructed for
further test including cell growth and the
expression of GFP.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/4182/wiki/engineering-success/4.png) FIG.4 PCR result of J23101-VVD fragment | ![](https://static.igem.wiki/teams/4182/wiki/engineering-success/5.png) FIG.5 PCR result of porin-VVD fragment |
| ![](https://static.igem.wiki/teams/4182/wiki/engineering-success/6.png) FIG.6 PCR result of linear vector pBBRMCS1 | ![](https://static.igem.wiki/teams/4182/wiki/engineering-success/7.png) FIG.7 Colony PCR verification of plasmid PAVVDH-J23101 |

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/8.png)

FIG.8 Colony PCR verification of plasmid
PAVVDH-porin

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/9.jpg)

FIG.9 Final plasmid map of blue light
induction regulatory system

### Test

To test expression of sfGFP of the three
plasmids, we develped a weak blue light
induction system, which is mainly consist of a
blue light plate and Pulse Width Modulation
(PWM) module powered by USB. The size of the
light plate is 20cm\*20cm, the blue wavelength is
470nm. As the intensity of the commonly used
blue light is higher than what we need in our
experiment, the PWM module was employed here to
adjust the intensity of light to about 5W/m2.

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/10.png)

FIG. 10 Design drawing of the self-made weak
blue light induction system

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/11.jpg)

FIG. 11 PWM module

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/4182/wiki/engineering-success/12.jpg) | ![](https://static.igem.wiki/teams/4182/wiki/engineering-success/13.jpg) |

FIG. 12 Self-made weak blue light induction
system

The recombinant DH5a cells harboring the
blue-light inducible plasmids were cultivated at
37℃ to OD600=0.6-0.8, then cells were exposed to
the self-made blue light induction system for 4
hours, and the control ones without blue-light
were covered by aluminium foil. The cell density
(OD600) and the fluorescent intensity of sfGFP
were detected every 1 h. The results are shown
in Figures 13-18.

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/c1.png)

FIG.13 mRNA level of VVD and sfGFP under
different promoters without blue light

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/c2.png)

FIG.14 The expression of green fluorescent
protein of strains with plasmid PAVVDH-Pc,
PAVVDH-J2301 and PAVVDH-porin

As shown in Figure 13, without blue-light
induction, a higher VVD transcription level was
observed when porin promoter was used to control
the expression of VVD-AraC fusion protein,
compare to J23101 promoter. It indicated the
tight and more precise regulation by porin
promoter. It is further proved in Figure 14 that
porin promoter exhibited a higher fluorescence,
a wider dynamic range and better sensitivity
when induced by blue light than the native PC
promoter and J23101 promoter. Therefore, the
plasmid PAVVDH-porin was selected for our
further studies.

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/16.png)

FIG.15 The cell growth of strain harboring
circuit with and without blue-light
induction.

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/17.png)

FIG. 16 The fluorescent intensity of
recombinant strain in induced and
non-induced groups

Figure 15 showed the similar cell growth of
recombinant strain with PAVVDH-porin circuit
under blue light or no blue light, indicating no
growth inhibition of blue-light. However, the
expression level of sfGFP varied in
induced-group and non-induced group as shown in
Figure 16, and significantly increased
fluorescent intensity can be observed with blue
light induction. The normalized fluorescent
intensity (sfGFP/OD600) was also shown in Figure
17, which revealed a constant increase of
normalized sfGFP with time, further directly
illustrating the efficient induction capacity of
the blue-light induction system. The induction
effect of blue-light was also confirmed by
confocal, and after blue-light induction,
numerous cells with green fluorescence were
observed in the microscopy (Figure 18).

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/c4.png)

FIG. 17 The normalized fluorescent intensity
of recombinant strain under blue-light
induction

Collectively, these results demonstrate the
efficiency of our blue-light induction circuit,
and porin promoter is proved to be the best one
among three promoters. Our newly developed weak
blue light induction device is also demonstrated
to be functional.

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/bcd.jpg)

FIG 18 Engineered cells was observed to show
green fluorescence after blue-light
induction

We also made a comprehensive analysis and
comparison of the effects of the fitting model
on the blue light induction system. A more
detailed explanation can be found on the
modeling page.

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/c3.png)

FIG 19 Modeling on blue-light induction system.

[A more detailed explanation can be found on
the modeling page.](https://2022.igem.wiki/xjtu-china/model)

Therefore, we successfully synthesized and
verified the modified blue light induction
regulatory system. The submitted composite
components are as follows:

BBa\_K4182000:VVD

BBa\_K4182001:Porin-eSD-VVD-AraC

BBa\_K4182002:J23101-eSD-VVD-AraC

BBa\_K4182003:Native Pc-eSD-VVD-AraC

### Learn

During the above Design-Build-Test Cycle,
actually we were always learning from the
Test results, and start another round of DBT
cycle.

#### 1. The truncated VVD (VVDH as shown above) was used after one DBT cycle for whole length VVD.

As shown in the figure, the conserved
domain of VVD starts at 36aa of N
terminus. So no blue-light responsive
effect when whole-length VVD were used,
maybe because of the incorrect structure
of whole length VVD. Thus truncated VVD
(VVDH) without N terminus 35aa was used
in our study, and achieved blue-light
inducible effect.

![](https://static.igem.wiki/teams/4182/wiki/engineering-success/20.png)

FIG 19 Structure analysis of VVD
protein

#### 2. A system with weak blue light was used instead of regular strength of light after another round of DBT cycle.

After failure of detection GFP
expression, we finally learn that the
high light intensity will cause a heavy
quenching effect, which will lead to the
low GFP intensity. Thus we reduced our
optical density from 200W/m2 to 5W/m2 by
our self-made weak light system, and
were able to detect GFP expression.

## Reference

1. [1] ROMANO E, BAUMSCHLAGER A, AKMERIÇ E B,
   et al. Engineering AraC to make it
   responsive to light instead of arabinose
   [J]. Nat Chem Biol, 2021, 17(7): 817-27.
2. [2] RAMAKRISHNAN P, TABOR J J. Repurposing
   Synechocystis PCC6803 UirS-UirR as a
   UV-Violet/Green Photoreversible
   Transcriptional Regulatory Tool in *E. coli*
   [J]. ACS Synth Biol, 2016, 5(7): 733-40.
3. [3] ONG N T, TABOR J J. A Miniaturized
   *E. coli* Green Light Sensor with
   High Dynamic Range [J]. Chembiochem, 2018,
   19(12): 1255-8.
4. [4] OHLENDORF R, VIDAVSKI R R, ELDAR A, et
   al. From dusk till dawn: one-plasmid systems
   for light-regulated gene expression [J]. J
   Mol Biol, 2012, 416(4): 534-42.
5. [5] LI X, ZHANG C, XU X, et al. A
   single-component light sensor system allows
   highly tunable and direct activation of gene
   expression in bacterial cells [J]. Nucleic
   Acids Res, 2020, 48(6): e33.
6. [6] JAYARAMAN P, DEVARAJAN K, CHUA T K, et
   al. Blue light-mediated transcriptional
   activation and repression of gene expression
   in bacteria [J]. Nucleic Acids Res, 2016,
   44(14): 6994-7005.
7. [7] DING Q, MA D, LIU G Q, et al.
   Light-powered *E. coli* cell division
   for chemical production [J]. Nat Commun,
   2020, 11(1): 2262.
8. [8] BAUMSCHLAGER A, AOKI S K, KHAMMASH M.
   Dynamic Blue Light-Inducible T7 RNA
   Polymerases (Opto-T7RNAPs) for Precise
   Spatiotemporal Gene Expression Control [J].
   ACS Synth Biol, 2017, 6(11): 2157-67.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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[* Background](#background)
[* Design Iterations](#design-iterations)
[* Basic Proof](#basic-proof)
[* Recombinase switch](#recombination-switch)
[* BCAA Sensor](#bcaa-sensor)

# Design

## Overview

1. Background
2. Design Iterations
3. Basic Proof
4. Recombinase Switch
5. BCAA Sensor

---

## Background

In order to determine how our probiotic should function, we conducted additional background research on the molecular basis of Maple Syrup Urine Disease (MSUD). We learned that mutations in the branched chain keto-acid dehydrogenase
(BCKDH) enzyme complex, which is responsible for breaking down branched chain keto-acids (BCKAs), cause the buildup of branched chain amino acids (BCAAs) and branched chain ketoacids (BCKAs) to toxic levels in MSUD patients.

We decided to have our probiotic bacteria overexpress BCKDH to break down excess BCAAs before they are absorbed into the bloodstream. We were initially drawn to *E. coli* Nissle as a chassis, being a well studied probiotic strain
of *E. coli* commonly used in synthetic biology and by iGEM teams. However, the human BCKDH enzyme is a very large complex consisting of multiple copies of each of 4 subunits encoded by different genes. *E. coli* lacks
this enzyme complex or similar machinery to break down BCAAs, so we would need to import all these sequences into *E. coli*, which would be rather difficult and potentially unstable. As a result, we instead turned to looking
at existing bacteria that already possess the BCKDH complex, and we found *B. subtilis*.

*B. subtilis* is a gram-positive bacterium commonly found in soil (1). *B. subtilis* is also a native inhabitant of the GI tract and a commonly used probiotic with proven health benefits (2). One unique feature of *B. subtilis* is that it is able to form spores and become metabolically dormant to survive in adverse environments, and many probiotics consist of *B. subtilis* in spore form. This allows it to survive the transit through the acidic environment
of the stomach, and then germinate in the nutrient-rich small intestine.

The bkd operon in *B. subtilis* encodes the BCKDH enzyme complex. Combined with a transaminase and various other downstream enzymes, *B. subtilis* is able to fully metabolize BCAAs down to the basic substrates of central
metabolism, such as acetyl CoA and succinyl CoA. As *B. subtilis* already has the bkd operon within its genome, we can simply use homologous recombination to replace the operon’s promoter with a constitutive one in order to
upregulate it. We also identified various other genes in *B. subtilis* responsible for importing and exporting BCAAs in order to enhance *B. subtilis’s* ability to uptake and retain BCAA’s.

Below is a simplified model of the relevant genetic circuitry within *B. subtilis*. Our genes of interest are

1. *bcaP*: a BCAA symporter
2. *ilvE* and *ilvK* which encode BCAT: BCAA transaminase, which catalyzes the first step in the breakdown of BCAAs
3. *bkd* operon: an operon that encodes branched chain keto-acid dehydrogenase (BCKDH), the enzyme complex that catalyzes the second step in the breakdown of BCAAs
4. *azlC* and *azlD*: BCAA exporters in the azlBCDEF operon

[![](https://static.igem.org/mediawiki/2021/6/64/T--MIT--design_1.png)](https://static.igem.org/mediawiki/2021/6/64/T--MIT--design_1.png%20)

Simplified model of genetic circuitry within *B. subtilis* made using BioRender

---

## Design Iterations

Our project had three main iterations:

## Basic Proof of Concept

In this first iteration, we wanted to prove that overexpressing these critical genes could lead to a measurable increase in BCAA uptake, so we designed parts for constitutive expression of these genes.

Design

 The four main components of this system are

1. Constitutive expression of the bcaP importer to import more BCAAs into the cell
2. Constitutive expression of ilvE/ilvK transaminase to break down BCAAs into alpha keto-acids
3. Constitutive expression of the bkd operon to break down alpha keto-acids
4. Knockout of azlCD exporters to prevent BCAAs from leaving the cell

[![](https://static.igem.org/mediawiki/2021/f/fd/T--MIT--design_2.png)](https://static.igem.org/mediawiki/2021/f/fd/T--MIT--design_2.png)

Simplified model of our modified B. Syruptilis genetic circuitry made using BioRender

Testing

 To transform *B. subtilis* with our DNA, we planned to have our parts integrate directly into the genome through homologous recombination. To confirm that we were able to execute this, we also designed parts that
expressed mApple, a red fluorescent protein, and KanR, a protein for kanamycin resistance. These same parts were also used in *E. coli* to check that our promoters were working as expected.

In total, our cloning and testing steps included the following experiments:

* Gibson cloning and traditional cloning to construct plasmid backbones
* Golden Gate to construct plasmids from our parts
* Colony PCR of both plasmid and genomic DNA to quickly check our Golden Gate products, as well as to check integration of our DNA into the *B. subtilis* chromosome
* Restriction digests to check our Golden Gate products prior to sequencing
* Transformation of E. coli to replicate our plasmid DNA
* Transformation into *B. subtilis*

Following successful transformation, we then assayed the ability of our *B. subtilis* to break down BCAAs, as well as *B. subtilis's* natural rate of BCAAs.

We performed the following experiments to assay *B. subtilis*:

* Test homologous recombination into *B. subtilis* with kanamycin selection marker
* BCAA concentration assay to determine the sensitive range of our assay and the best starting concentration of BCAAs with which to perform subsequent assays
* BCAA time point assay to compare the rate of BCAA breakdown between wild-type and probiotic *B. subtilis*

---

## Recombinase switch for constitutive expression

This next design iteration keeps the basis of overexpressing genes but adds in additional regulatory controls. As we are engineering *B. subtilis* to overexpress many large protein complexes, it is critical to maintain fitness
of the organism while it is being manufactured or traveling within the GI tract. After much discussion with our mentors, we decided to induce the expression of a recombinase to switch on constitutive expression of these proteins
at the right time.

What is a recombinase?

 There are two types of recombinases: tyrosine recombinases (ex. Cre) and serine recombinases (ex. PhiC31). Recombinases recognize certain sequences in the DNA (ex. loxP and attP/B), and act on the DNA in between them. Generally,
this takes the form of either an excision or a reversion of the intervening sequence, depending on the directions in which the flanking sites are facing. Excision is useful for permanently turning on or off a gene, while an inversion
can function as a switch that can be adjusted depending on the condition of the cell.

[![](https://static.igem.org/mediawiki/2021/7/7e/T--MIT--design_3.png )](https://static.igem.org/mediawiki/2021/7/7e/T--MIT--design_3.png%20)

Mechanism of the excision (a) and inversion (b) of DNA flanked with loxP sites by Cre recombinase. [Source](https://link.springer.com/article/10.1007/s11914-018-0455-7/figures/1)

Design

 We placed the Cre recombinase under the inducible control of a pTet/tetR/ATC system in order to turn on the constitutive expression of bcaP, ilvE/ilvK, and the bkd operon in the presence of doxycycline.

[![](https://static.igem.org/mediawiki/2021/a/a5/T--MIT--design_4.png )](https://static.igem.org/mediawiki/2021/a/a5/T--MIT--design_4.png%20)

In our system, expression of Cre recombinase is induced by doxycycline. Picture created on BioRender

Cre recognizes specific loxP sites in DNA, in our case the lox71 and lox66 sites. There are two ways in which Cre can function, depending on how these sites are oriented:

1. If the loxP sites face the same direction, the region of DNA between the sites is excised as a circular piece of DNA.
2. If the loxP sites face opposite directions, the region of DNA in between the sites is inverted.

Using these properties, we have created a few different constructs for controlling constitutive expression:

 Constitutive expression of bkd operon:

1. The native promoter is excised and replaced with a constitutive promoter

[![](https://static.igem.org/mediawiki/2021/7/71/T--MIT--design_5.png)](https://static.igem.org/mediawiki/2021/7/71/T--MIT--design_5.png)

The composite part that controls this one way recombinase switch consists of Pveg - lox71 - terminator - bkd operon native promoter - lox66. Cre recombinase excises the sequence flanked with lox71 and lox66 sites, leaving
the bkd operon under constitutive control by Pveg. Picture created on BioRender

2. The native promoter is initially in place, while the constitutive promoter is inverted. Inversion of both these promoters swaps their positions, placing the constitutive promoter in charge of the operon

[![](https://static.igem.org/mediawiki/2021/8/8c/T--MIT--design_6.png)](https://static.igem.org/mediawiki/2021/8/8c/T--MIT--design_6.png)

This reversible recombinase switch consists of lox71 - bkd operon native promoter - reverse complement of Pveg - inverted lox66. Cre recombinase inverts the sequence between the opposing loxP sites, allowing the system to be
under constitutive control by Pveg when induced by dox. Inducing the system with dox a second time will reverse the system back to its original state. Picture created on BioRender

**Constitutive expression of bcaP and ilvE/ilvK**

 Inversion of a reversed constitutive promoter results in constitutive expression

[![](https://static.igem.org/mediawiki/2021/9/9e/T--MIT--design_7.png)](https://static.igem.org/mediawiki/2021/9/9e/T--MIT--design_7.png)

This reversible recombinase switch consists of lox71 - reverse complement of Pveg - inverted lox66. Cre recombinase inverts the sequence between the opposing loxP sites, allowing the system to be under constitutive control
by Pveg when induced by dox. Inducing the system with dox a second time will reverse the system back to its original state. Picture created on BioRender

Testing

 In order to test the Cre recombinase switch, we first have to verify that Cre works as expected in *B. subtilis*. We designed a construct of mApple2 surrounded by lox71 and lox66 sites so we can test whether Cre excises
mApple2 from the genome and note the time it takes for this process to occur. Additionally, we will continue to use the BCAA assay to test the rate of BCAA uptake into *B. subtilis* before and after adding dox to the system
to induce the expression of Cre.

---

## BCAA Sensor Using BRET and Optogenetic System

Our final conceptual design is a fully autonomous system capable of switching on constitutive expression of our genes of interest in response to fluctuating BCAA levels within *B. subtilis*. This is better than the recombinase
approach as *B. subtilis* would be able to regulate its own gene expression and we would not need to induce it with dox or some other small molecule.

What is BRET?

 BRET is a system that utilizes one bioluminescent (donor) and one fluorescent protein (acceptor). Fluorescent proteins have defined excitation and emission wavelengths. After absorbing light with a wavelength within their
excitation range, fluorescent proteins can then emit light in a separate wavelength, which can then be detected by a microscope. We can take advantage of this by coupling a fluorescent protein with a bioluminescent protein that
emits light at the excitation wavelength. When these proteins are in close proximity, light emitted from the fluorescent protein may be detected.

Design

 While conducting background research on promoters and systems that respond to different levels of BCAAs, we came across oLIVe, a BCAA biosensor designed to exhibit FRET (BRET with a donor that is a fluorescent protein). oLIVe
consists of livJ, a protein that binds BCAAs, with fluorescent proteins fused with linkers in regions of livJ that allow all three proteins to be folded properly and exhibit correct behaviours. The binding of a BCAA to livJ triggers
a conformation shift that brings the fluorescent proteins in close enough proximity for FRET to occur.

[![](https://static.igem.org/mediawiki/2021/4/48/T--MIT--design_8_cropped.png)](https://static.igem.org/mediawiki/2021/4/48/T--MIT--design_8_cropped.png)

The oLIVe FRET system consists of a cyan fluorescent protein donor that excites the yellow fluorescent protein acceptor when a BCAA binds. [Source](https://pubs.acs.org/doi/10.1021/acssensors.9b02067)

In a human system, it is undesirable to have to excite the initial donor fluorescent protein at a similar wavelength via FRET, hence the decision to use BRET. In our theoretical system, RLuc8.6 luciferase (em 535 nm) and TurboFP635
fluorescent protein (em 635 nm) are inserted in opposite regions of livJ. Initially, only RLuc8.6 exhibits bioluminescence. When the livJ protein binds BCAA, livJ undergoes a conformational change, and the two fluorescent proteins
are brought closer. This conformational change thus places RLuc8.6 and TurboFP635 in close proximity to each other and allows BRET to occur, and emission from RLuc8.6 excites TurboFP635, and allows TurFP635 to fluorescence instead
of RLuc8.6.

Next, a separate optogenetic circuit may be used to detect the specific wavelength of light emitted by TurboFP635 and trigger gene expression. Here, we adopt the CcaSR system developed by the Voigt lab in *E. coli*, further adapted
to *B. subtilis* by the Tabor lab at Rice University. CcaSR is able to turn on gene expression when induced by light at 535 nm and turn off expression at 670 nm. For our purposes, we need default activation at 535 nm to repress
our genes of interest, and inactivation of CcaSR at 670 nm to cease repression of our genes, leading to expression. This is due to an inability to have a default ‘off’ system at 670 nm, as BRET cannot have a lower energy state
FP (TurboFP635 emitting at 635 nm) excite a higher energy state (RLuc8.6 emitting at 535 nm).

[![](https://static.igem.org/mediawiki/2021/b/b2/T--MIT--design_9.png)](https://static.igem.org/mediawiki/2021/b/b2/T--MIT--design_9.png)

The CcaSR system turns on gene expression when induced with light with a wavelength of 535 nm and turns off gene expression when induced with light with a wavelength of 670 nm.[Source](https://www.nature.com/articles/s41467-019-10906-6/figures/1)

Putting these two systems together, we would configure BRET with RLuc8.6 and TurboFP635 both fused to livJ. The default ‘on’ state with RLuc8.6 (em 535 nm) activity induces CcaSR expression of a repressor that represses constitutive
expression of our genes of interest. When livJ binds BCAA, it undergoes a conformational change to excite TurboFP635 (em 635 nm) to inactivate expression of the repressor.

Testing

 To test that the optogenetic system works in *B. subtilis*, we must confirm multiple things:

* Whether CcaSR is inducible by correct wavelengths of light
* Whether a livJ fusion protein is capable of inducing BRET upon BCAA binding
* Whether the two systems can work in conjunction with one another, and how this system can be tuned to express our genes at the appropriate levels

---

## References

1. Castillo-Hair, S. M., Baerman, E. A., Fujita, M., Igoshin, O. A., & Tabor, J. J. (2019). Optogenetic control of Bacillus subtilis gene expression. Nat Commun, 10(1). 10.1038/s41467-019-10906-6
2. Dallas, S. L., Xie, Y., Shiflett, L. A., & Ueki, Y. (2018). Mouse Cre Models for the Study of Bone Diseases. Curr Osteoporos Rep, 16(4), 466–477. 10.1007/s11914-018-0455-7
3. Yoshida, T., Nakajima, H., Takahashi, S., Kakizuka, A., & Imamura, H. (2019). OLIVe: A Genetically Encoded Fluorescent Biosensor for Quantitative Imaging of Branched-Chain Amino Acid Levels inside Single Living Cells. 10.1021/acssensors.9b02067.s002

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