iGEM 2023 — Team sdu-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 %} {% block
header %}

Notebook

2023 SDU-CHINA

{% endblock %} {% block page\_content %}

## TIMELINE

### SDU-CHINA

![](https://static.igem.wiki/teams/4583/wiki/notes/nt-4.jpg)

## 01 Selection and Characterization of QS system

[Week1：11.25-12.1](https://static.igem.wiki/teams/4583/wiki/notes/week1-11-25-12-1.pdf)

[Week2：12.2-12.8](https://static.igem.wiki/teams/4583/wiki/notes/week2-12-2-12-8.pdf)

![](https://static.igem.wiki/teams/4583/wiki/notes/nt-2.jpg)

## 02 Selection of Late Stationary Phase Promoter

[Week3 ：12.9-12.15](https://static.igem.wiki/teams/4583/wiki/notes/week3-12-9-12-15.pdf)

[Week4](https://static.igem.wiki/teams/4583/wiki/notes/week4-docx.pdf)

[Week5](https://static.igem.wiki/teams/4583/wiki/notes/week5-docx.pdf)

[Week6 ：3.13-3.19](https://static.igem.wiki/teams/4583/wiki/notes/week6-3-13-3-19-docx.pdf)

[Week7 3.20-3.26](https://static.igem.wiki/teams/4583/wiki/notes/week7-3-20-3-26.pdf)

[Week8 4.1-4.7](https://static.igem.wiki/teams/4583/wiki/notes/week8-4-1-4-7.pdf)

[Week9 4.8-4.14](https://static.igem.wiki/teams/4583/wiki/notes/week9-4-8-4-14.pdf)

[Week10 4.22-4.28](https://static.igem.wiki/teams/4583/wiki/notes/week10-4-22-4-28.pdf)

![](https://static.igem.wiki/teams/4583/wiki/notes/nt-3.jpg)

## 03 Characterization of Late Stationary Promoter

[Week11 4](https://static.igem.wiki/teams/4583/wiki/notes/week11-4.pdf)

[Week14 6](https://static.igem.wiki/teams/4583/wiki/notes/week14-6.pdf)

[Week15](https://static.igem.wiki/teams/4583/wiki/notes/week15.pdf)

[Week16](https://static.igem.wiki/teams/4583/wiki/notes/week16.pdf)

![](https://static.igem.wiki/teams/4583/wiki/notes/nt-5-1.jpg)

## 04 Selection and Characterization of Lysis system

[Week12](https://static.igem.wiki/teams/4583/wiki/notes/week12.pdf)

[Week13 5.26-6.1](https://static.igem.wiki/teams/4583/wiki/notes/week13-5-26-6-1.pdf)

[Week17](https://static.igem.wiki/teams/4583/wiki/notes/week17.pdf)

[Week18](https://static.igem.wiki/teams/4583/wiki/notes/week18.pdf)

[Week19 7](https://static.igem.wiki/teams/4583/wiki/notes/week19-7.pdf)

[Week20 7](https://static.igem.wiki/teams/4583/wiki/notes/week20-7.pdf)

[Week21](https://static.igem.wiki/teams/4583/wiki/notes/week21.pdf)

[Week22](https://static.igem.wiki/teams/4583/wiki/notes/week22.pdf)

[Week23](https://static.igem.wiki/teams/4583/wiki/notes/week23.pdf)

[Week24](https://static.igem.wiki/teams/4583/wiki/notes/week24.pdf)

[Week25](https://static.igem.wiki/teams/4583/wiki/notes/week25.pdf)

![](https://static.igem.wiki/teams/4583/wiki/notes/nt-1.jpg)

## 05 PHB Fermentation Analysis

[Week26](https://static.igem.wiki/teams/4583/wiki/notes/week26.pdf)

[Week27](https://static.igem.wiki/teams/4583/wiki/notes/week27.pdf)

[Week28](https://static.igem.wiki/teams/4583/wiki/notes/week28.pdf)

[Week29](https://static.igem.wiki/teams/4583/wiki/notes/week29.pdf)

{% endblock %}

{% 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
%} {% block header %}

Engineering

2023 SDU-CHINA

{% endblock %} {% block page\_content %}

[**Introduction**](#intro)
[**Three-layer dynamic regulation model**](#TL)
[**1st iteration**](#1st)
[**2nd iteration**](#2nd)
[**3rd iteration**](#3rd)

[**References**](#ref)

* Introduction

We aim to implement **cell intelligence** to boost the production
of PHB. **Cell intelligence (CI)** refers to
**the automation of the production process**, resulting in
increased productivity and reduced costs. Our hardware and software
have been specifically designed to attain this objective. We
followed the **DBTL principle**, developed our
**three-layer dynamic regulation model**.

* Three-layer dynamic regulation model

* 2.4 Three-Layer dynamic regulation model

![The following combinations are transformed into both L19 and L31](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

|  | Catalog | Description | length | Name |
| --- | --- | --- | --- | --- |
| 01 | Composite | PesaS-B0034-GFP-PesaRwt-B0034-mKate-PYU3-BFP | 2941bp | [BBa\_K4583027](http://parts.igem.org/Part%3ABBa_K4583027) |
| 02 | Composite | PesaS-B0034-GFP-PesaRc-B0034-mKate-PYU3-BFP/td | 2655bp | [BBa\_K4583031](http://parts.igem.org/Part%3ABBa_K4583031) |
| 03 | Composite | PesaS-B0034-GFP-PesaRwt-B0034-mKate-PYU7-BFP | 2944bp | [BBa\_K4583032](http://parts.igem.org/Part%3ABBa_K4583032) |
| 04 | Composite | PesaS-B0034-GFP-PesaRc-B0034-mKate-PYU7-BFP | 2658bp | [BBa\_K4583033](http://parts.igem.org/Part%3ABBa_K4583033) |
| 05 | Composite | PesaS-B0034-GFP-PesaRp-B0034-mKate-PYU7-BFP | 2944bp | [BBa\_K4583034](http://parts.igem.org/Part%3ABBa_K4583034) |
| 06 | Composite | PesaS-B0034-GFP-PesaRwt-B0034-mKate-PYU92-BFP | 2940bp | [BBa\_K4583035](http://parts.igem.org/Part%3ABBa_K4583035) |
| 07 | Composite | PesaS-B0034-GFP-PesaRp-B0034-mKate-PYU16-BFP | 2941bp | [BBa\_K4583036](http://parts.igem.org/Part%3ABBa_K4583036) |

* 1st iteration

* Goal

Overall, we would like to divide the process of PHB production by
E. coli into three stages: **the growth stage**,
**the production stage** and **the product release stage**.
In this way, E. coli can be fed first, and then work at full
capacity; when their "stomach" is full of PHB, we crack it and
release the products automatically. We plan to use
**the Esa I/R quorum sensing system** to **switch** between
growth and production phases. **A promoter** expressed in the
late stationary phase, **linked to a lysis gene**, will be used
to control bacterial lysis at the end of the production phase.

* Design 1.0

**Three-layer Dynamic Regulation Model**

![Fig.1: Schematic diagram of the Three-layer Dynamic Regulation Model](https://static.igem.wiki/teams/4583/wiki/project-description/8.jpg)

**Fig.1 |** Schematic diagram of the Three-layer Dynamic
Regulation Model

**Growth Phase and Production Phase**

We chose to **use the Esa I/R system** to control the switch
between Growth Phase and Production Phase.
**The EsaI/R QS system** is homologous to
**the LuxI/R QS system**, but EsaR can act as
**both** transcriptional activator and repressor[1]. Our work
was based on 3 strains that that already have the system (see
Experiments for more details). **At low cell density**, the
PesaR is turned off and the PesaS is turned on.
**At high cell density**, the PesaR is turned on and the
PesaS is turned off (see Experiments for more details). We
firstly used **fluorescence** to characterize.

![Fig.2: Genetic Circuit for Growth phase and Production Phase](https://static.igem.wiki/teams/4583/wiki/engineering/design1-1.png)

**Fig.2 |** Genetic Circuit for Growth phase and Production
Phase

**Product-release Phase**

We firstly used **4** stationary phase promoters (Pfic, P1.1,
P2.1, P3.1)[2].

![Fig.3: Genetic Circuit for product-release phase](https://static.igem.wiki/teams/4583/wiki/engineering/design1-2.png)

**Fig.3 |** Genetic Circuit for product-release phase

* Build 1.0

![Transferring process](https://static.igem.wiki/teams/4583/wiki/engineering/build1.png)

Combining the different plasmids and transferring them into E.
coli, we obtained the following bacteria containing two
plasmids.

These **6** strains are for QS- switch characterization:

* These 6 strains are for QS- switch characterization

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

| Strains | Plasmid 1 | Plasmid 1 | Name |
| --- | --- | --- | --- |
|  |  | pCL-PesaRwt-mkate | L19SRw |
| L19 | pCL-PesaS-GFP(LVA) | pCL-PesaRc-mkate | L19SRc |
|  |  | pCL-PesaRp-mkate | L19SRp |
|  |  | pCL-PesaRwt-mkate | L31SRw |
| L31 | pCL-PesaS-GFP(LVA) | pCL-PesaR-cmkate | L31SRc |
|  |  | pCL-PesaRp-mkate | L31SRp |

The following combinations are transformed into both
**L19 and L31**. The total number is **24**.

* The following combinations are transformed into both L19 and
  L31

![The following combinations are transformed into both L19 and L31](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

| Plasmid 1 | Plasmid 2 | | | |
| --- | --- | --- | --- | --- |
| PesaR | Pfic | P1.1 | P2.1 | P3.1 |
| PesaR-wt | RwF | Rw1.1 | Rw2.1 | Rw3.1 |
| PesaR-c | RcF | Rc1.1 | Rc2.1 | Rc3.1 |
| PesaR-p | RpF | Rp1.1 | Rp2.1 | Rp3.1 |

* Test 1.0

**QS-switch Characterization**

We used a **Multi-Detection Microplate Reader** (Synergy HT,
Biotek, U.S.) to detect the fluorescence.

![Fig.4: Characterization of QS-switch promoter](https://static.igem.wiki/teams/4583/wiki/engineering/test1-1.png)

**Fig.4 |** Characterization of QS-switch promoter

We found that the characterization results for all six
combinations were as expected: **At low cell density**, the
PesaR is turned off and the PesaS is turned on, while
**at high cell density**, the PesaR is turned on and the
PesaS is turned off (see Experiments for more details).

**Stationary Phase Promoter Characterization**

![Fig.5: Characterization of Pic, P1.1, P2.1, P3.1and PesaRwt, PesaRc, PesaRp](https://static.igem.wiki/teams/4583/wiki/engineering/test1-2.png)

**Fig.5 |** Characterization of Pic, P1.1, P2.1, P3.1and
PesaRwt, PesaRc, PesaRp

We found that **combinations were not as expected**: no
significant temporal separation of expression occurred.

* Learn 1.0

The QS system we use meets our needs,
**but the stationary phase promoter is expressed too early**.
We need to find the promoter that is
**expressed at late stationary phase**.

* 2nd iteration

* Goal

In the 2nd iteration, we aimed to
**find promoters expressed in the late stationary phase** and
**characterize** them.

* Design 2.0

This time, by reading scientific articles, we found
**four promoters**
expressed in the late stationary phase (PYU3, PYU7, PYU16,
PYU92)[3].

![Fig.6: Genetic circuit for the late stationary phase promoter](https://static.igem.wiki/teams/4583/wiki/engineering/design2.png)

**Fig.6 |** Genetic circuit for the late stationary phase
promoter

* Build 2.0

Using the same steps in the 1st iteration build 1.0, we
constructed the following **24** strains.

* Our 24 strains

![Our 24 strains](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

| Plasmid 1 | Plasmid 2 | | | |
| --- | --- | --- | --- | --- |
| PesaR | PYU3 | PYU7 | PYU16 | PYU92 |
| PesaR-wt | PesaRwt-PYU3 | PesaRwt-PYU7 | PesaRwt-PYU16 | PesaRwt-PYU92 |
| PesaR-c | PesaRc-PYU3 | PesaRc-PYU7 | PesaRc-PYU16 | PesaRc-PYU92 |
| PesaR-p | PesaRp-PYU3 | PesaRp-PYU7 | PesaRp-PYU16 | PesaRp-PYU92 |

* Test 2.0

**Stationary Phase Promoter Characterization—2.0**

![Fig.7: Some characterization results of PYU promoter and PesaR](https://static.igem.wiki/teams/4583/wiki/engineering/test2-10.png)
![Fig.7: Some characterization results of PYU promoter and PesaR](https://static.igem.wiki/teams/4583/wiki/engineering/test2-20.png)

**Fig.7 |** Some characterization results of PYU promoter and
PesaR

These are the eleven groups out of 24 combinations that
fulfilled the expected criteria: there was a
**significant difference** in the **expression times** of
the two promoters.

* Learn 2.0

In the 2nd iteration, we find promoters that meet the need based
on the results of the previous iteration and characterize them
in our system. Out of 24 combinations, we found one combination
that met the expectation. This means that
**each part of our model has been successfully characterized
separately**. We hope to further validate our system within the same
bacteria, which will be more rigorous and scientific.

* 3rd iteration

* Goal

In the previous iterations, we have validated the feasibility of
each component separately. However, our advisors pointed out that
**we need to characterize the individual components inside the
same bacterium**, which would be more rigorous and scientifically sound.

* Design 3.0

![Using BFP for characterization](https://static.igem.wiki/teams/4583/wiki/engineering/design3-1.png)

In this iteration, we decide to transform **3** plasmids
combinations into L19 and L31. We used **BFP** for
characterization of PYU promoter.

![Fig.8: Genetic circuit for the late stationary phase promoter (linked with BFP gene)](https://static.igem.wiki/teams/4583/wiki/engineering/design3-2.png)

**Fig.8 |** Genetic circuit for the late stationary phase
promoter (linked with BFP gene)

* Build 3.0

We construct the following **10** strains that each contains
3 plasmids based on 1st and 2nd iteration:

* Our 10 strains

![Our 10 strains](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

| No. | Name | Plasmid 1 | Plasmid 2 | Plasmid 3 |
| --- | --- | --- | --- | --- |
| 01 | L19-PesaS-PesaRwt-PYU3 | pCL-PesaS | pCL-PesaRwt | PYU3 |
| 02 | L19-PesaS-PesaRwt-PYU7 | pCL-PesaS | pCL-PesaRwt | PYU7 |
| 03 | L19-PesaS-PesaRwt-PYU92 | pCL-PesaS | pCL-PesaRwt | PYU92 |
| 04 | L19-PesaS-PesaRc-PYU3 | pCL-PesaS | pCL-PesaRc | PYU3 |
| 05 | L19-PesaS-PesaRc-PYU7 | pCL-PesaS | pCL-PesaRc | PYU7 |
| 06 | L19-PesaS-PesaRp-PYU7 | pCL-PesaS | pCL-PesaRp | PYU7 |
| 07 | L31-PesaS-PesaRwt-PYU3 | pCL-PesaS | pCL-PesaRwt | PYU3 |
| 08 | L31-PesaS-PesaRwt-PYU92 | pCL-PesaS | pCL-PesaRwt | PYU92 |
| 09 | L31-PesaS-PesaRp-PYU7 | pCL-PesaS | pCL-PesaRp | PYU7 |
| 10 | L31-PesaS-PesaRp-PYU16 | pCL-PesaS | pCL-PesaRp | PYU16 |

* Test 3.0

**Three-Layer Dynamic Regulation Model Characterization**

Here are the results:

![Fig.9: Characterization results of Three-Layer Dynamic Regulation Model Characterization ](https://static.igem.wiki/teams/4583/wiki/engineering/test3-1.png)
![Fig.9: Characterization results of Three-Layer Dynamic Regulation Model Characterization ](https://static.igem.wiki/teams/4583/wiki/engineering/test3-2.png)

**Fig.9 |** Characterization results of Three-Layer Dynamic
Regulation Model Characterization

![Fig.10: The results of L19-PesaRwt-PYU3 strain](https://static.igem.wiki/teams/4583/wiki/engineering/test3-3.png)

**Fig.10 |** The results of L19-PesaRwt-PYU3 strain

* Learn 3.0

After three iterations,
**we successfully developed the model**. This model worked
well in E. coli MG1655. Subsequently, we applied it to the
production of PHB.

* References

![Fig.6 The formula we use to assess our web accessibility](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

|  |
| --- |
| 1. Minogue, T.D., et al., The autoregulatory role of EsaR, a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function. Mol Microbiol, 2002. 44(6): p. 1625-35. |
| 2. Jaishankar, J. and P. Srivastava, Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid, 2020. 109: p. 102491. |
| 3. Talukder, A.A., et al., RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli. FEBS Lett, 1996. 386(2-3): p. 177-80. |

{% endblock %}

{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block
lead %}Describe the research, experiments, and protocols you used in your iGEM
project.{% endblock %} {% block header %}

Protocols

2023 SDU-CHINA

{% endblock %} {% block page\_content %}

[**01 Preparation**](#01)
[**02 Main experimental:**](#02)
[**03 Fermentation experiment**](#03)
[**04 Others**](#04)

* 01 Preparation

* 1.1 Medium

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

**Equipment**

- Conical bottle.
- Test tube.
- dd Water.
- Medium component.

**Equipment**

- LB medium
- 1L dd Water.
- 10g Tryptone.
- 5g Yeast extract.
- 10g NaCl.

**LB solid medium**

- LB liquid medium.
- 1.5-2% AGAR powder.

**Fermentation medium**

- 1L dd Water.
- 2g Yeast powder.
- 1g NaCl.
- 1g NH4Cl.
- 15.6g Na2HPO4·12H2O.

**Protocol**

After sterilization, 200uLMgSO4 solution (1M),100uLCaCl2 solution
(0.1M) and 4mL50%Glu solution were added every 100mL.

* 1.2 PCR System

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

**Equipment**

- PCR instrument.
- PCR tubule.
- High fidelity polymerase.
- dNTPs.
- 2×Buffer.
- dd Water.
- Primer.

**Colony PCR system**

- 10uL Taq Enzyme mix mixing system.
- 10uL dd Water.
- 1uL Primer 1 (10uM).
- 1uL Primer 2 (10uM).
- 1uL Template.

**PCR reaction system (50 uL)**

- 1-10ng Template.
- 2uL Primer 1 (10uM).
- 2uL Primer 2 (10uM).
- 25uL 2×Buffer.
- 1uL dNTPs.
- 1uL High fidelity polymerase.
- Up to 50uL ddH2O.

* 02 Main experimental

* 2.1 Electroporation

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

**Equipment**

- 1mm electroporation cuvettes.
- Electroshock apparatus.
- Strain to be transferred.
- Plasmid to be transferred.
- 10% glycerol.
- LB medium.

**Protocol**

**1.** The recipient bacteria (DH5a or DH10B) were inoculated
overnight with 1% inoculated in LB medium and cultured overnight
in a shaking bed at 37℃.

**2.** 200uL of overnight culture was transferred to 5mL LB
medium, and the culture was violently shaken on a shaking table
at 37℃ until OD600 = 0.6 (about 2.5-3 hours).

**3.** Absorb 1.5mL cultured bacterial solution into 1.5mL
centrifuge tube.

**4.** Centrifuge at 12000rpm at 4℃ for 5 min.

**5.** Discard the supernatant, add 1000 uL 10% glycerin,
gently suck up and down with a pipette gun to make the cells
re-suspend.

**6.** Centrifuge at 12000rpm at 4 ° C for 5 min.

**7.** Discard the supernatant, add 1000 uL cold 10%
glycerin, gently suck up and down with a pipette gun to make the
cells re-suspend.

**8.** Centrifuge at 12000rpm at 4 ° C for 5 min.

**9.** Discard the supernatant, add 200uL10% glycerin, gently
draw up and down with a pipette gun to make the cells
re-suspend.

**10.** Add 5-10 uL plasmid to each cell, gently suck and
evenly with a pipette.

**11.** The converter selects 1800V as the output voltage.

**12.** Convert the cup, immediately press the button to
shock.

**13.** Immediately add 1mL LB medium to the transformation
cup to resuspension cells.

**14.** The cells were transferred into a suitable culture
tube and cultured at 37℃ for 1 hour.

**15.** Absorb a suitable volume of bacterial liquid coated
with the finished antibiotic medium plate.

**16.** Overnight culture at 37℃ and observation results.

* 2.2 Extracting Plasmid

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

**Equipment**

- Omega plasmid extraction kit.

**Protocol**

**1.** Isolate a single colony from a freshly streaked
selective plate, and inoculate a culture of 1-5 mL LB medium
containing the appropriate selective antibiotic. Incubate
for~12-with a volume of at least 4 times the volume of the
culture. It is strongly recommended that an endA negative strain
of E. coli be used for routine plasmid isolation. Examples of
such strains include DH5a® and JM109®.

**2.** Centrifuge at 10,000 x g for 1 minute at room
temperature.

**3.** Decant or aspirate and discard the culture media.

**4.** Add 250 uL Solution I/RNase A. Vortex or pipet up and
down to mix thoroughly. Complete resuspension of cell pellet is
vital for obtaining good yields.

**Note:** RNase A must be added to Solution I before use.
Please see the instructions in the Preparing Reagents section
on Page 6.

**5.** Transfer suspension into a new 1.5 mL microcentrifuge
tube.

**6.** Add 250 uL Solution Il. Invert and gently rotate the
tube several times to obtain a clear lysate. A 2-3 minutes
incubation may be necessary.

**Note:** Avoid vigorous mixing as this will shear
chromosomal DNA and lower plasmid purity. Do not allow the
lysis reaction to proceed more than 5 minutes. Store Solution
Il tightly capped when not in use to avoid acidification from
CO, in the air.

**7.** Add 350 uL Solution Ill. Immediately invert several
times until a flocculent white -precipitate forms.

**Note:** It is vital that the solution is mixed thoroughly
and immediately after the addition of Solution Ⅲ to avoid
localized precipitation.

**8.** Centrifuge at maximum speed (≥13,000 x g) for 10
minutes. A compact white pellet will form. Promptly proceed to
the next step.

**9.** Prepare the vacuum manifold according to
manufacturer's instructions.

**10.** Connect the HiBind® DNA Mini Column to the vacuum
manifold.

**Optional Protocol for Column Equilibration**

1. Add 100 uL3M NaOH to the HiBind® DNA Mini Column.

2. Turn on the vacuum source to draw the NaOH through the
column.

3. Turn off the vacuum.

**11.** Transfer the cleared supernatant from Step 8 by
CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be
careful not to disturb the pellet and that no cellular debris is
transferred to the HiBind® DNA Mini Column.

**12.** Turn on the vacuum source to draw the sample through
the column.

**13.** Turn off the vacuum.

**14.** Add 500 uL HBC Buffer.

**Note:** HBC Buffer must be diluted with isopropanol
before use. Please see Page 6 for instructions.

**15.** Turn on the vacuum source to draw the buffer through
the column.

**16.** Turn off the vacuum.

**17.** Add 700 uL DNA Wash Buffer.

**Note:** DNA Wash Buffer must be diluted with 100% ethanol
prior to use. Please see Page 6 for instructions.

**18.** Turn on the vacuum source to draw the buffer through
the column.

**19.** Turn off the vacuum.

**20.** Repeat Steps 17-19 for a second DNA Wash Buffer wash
step.

**21.** Transfer the HiBind® DNA Mini Column to a 2 mL
Collection Tube.

**22.** Centrifuge the empty HiBind® DNA Mini Column for 2
minutes at maximum speed to dry the column matrix.

**Note:** It is important to dry the HiBind® DNA Mini
Column matrix before elution. Residual ethanol may interfere
with downstream applications.

**23.** Transfer the HiBind® DNA Mini Column to a clean 1.5
mL microcentrifuge tube.

**24.** Add 30-100 uL Elution Buffer or sterile deionized
water directly to the center of the column membrane.

**25.** Let sit at room temperature for 1 minute.

**26.** Centrifuge at maximum speed for 1 minute.

**Note:** This represents approximately 70% of bound DNA.
An optional second elution will yield any residual DNA, though
at a lower concentration.

**27.** Store DNA at -20℃.

* 2.3 Gibson assembly

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

**Equipment**

- PCR instrument.
- PCR recovery kit.
- Metal bath.
- Assembly mix.
- EP tube.

**Protocol**

**1.** The target fragment was obtained by PCR.

**2.** DpnⅠ treatment.
0.5uL DpnⅠ enzyme solution was
added into the 50uL system at 37℃ for 1h.

**3.** PCR recovery.

**1.** The reaction liquid added to the column and
centrifuged at 13000rpm for 1min.

**2.** Add 500uL HBC, centrifuge at 13000rpm for 1 min.

**3.** Add 700uL DNA WASH Buffer, centrifuge at 13000rpm
for 1min, repeat twice.

**4.** Dry empty tube for 2min, replace EP tube, add
20-80uL ddH2O or TE buffer at 65℃, centrifuge at 13000rpm for
1min.

**4.** Linkage reaction.
The ligation reaction system is maintained at 50°C for 30-60
minutes to complete the ligation reaction.

**Linkage reaction system (20uL)**

- 8.7uL Target fragment 1
- 1.3uL Target fragment 2.
- 10uL 2×assembly mix.

**5.** The linking solution containing the recombinant
plasmid is converted into DH5α.

* 2.4 Chemical Transformation of Plasmids into DH5α

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

**Equipment**

- Commodity DH5α.
- Ice.
- Water bath.
- LB medium.

**Protocol**

**1.** Take the thawed-sensitive state DH5a on ice, add
1ug/uL plasmid 1uL into the 50uL receptive state, and place it
on ice for 30min.

**2.** Heat shock 90s in 2.42°C water bath, quickly
transferred to the ice for 2min.

**3.** 1mL LB medium was added into the plasmid-containing
receptive state of each tube on a super-clean table, shaken at
37℃ for 1h.

**4.** LB AGAR culture dishes were taken from the super-clean
table, and 20mL AGAR medium (containing transformed plasmid
resistant antibiotics) was poured into each dish to coagulate.

**5.** Centrifuge at 5.12000 rpm, discard the supernatant,
take 100uL of invert bacteria coated plate, and invert it in 37℃
constant temperature incubator for 12-16 hours, and observe the
colony growth the next day.

**6.** Stripe separation and purification, single colonies
were selected in liquid medium, culture, bacteria liquid PCR,
and storage strains.

* 03 Fermentation experiment

* 3.1 Fermentation Method

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

**Equipment**

- Spectrophotometer.
- Glucose tester.
- Gas chromatography.
- Shaker.
- dd Water.
- Conical bottle.
- Fermentation medium.
- Strain to be fermented.

**Protocol**

**1.** 1% inoculated in 100mL fermentation medium at 37℃ at
250rpm.

**Note:** Three parallel experiments were conducted for each
strain.

**2.** Samples were taken every 3-4 hours in the first 20
hours, OD600 and glucose concentrations were measured every 6-10
hours after 20 hours, and PHB concentrations were measured after
11 hours.

**Note:** During this period, glucose is supplemented to
approximately 20g/L in the fermentation broth based on glucose
concentration data.

* 04 Others

* 4.1 Plasmid carrying resistance

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

- pCL-PesaS-GFP (LVA) is Kan.
- pCL-PesaR-B0034-mkate, pCL-PesaRp-B0034-mkate,
  pCL-PesaRc-B0034-mkate indicates the Spc.
- The PACYC series of plasmids were resistant to Cm and later
  changed to Spc.
- pUC-PesaR-B0034-PHBcab is Amp resistant.
- L19-B0034-gltA and L31-B0034-gltA are Cm resistant.

* 4.2 Strain preservation method

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

- 800uL bacterial solution +200uL glycerin stored in -20℃
  refrigerator.

* 4.3 Characterization and inoculation amount before
  fermentation

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

- 1% dose of bacteria.
- 24-well plate 1mL medium, 12-well plate 3mL, 96-well plate 100uL.

* 4.4 Preparation method of antibiotics

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

| Name | Preparation method | Sterilization method | Concentration |
| --- | --- | --- | --- |
| Amp | 0.5g+5mL dd water | Filtration | 1‰ |
| Kan | 0.5g+10mL dd water | Filtration | 1‰ |
| Cm | 1.02g+30mL anhydrous ethanol | Filtration | 0.5‰ |
| Spc | 1.25g+25mL dd water | Filtration | 0.5‰ |

* 4.5 Electrophoretic detection

![These 6 strains are for QS- switch characterization](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

**1.** Accurately prepared 1% agarose gel, fully boiling,
cooling to about 60℃ can be added to the coloring agent GelRed
(highly sensitive, safe and highly stable fluorescent nucleic
acid dye). Sampling, electrophoresis.

**2.** After electrophoresis, the imaging was observed under
the gel imager.

{% endblock %}

{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block lead
%}You can describe the results of your project and your future plans here.{%
endblock %} {% block header %}

Results

2023 SDU-CHINA

{% endblock %} {% block page\_content %}

[**Introduction**](#intro)
[**01 Selection of QS system and bacterial strains**](#01)

[**02 The characterization of Esa I/R QS system**](#02)

[**03 Selection and characterization of stationary phase promoter**](#03)

[**04 Characterization of Three-layer dynamic regulation system**](#04)

[**05 Selection and Characterization of lysis gene**](#05)

[**06 Validation of the effectiveness of PHB synthesis genes**](#06)

[**07 PHB fermentation analysis**](#07)

[**Failures & Experiences**](#F&E)
[**References**](#ref)

* **Introduction**

. On this page, we describe the most important results that portray
our journey while designing our project.

* 01 Selection of quorum sensing system and bacterial strains

* 1.1 Esa I/R Quorum Sensing (QS) system

**Quorum Sensing**is a way for cells to regulate downstream gene
expression based on their own density. The concentration of the
signaling molecule - **AHL** - secreted by the cell increases as
the cell density increases. When the concentration of AHL reaches a
certain level, it can bind to the corresponding binding protein and
alter the expression of downstream genes.

**The Esa I/R system** is quite special from traditional QS
system. The EsaI/R QS system is homologous to the LuxI/R QS system
and originates the maize pathogen--Pantoea stewartii subsp.
stewartia. EsaR can act as
**both transcriptional activator and repressor**.

![Esa I/R system](https://static.igem.wiki/teams/4583/wiki/result/esa.jpg)

**Fig.1 |** Esa I/R system

* 1.2 Our strains and Previous work

Our project builds on what has been done before. In 2020, Fei Gu et.
al succeeded in **redirecting the metabolic flow**of E. coli
using the Esa I/R system[1]. And they have applied the QS switch in
the production of PHB. The Stains that they used come from another
study[2]. Our project goes one step further. We have
**re-characterized the system** and
**added an auto-lysis system**.This constitutes
**a three-layer dynamic regulation model** that enables the
separation of the bacterial growth phase, the production phase and
the product-release phase. **L19** and **L31** were engineered
E. coli MG1655, which already have Esa R/I system in them. They have
different regulatory thresholds (Fig.2), and we chose L19 and L31 as
our strains.

![switching-od-of-different-strains](https://static.igem.wiki/teams/4583/wiki/result/switching-od-of-different-strains.png)

**Fig.2 |** Switching OD of different strains[2]

* 02 The characterization of Esa I/R quorum sensing system

* 2.1 Design

We transformed pCL-PesaS-GFP (LVA) into E. coli L19 and L31 in
combination with pCL-PesaRwt-mkate, pCL-PesaRc-mkate, and
pCL-PesaRp-mkate, respectively. We repeatedly characterized the Esa
I/R QS system by detecting fluorescence intensity to reflect
promoter expression intensity. **Green fluorescence** was used to
reflect the transcriptional expression intensity of **PesaS**,
and **red fluorescence** represented the transcriptional
expression intensity of **PesaR**. In this way, the most suitable
models for growth and production stages were screened. Here are the
strains we constructed.

**Table.1 |** Strains for QS switch characterization

| ITEM | MEANING | CONNT | UNITS |
| --- | --- | --- | --- |
| L19 | pCL-PesaS-GFP(LVA) | pCL-PesaRwt-mkate | L19SRw |
| pCL-PesaRc-mkate | L19SRc |
| pCL-PesaRp-mkate | L19SRp |
| L31 | pCL-PesaS-GFP(LVA) | pCL-PesaRwt-mkate | L31SRw |
| pCL-PesaRc-mkate | L31SRc |
| pCL-PesaRp-mkate | L31SRp |

* 2.2 Results

![the-results-of-qs-system-characterization](https://static.igem.wiki/teams/4583/wiki/result/the-results-of-qs-system-characterization.png)

**Fig.3 |** The results of QS system characterization

* 2.3 Analysis of results

Through preliminary characterization, we can clearly see that
**PesaS and PesaR have a distinct sequential expression time
sequence at different stages**. The **PesaS** transcript expression
**peaked in the first 8 hours**, especially in the 4th-6th hours,
while PesaR peaked and stabilized after 10-12 hours (except for
PesaR-p).

We hoped that by comparing the before and after differences in the
expression of the two, we would be able to enter into the growth
mode during the propagation of the strains faster, and the
boundaries between the production mode and the growth mode would be
clearer, with a quicker and more complete transition, and the
results performed very well for both.

* 03 Selection and characterization of stationary phase promoter

* 3.1 Why we need stationary phase promoter and its advantages

If artificial induction is used to make the bacteria lysed, the
inducer is not only **expensive**, but also produces
**strong toxicity** to the cells. If a general constitutive
promoter is used to express the cleavage gene, it will cause the
**bacterial metabolic burden** and **great growth pressure**.

So, we planned to use **stationary phase promoter** to control
the lysis gene. Bacteria undergo significant changes in their
physiological state after entering the plateau phase. The stationary
phase promoter endogenously produces more
**σ factors**
required for the stationary phase when bacteria enter the
exponential phase, and it can direct bacteriophage RNA polymerase to
transcribe specific genes during the stationary phase. Because the
signal required for the stationary phase promoter is produced
**endogenously**, it is not toxic to the bacteria. It is also
**dynamically regulated** and does not cause metabolic stress or
growth problems.

* 3.2 Selection and Characterization results of first batch
  promoters (Pflic, P1.1, P2.1, P3.1)

We combined PACYC-Pflic-GFP, PACYC-P1.1-GFP, PACYC-P2.1-GFP, and
PACYC-P3.1-GFP plasmids (these plasmids were given by Xin Jin, our
advisor) with pCL-PesaRwt, pCL-PesaRc, and pCL-PesaRp plasmids
(These plasmids were given by Fei Gu, our advisor), respectively,
and electrotransformed them into L19 and L31. In total,
**we constructed 24 strains** (Table 2)

We then used a Multi-Detection Microplate Reader (Synergy HT,
Biotek, U.S.) to detect the red (excitation at 585 nm and emission
at 640 nm) fluorescence and green (excitation at 485 nm and emission
at 528 nm) fluorescence.

**Table.2 |** Plasmids combinations for first screening of
promoters

| ITEM | MEANING | CONNT |
| --- | --- | --- |
| L19 | pCL-PesaRwt-mkate | PACYC-Pfic-GFP |
| PACYC-P1.1-GFP |
| PACYC-P2.1-GFP |
| PACYC-P3.1-GFP |
| pCL-PesaRp-mkate | PACYC-Pfic-GFP |
| PACYC-P1.1-GFP |
| PACYC-P2.1-GFP |
| PACYC-P3.1-GFP |
| pCL-PesaRc-mkate | PACYC-Pfic-GFP |
| PACYC-P1.1-GFP |
| PACYC-P2.1-GFP |
| PACYC-P3.1-GFP |
| L31 | pCL-PesaRwt-mkate | PACYC-Pfic-GFP |
| PACYC-P1.1-GFP |
| PACYC-P2.1-GFP |
| PACYC-P3.1-GFP |
| pCL-PesaRpt-mkate | PACYC-Pfic-GFP |
| PACYC-P1.1-GFP |
| PACYC-P2.1-GFP |
| PACYC-P3.1-GFP |
| pCL-PesaRc-mkate | PACYC-Pfic-GFP |
| PACYC-P1.1-GFP |
| PACYC-P2.1-GFP |
| PACYC-P3.1-GFP |

![](https://static.igem.wiki/teams/4583/wiki/result/2.png)
![The characterization results of Pfic, P1.1, P2.1, and P3.1 in L19](https://static.igem.wiki/teams/4583/wiki/result/the-characterization-results-of-pfic-p1-1-p2-1-and-p3-1-in-l19.png)

**Fig.5 |** The characterization results of Pfic, P1.1, P2.1,
and P3.1 in L19

![The characterization results of Pfic, P1.1, P2.1, and P3.1 in L31                   ](https://static.igem.wiki/teams/4583/wiki/result/the-characterization-results-of-pfic-p1-1-p2-1-and-p3-1-in-l31.png)

**Fig.6 |** The characterization results of Pfic, P1.1, P2.1,
and P3.1 in L31

The expression time was found
**to be close to that of PesaR**, with a maximum difference of
only 2-4 hours to peak (Fig.4). Also, expression of this promoter
is **very rapid**, with
**peak expression in less than 4 hours** (Fig5, Fig.6). This
means that by constructing an auto-lysis system with such a
stationary phase promoter, our engineered bacteria will
**only have a few hours to produce PHB**, not taking into
account the metabolic disruption and reduced yield caused by the
timing being too close to meet our expectations. : (

We also found that Pfic, P1.1, P2.1 and P3.1
**have different levels of expression** (Fig5, Fig.6)[3]. This
difference is particularly evident in L19 (Fig5, Fig.6).

* 3.3 Selection and Characterization results of second batch
  promoters (PYU3, PYU7, PYU16, and PYU92)

So, by reading the scientific articles, we selected the late
stationary phase promoters of **PYU3, PYU7, PYU6** and
**PYU92**
for the second screening[4]. We combined them with PesaR and
characterized them. In this batch we also constructed 24 bacteria
(table 3).

**Table.3 |** Plasmids combinations for second screening of
promoters

| Strain | Plasmid 1 | Plasmid 2 |
| --- | --- | --- |
| L19 | pCL-PesaRwt-mkate | PACYC-PYU3-GFP |
| PACYC-PYU7-GFP |
| PACYC-PYU16-GFP |
| PACYC-PYU92-GFP |
| pCL-PesaRp-mkate | PACYC-PYU3-GFP |
| PACYC-PYU7-GFP |
| PACYC-PYU16-GFP |
| PACYC-PYU92-GFP |
| pCL-PesaRc-mkate | PACYC-PYU3-GFP |
| PACYC-PYU7-GFP |
| PACYC-PYU16-GFP |
| PACYC-PYU92-GFP |
| L31 | pCL-PesaRwt-mkate | PACYC-PYU3-GFP |
| PACYC-PYU7-GFP |
| PACYC-PYU16-GFP |
| PACYC-PYU92-GFP |
| pCL-PesaRp-mkate | PACYC-PYU3-GFP |
| PACYC-PYU7-GFP |
| PACYC-PYU16-GFP |
| PACYC-PYU92-GFP |
| pCL-PesaRc-mkate | PACYC-PYU3-GFP |
| PACYC-PYU7-GFP |
| PACYC-PYU16-GFP |
| PACYC-PYU92-GFP |

![Ten valid combinations of late stationary promoter characterization ](https://static.igem.wiki/teams/4583/wiki/result/ten-valid-combinations-of-late-stationary-promoter-characterization1.png)
![Ten valid combinations of late stationary promoter characterization ](https://static.igem.wiki/teams/4583/wiki/result/result111.jpg)

**Fig.7 |** Ten valid combinations of late stationary
promoter characterization

From the 24 characterization results, the above ten combinations
with significant expression time differences were selected and
prepared for further screening (Fig. 7).

In several of these combinations, we found, for example,
L19-PesaRwt-PYU7, that the two promoters peaked in their
respective expressions at times that
**differed by more than 10 hours**The results show that
**the production and cleavage phases can be temporally
uncoupled**Therefore, the characterization results have met our requirement
-
**the production phase is separated from the product-release
phase**. : )

* 3.4 Conclusion

In the end,
**the late steady state promoters PYU3, PYU7, PYU16 and PYU92**
were selected as our ideal promoters. We also selected
**ten combinations** that met our expectations to further build
the three-layer dynamic regulation system.

* 04 Characterization of Three-layer dynamic regulation system

In the previous section, we first verified that
**the Growth Phase is temporally decoupled from the Production
Phase**, and then verified that
**the Production Phase and the Product-release Phase are temporally
decoupled**.

To be more scientific, we decided to **co-characterize**  the
growth phase, the production phase and the product launch phase, and
to check that all three were **expressed in chronological** order
and **at the required time**.

Due to the certain red-green crosstalk problem during detection, we
constructed PACYC-PYU3-BFP, PACYC-PYU7-BFP, PACYC-PYU16-BFP,
PACYC-PYU92-BFP plasmids by ligating the blue fluorescent protein gene
BFP with PYU3,7,16,92 promoter (Fig.8, Fig9) in order to make the
results clearer and the overall expression time sequence. We
constructed 10 strains that each have 3 plasmids (table 4).

![Genetic circuit of PACYC-PYU3-BFP ](https://static.igem.wiki/teams/4583/wiki/result/genetic-circuit-of-pacyc-pyu3-bfp.png)

**Fig.8 |** Genetic circuit of PACYC-PYU3-BFP

![BFP fragment](https://static.igem.wiki/teams/4583/wiki/result/bfp-fragment.png)

**Fig.9 |** BFP fragment

**Table.4 |**  Plasmids combinations of 3-plasmids characterization

| Strain | Plasmid 1 | Plasmid 2 | Plasmid 3 |
| --- | --- | --- | --- |
| L19 | pCL-PesaSGFP (LVA) | pCL-PesaRwt-mkate | PACYC-PYU3-BFP |
| pCL-PesaSGFP (LVA) | PACYC-PYU7-BFP |
| pCL-PesaSGFP (LVA) | PACYC-PYU92-BFP |
| pCL-PesaSGFP (LVA) | pCL-PesaRwt Pmkate | PACYC-PYU7-BFP |
| pCL-PesaSGFP (LVA) | pCL-PesaRc-mkate | PACYC-PYU3-BFP |
| pCL-PesaSGFP (LVA) | PACYC-PYU7-BFP |
| L31 | pCL-PesaSGFP (LVA) | pCL-PesaRwt-mkate | PACYC-PYU3-BFP |
| pCL-PesaSGFP (LVA) | PACYC-PYU92-BFP |
| pCL-PesaSGFP (LVA) | pCL-PesaRpmkate | PACYC-PYU7-BFP |
| pCL-PesaSGFP (LVA) | PACYC-PYU16-BFP |

* 4.1 The results of characterization

![](https://static.igem.wiki/teams/4583/wiki/result/the-characterization-results-of-three-layer-dynamic-regulation-model1.png)
![ The characterization results of Three-layer dynamic regulation model](https://static.igem.wiki/teams/4583/wiki/result/the-characterization-results-of-three-layer-dynamic-regulation-model2.png)

**Fig.10 |**  The characterization results of Three-layer
dynamic regulation model

![The model of L19-PesaRwt-PYU3](https://static.igem.wiki/teams/4583/wiki/result/the-model-of-l19-pesarwt-pyu3.png)

**Fig.11 |** The model of L19-PesaRwt-PYU3

* 4.2 Analysis

We can see from this that the stationary phase begins to be
expressed when the production phase is at its peak and the
separation of the production phase from the desired lysis phase has
been achieved. This means that
**the growth phase of the bacteria is barely affected** and
**the bacteria have more than enough time for the production
phase**. It was only **after 40 hours** that the bacteria entered the
expected lysis peak and lysed in droves.

* 4.3 conclusion

We built three different modules,**the growth module** ,**the production module**
, and **the product release module**, which form our
**Three-layer dynamic regulation model**. we expressed them in
the same bacterium, and the results were very good.

* 05 Selection and Characterization of lysis gene

* 5.1 Selection of lysis system

* **Lambda phage lysis gene cassette[5] (hereinafter referred
  to as SRRz System)**

When **holin monomers** are isolated from antiholins that
inhibit holin function, their hydrophobic structural domains are
inserted into the cell membrane and then aggregate to form a more
ordered assembly in a pore as large as ~500 kDa, allowing proteins
to cross the cell membrane. Endolysins accumulated in the
cytoplasm can then be released into the periplasm to degrade
peptidoglycan in the cell wall and, in addition, the Rz/Rz1
complex of the λ phage promotes fusion of the inner membrane (IM)
and outer membrane (OM), which in turn pushes the OM away from the
murein layer, removing the final barrier.

* **Phi X174 phage lysis system[6] (hereafter referred to as
  the E system)**

**Protein E** of phi174 is a hydrophobic membrane protein
consisting of 91 amino acids that itself lacks enzymatic activity.
It is hypothesized to function by triggering endogenous murein
hydrolase in the host to
**form a transmembrane structure**
that spans the inner and outer membranes.

* 5.2 Plasmid construction

We cloned the backbone part on the PACYC plasmid except the GFP
fragment by PCR using **the Gibson method**, leaving a short
homologous sequence with the lysis gene. At the same time, we
obtained the SRRz gene by company synthesis and cloned the SRRz gene
fragment containing the homologous arm of PACYC; the E gene fragment
containing the homologous arm of PACYC was cloned by PCR on Phi X174
plasmid, and the PACYC backbone fragment and the cleaved gene
fragment were seamlessly joined under the action of recombinase.
PACYC-PYU3, 7, 16, 92-SRRz and PACYC-PYU3, 7, 16, 92-E plasmids were
constructed (Fig. 12). All gene recombinations in the experiment
were performed by the Gibson method.

![Map of lysis plasmid construction](https://static.igem.wiki/teams/4583/wiki/result/map-of-lysis-plasmid-construction-l.png)

![Map of lysis plasmid construction](https://static.igem.wiki/teams/4583/wiki/result/map-of-lysis-plasmid-construction-r.png)

**Fig.12 |**  Map of lysis plasmid construction

* 5.3 First characterization of lysis gene

We transformed the constructed plasmids into L19 and L31 to observe
its lysis effect. The left side of the figure shows L19 containing
the cleavage plasmid, and the right side does not contain the
cleavage plasmid. After 1h of resting, it can be seen that
**the left side** has been **cleaved and clarified**, and
**the right side** is
**turbid, and the bacterium is sinking**
(Fig. 13).

![Comparison of lysis effect](https://static.igem.wiki/teams/4583/wiki/result/comparison-of-lysis-effect.jpg)

**Fig.13 |** Comparison of lysis effect

* 5.4 Second characterization of lysis gene and RBS of different
  strength

Due to incomplete lysis in L19 and L31, we utilized the
**Gibson self-assembly method** and designed primers to add RBS
(BBa\_B0031, BBa\_B0032, BBa\_B0033, and BBa\_B0034) of different
intensities to the promoter regions of PYU3, PYU7, PYU16, PYU92 with
intensities of 0.010. 0.07,0.3, and 1 (Fig. 14). Due to time and
effort constraints, we did not succeed in constructing all plasmids
(Fig. 15).

![Genetic circuit of lysis system](https://static.igem.wiki/teams/4583/wiki/result/genetic-circuit-of-lysis-system.png)

**Fig.14 |** Genetic circuit of lysis system

![Electrophoretic validation results](https://static.igem.wiki/teams/4583/wiki/result/electrophoretic-validation-results.png)

**Fig.15 |** Electrophoretic validation results

Due to primer design problems, PCR extension length setting, or
assembly errors, etc., the RBS substitution was not completely done
successfully, so the plasmids verified to be working successfully in
the following table were used for the next experiments.

**Table.5 |** Valid plasmids

| Strain | Plasmid 1 | Plasmid 2 | Plasmid 3 | Lysis gene |
| --- | --- | --- | --- | --- |
| L19 | PACYC | PYU3 | B0031 | E |
| PACYC | B0032 | E |
| PACYC | B0033 | E |
| PACYC | B0034 | E |
| L19 | PACYC | PYU7 | B0031 | E |
| PACYC | B0032 | E |
| PACYC | B0033 | E |
| PACYC | B0034 | E |
| L19 | PACYC | PYU16 | B0031 | E |
| PACYC | B0032 | E |
| PACYC | B0033 | E |
| PACYC | B0034 | E |
| L19 | PACYC | PYU7 | B0031 | E |
| PACYC | B0032 | E |
| PACYC | B0033 | E |
| PACYC | B0034 | E |
| L19 | PACYC | PYU3 | B0031 | SRRz |
| PACYC | B0032 | SRRz |
| L19 | PACYC | PYU16 | B0031 | SRRz |
| PACYC | B0034 | SRRz |
| L31 | PACYC | PYU92 | B0031 | SRRz |
| PACYC | B0032 | SRRz |
| PACYC | B0034 | SRRz |

Next, we transformed them to L19,L31 for
**characterization**:

![The results of lysis system characterization](https://static.igem.wiki/teams/4583/wiki/result/the-results-of-lysis-system-characterization.png)

**Fig.16 |** The results of lysis system characterization

**PYU16-B0034-SRRz** and **PYU92-B0034-SRRz** were finally
selected as the ideal lysis systems (Fig. 16). They have the
advantages of **higher efficient lysis capacity** and
**low impact on strain growth**.

* 06 Validation of the effectiveness of PHB synthesis genes

**Nile red** has fluorescent properties, so it can be used as a
fluorescent dye for staining microplastics, lipids and proteins, etc.
It is commonly used in fluorescence microscopy and flow cytometry to
detect intracellular lipid droplets. Through Nile red staining and
fluorescence microscopy of bacterial cells containing PHB and non-PHB
lipid storage substances, Nile red is a good fluorescent stain for
**lipid substances** stored in bacterial cells with high
sensitivity[7].

* 6.1 Method

Nile red dye powder, prepared as a solution using dimethyl
sulfoxide, was added to the medium at a working concentration of 0.5
μg/ml and plates were poured. We set up two groups:
**the first group** used Nile Red plates, with PHB-producing
strains and non-PHB-producing strains delineated on the plates;
**the second group** used PHB-producing strains, delineated on
Nile Red and non-Nile Red plates. Observations were made using a UV
imager after 48 hours of incubation.

![Nile Red Plate：PHB-producing strain vs. non-PHB-producing strain](https://static.igem.wiki/teams/4583/wiki/result/17.jpg)

**Fig.17 |** Nile Red Plate：PHB-producing strain vs.
non-PHB-producing strain

![PHB-producing strains streaked on Nile red plate and non-Nile red plate control](https://static.igem.wiki/teams/4583/wiki/result/18.jpg)

**Fig.18 |** PHB-producing strains streaked on Nile red plate
and non-Nile red plate control

* 6.2 Analysis

From the figure, we can see that
**the PHB-producing strains can be observed to fluoresce**
while the non-PHB-producing strains cannot fluoresce under UV
irradiation. And Nile Red works very effectively because there is no
fluorescence on the non-Nile Red plate. This is proof that
**our PHBcab gene is effective**.

* 07 PHB fermentation analysis

* 7.1 strains construction

**Table.6 |** Strains for fermentation analysis

| No. | Strains | Note |
| --- | --- | --- |
| **Group1** | L19: PesaS-B0034 (integrated into the genome), pUC-PesaRwt-PHBcab, PACYC-PYU16-0034-SRRz； | Treatment group |
| **Group2** | L31: PesaS-B0034(integrated into the genome), pUC-PesaRwt- PHBcab，PACYC-PYU92-0034-SRRz |
| **Group3** | L19: PesaS-B0034(integrated into the genome), pUC-PesaR- PHBcab | Control group |
| **Group4** | L31: PesaS-B0034(integrated into the genome), pUC-PesaR- PHBcab |
| **Group5** | L19: pUC-Pcon- PHBcab |
| **Group6** | L31: pUC-Pcon- PHBcab |
| **Group7** | L19: pUC-Pcon- PHBcab, PACYC-PYU16-B0034-SRRz |
| **Group8** | L31: pUC-Pcon- PHBcab, PACYC-PYU92-B0034-SRRz |

![Stains construction](https://static.igem.wiki/teams/4583/wiki/result/stains-construction02.jpg)

**Fig.19 |** Stains construction

* 7.2 Fermentation conditions

Single colonies were transferred to 5mL of Luria−Bertani (LB) Broth
with appropriate antibiotics. And they were culturing at 220rpm/ min
and 37℃for 8h. 1% seeds were inoculated in M9 medium supplemented
with 22g/L glucose.

**Table.7 |** 1L M9 medium

| Material | Volume/Mass |
| --- | --- |
| **ddH2O** | 1L |
| **Yeast extraction** | 2g |
| **NaCl** | 1g |
| **NH4Cl** | 1g |
| **Na2HPO4·12H2O** | 15.6g |
| **KH2PO4** | 3g |

![Sampling](https://static.igem.wiki/teams/4583/wiki/result/sampling.jpg)

**Fig.20 |** Sampling

* 7.3 Measurement

Samples were taken at 3,7,11,14,24,30,38,48h for OD600 and glucose
concentrations, and PHB concentrations were measured at the same
time from the **11th hour** onwards (Fig. 15).

* **Glucose**

After diluting the samples 50-fold (Fig. 16), the glucose
concentration was measured using a glucose tester and glucose was
replenished to 20 g/L at 14h and 25.5h of fermentation. Using our
hardware can easily complete this work. The raw data is in the
Notebook.

![Dilution of samples for glucose detection](https://static.igem.wiki/teams/4583/wiki/result/dilution-of-samples-for-glucose-detection02.jpg)

**Fig.21 |**  Dilution of samples for glucose detection

* **OD600**

Using spectrophotometer to measure OD600(Fig. 17). The raw data is
in the notebook.

![Spectrophotometer](https://static.igem.wiki/teams/4583/wiki/result/spectrophotometer.jpg)

**Fig.22 |** SpectrophotometerSpectrophotometer

* **PHB**

To test the PHB content, we took 2.85mL of culture solution for each
group and added 0.15mL of chloroform (5%). Mix by gently inverting up
and down, then centrifuge at 3400xg for 8 minutes at 4°C. Remove the
chloroform-PHB phase (lower layer) in a glass vial with a pipette gun.
To it, 150ul of sulfuric acid was added, 850ul of methanol was added,
1000 µl of chloroform was added, and oil bath was used for 1 h. It was
taken out, cooled down to room temperature and then 1ml of dd water
was added, and the shaker was shaken for 30 s. It was left to stand
for more than 30 min for layering, and 80ul-150ul of the chloroform
layer was taken into the gas-phase vial. Determination was carried out
by **gas chromatography**[8].

![Gas chromatography](https://static.igem.wiki/teams/4583/wiki/result/gas-chromatography.jpg)

**Fig.23 |** Gas chromatography

However, when we tested it, the results showed that
**there was no PHB**. Since we had previously performed a
step-by-step validation to ensure that each part could function
properly, we suspected that there was a problem with the method of
extracting PHB or the method of conducting the test.

In order to verify our conjecture, we took 1mL of the 48h culture
solution and centrifuged it, then removed the supernatant and took a
small amount of the bacterium and observed it under the fluorescence
microscope (Fig. 19 and Fig. 20). We found that in Group 3 bacteria,
**there were many glowing red particles**, which were intracellular
PHB (Fig. 20), while in Group 1 bacteria, there were few red
particles, which represented that most of the PHB had been released
(Fig. 19). This suggests that
**our three-layer model is valid, it's the detection method that's
wrong**.

So, we will need to validate our results in a different way.

![ Group1 (has lysis system)](https://static.igem.wiki/teams/4583/wiki/result/group1-has-lysis-system-2.jpg)

**Fig.24 |**  Group1 (has lysis system)

![Group3 (does not have lysis system)](https://static.igem.wiki/teams/4583/wiki/result/group3-does-not-have-lysis-system-2.jpg)

**Fig.25 |** Group3 (does not have lysis system)

We then added 3 ml of chloroform to 15 mL of culture medium at 48 h
and centrifuged it for 8 min at 4°C, which was used to extract
extracellular PHB. 100 μL of the chloroform layer was then taken, and
0.5 μL of Nile Red-stained chloroform layer (working concentration 0.5
μg/ml) was added and measured by fluorescence intensity using a
Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) (Fig.
21).

![Using fluorescence intensity to measure PHB](https://static.igem.wiki/teams/4583/wiki/result/using-fluorescence-intensity-to-measure-phb-content.png)

**Fig.26 |** Using fluorescence intensity to measure PHB content

![results](https://static.igem.wiki/teams/4583/wiki/result/results.png)

**Fig.27 |** results

All of these results demonstrate that the extracellular PHB production
of the strains containing the three-layer regulatory system was
**much higher** than that of the strains without the addition of
the lysis system or without the use of the Quorum Sensing system to
regulate the metabolic flow.

* Failures & Experiences:

1. Transformation：

The failure rate of simultaneous transfer of two plasmids during
electroporation was significantly higher, which was later switched to
**one by one** with a higher success rate.

It is especially important to prepare receptor cells for
transformation plasmid experiments, and
**culturing them to the pre-exponential stage** (2 hours of
transfection) can improve transformation efficiency.

**The recovery culture should not be too long**. We have had
transformation failures due to too long recovery cultures.

E. coli can usually appear colonies in one day. When it appears to
take more than 24h to grow a colony,
**this colony may not be E. coli**. The first fermentation of this
experiment was re-tested for a second eletroporation and fermentation
analysis because the yeast colonies were picked resulting in no
fermentation product being detected in the end.

2. **The amount of bacterial solution** when applying the plate
**should not be too much**, it will result in growing into one
layer (all bacteria in this experiment are prone to grow into a layer,
except for the one containing a strong cleavage gene).

3. The strain needs to be
**re-cultured and preserved every 3 months**. If the time is too
long, the activity of the bacteria will be affected.

4. When using the **Gibson assembly method** for seamless ligation
of gene fragments, **try changing primers** if you encounter a
difficult ligation situation or if the pcr product is difficult to
obtain.

5. When extracting the plasmid, the incubation time needs to depend on
the situation. E. coli DH5α with **lysogeny plasmid** needs to
extract the plasmid **around 4 hours of growth**, otherwise
**the plasmid is lost** due to bacterial lysis resulting in large
amounts of content being discharged.

6. Our experiments required the measurement of extracellular PHB,
which did not work well despite being mentioned in some scientific
articles. We need to further explore the experimental conditions.

* Refences

* References

![Fig.6 The formula we use to assess our web accessibility](https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png)

|  |
| --- |
| 1. Gu, F., et al., Quorum Sensing-Based Dual-Function Switch and Its Application in Solving Two Key Metabolic Engineering Problems. ACS Synth Biol, 2020. 9(2): p. 209-217. |
| 2. Gupta, A., et al., Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit. Nat Biotechnol, 2017. 35(3): p. 273-279. |
| 3. Jaishankar, J. and P. Srivastava, Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid, 2020. 109: p. 102491. |
| 4. Talukder, A.A., et al., RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli. FEBS Lett, 1996. 386(2-3): p. 177-80. |
| 5. Gao, Y., et al., Inducible cell lysis systems in microbial production of bio-based chemicals. Appl Microbiol Biotechnol, 2013. 97(16): p. 7121-9. |
| 6. Barrell, B.G., G.M. Air, and C.A. Hutchison, 3rd, Overlapping genes in bacteriophage phiX174. Nature, 1976. 264(5581): p. 34-41. |
| 7. Greenspan, P., E.P. Mayer, and S.D. Fowler, Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol, 1985. 100(3): p. 965-73. |
| 8. Borrero-de Acuña, J.M., et al., A novel programmable lysozyme-based lysis system in Pseudomonas putida for biopolymer production. Sci Rep, 2017. 7(1): p. 4373. |

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