iGEM 2023 — Team westcentral-hs

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**DAY1/2023.7.31**

We
shared and discussed the content of the iGEM program and received
training on laboratory safety. Then, we prepared LB liquid medium and
LB solid medium.
![](https://static.igem.wiki/teams/4842/wiki/cimafen/1.png)

**DAY2/2023.8.1**

We
learned the basic principles and procedures of molecular cloning, as
well as the use of Snapgene software.

**DAY3/2023.8.2**

We
learned the principles and operations of plasmid extraction and
extracted the pET28a-mCherry and pETM6-pnar plasmids.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/2.png)

**DAY4/2023.8.3**

We
double-digested pET28a-mCherry and pETM6-pnar with *Xho*I
and *Avr*II
restriction enzymes, respectively. Then, we
performed agarose gel electrophoresis to verify the fragments.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/3.png)

**DAY5/****2023.****8.4**

We
learned the principles and operations of gel extraction and fragment
recovery of the cut strips.

**DAY6/****2023.****8.5**

After
obtaining the target fragment, we ligated the two fragments using T4
ligase, an enzyme ligation reaction that needs to be performed
overnight.

**DAY7/****2023.****8.6**

We
transformed the recombination product into the *E.
coli* DH5α
competent cells, performed a plate-coating operation, and incubated
the plates overnight.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/4.png)

**DAY8/****2023.****8.7**

Since
yesterday's experiment failed, the steps of yesterday's experiment
were repeated today.

**DAY9/****2023.****8.8**

We
identified the transformants by colony PCR and inoculated the
transformants with the correct bands.

**DAY10/****2023.****8.9**

We
used the kit to extract plasmids from overnight cultures for later
construction of the RBS mutant library.

**DAY11/****2023.****8.10**

We
learned how to design mutants for RBS with the RBS calculator
(https://salislab.net/software/). Primers with mutant sites were
designed for subsequent whole plasmid PCR.

**DAY12/****2023.****8.11**

Today,
we obtained the RBS1-RBS8 fragments by whole plasmid PCR, blunted the
ends of the products, and ligated them overnight with T4 enzyme,
respectively.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/5.png)

**DAY13/****2023.****8.12**

We
transformed the recombination
product into *E.
coli* BL21(DE3)
competent
cells, spread the plates, and incubated the plates overnight.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/6.png)

**DAY14/****2023.****8.13**

We
identified the transformants by colony PCR and inoculated the
transformants with the correct bands.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/7.png)

**DAY15/****2023.****8.14**

We
inoculated the transformants verified by colony PCR for tomorrow's
fluorescence intensity test.

**DAY16/****2023.****8.15**

We
performed fluorescence intensity tests on the obtained RBS mutant
library to determine which RBS sequences have higher translation
efficiency.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/8.png)

**DAY17/****2023.****8.16**

Organize
experimental results and write wiki materials.

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© 2023 - Content on this site is licensed under a Creative Commons Attribution 4.0 International license.
The repository used to create this website is available at gitlab.igem.org/2023/westcentral-hs.

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**Overview****：**

(*S*)-equol
is a highly bioactive metabolite with antioxidant capacity that
prevents and alleviates cardiovascular diseases, skin aging, hair
loss, obesity, and other hormone-related conditions[1].
However, only 25-50% of the global population harbors the specialized
gut microflora required to naturally produce (*S*)-equol,
and merely a small fraction of these “equol producers”
generate therapeutically effective doses during menopause[2].
To enable low-cost (*S*)-equol
production through synthetic biology, our team aimed to regulate
biosynthesis by modulating (*S*)-equol
enzyme expression at the translational level via RBS optimization. We
executed two engineering design (DBTL) cycles:

1)
Designing and constructing the pETM6-pnar-mCherry plasmid and
evaluating its expression.

2)
Designing and constructing a pETM6-pnar-RBS (1-8)-mCherry mutant
library and assessing the regulatory effects of different RBS mutants
on protein expression.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/figure-1.jpg)

**Figure
1 Design diagram of this project.**

**Cycle
1:** **Construction
of the plasmid****pETM6-pnar-mCherry**

**Design
1:**

We
chose the pETM6-pnar plasmid as a vector, in which the *nar*promoter
is anaerobically inducible, enabling induction at any cell growth
stage. For the model protein, we chose mCherry, which is the most
commonly used red fluorescent protein with great photostability and
is ideal for tracking expression levels. We designed the expression
frame of pETM6-pnar-mCherry (Figure 2), and after obtaining this
plasmid, we tested whether the *nar*
promoter could successfully initiate the transcription of target
proteins under anaerobic conditions.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/heart/2222.png)

**Figure
2 Expression frame of the pETM6-****pnar-mCherry.**

**Build
1:**

To construct plasmid pETM6-pnar-mCherry, we first double-digested
pET28a-mCherry and pETM6-pnar with *X**ho*I and *Avr*II
restriction enzymes, respectively (Figure 3A). After recycling the
target fragments, we ligated the mCherry fragment with the pETM6-pnar
vector using T4 DNA ligase to obtain the complete recombinant plasmid
(Figure 3B). We then transformed the recombinant plasmid into *E.
coli* DH5α competent cells. After overnight culture,
positive transformants grew on LB plates. We identified them by
colony PCR to identify the transformant with the correct band and
inoculated it for subsequent plasmid extraction.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/figure-3.jpg)

**Figure
3** **Construction
result of the plasmid****pETM6-pnar-mCherry.**

**Test
1:**

We inoculated positive transformants, followed by fluorescence
intensity testing to measure mCherry expression after induction of
expression under hypoxic conditions. As shown in Figure 4, as culture
time increased, the fluorescence intensity of pETM6-pnar-mCherry
cells became stronger, while no fluorescence was detected in
pETM6-pnar cells. This indicates successful mCherry expression
induction by the *nar* promoter.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/heart/4444.png)

**Figure
4** **Fluorescence
intensity assay results of****pETM6-pnar-mCherry.**

**Learn
1:**

The
anaerobically induced *nar*
promoter successfully induced mCherry expression, and its expression
could be evaluated by fluorescence intensity. We demonstrated that
mCherry fluorescence intensity (*i.e*.,
expression level) exhibited a good linear relationship with induction
time, and pETM6-pnar without mCherry did not produce fluorescence.
This reflects the reliability of this method for subsequent testing
of the regulatory effects of different RBS sequences on protein
expression levels.

**Cycle
2:** **Construction
of the pETM6-pnar-RBS (1-8)-mCherry****mutant library**

**Design
2:**

The
role of RBS is to help the ribosome properly bind with messenger RNA
(mRNA) and initiate protein synthesis. The sequence of RBS can be
adjusted to control the binding efficiency between the ribosome and
mRNA, thereby affecting the protein expression level. To test the
regulation of protein expression by the series of RBS sequences
downstream of the *nar*
promoter, we mutated the RBS sequence on the pETM6-pnar-mCherry
plasmid. We designed eight RBS mutants using the RBS Calculator
(<https://salislab.net/software/>)
and tested the regulation of protein expression by different RBS
mutants.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/figure-5.jpg)

**Figure
5 Design diagram of DBTL cycle 2.**

**Build
2:**

We performed whole plasmid PCR on pETM6-pnar-mCherry using eight
synthesized primers with mutation sites, and the results showed that
we successfully amplified plasmids with different RBS sequences to
obtain the pETM6-pnar-RBS (1-8)-mCherry mutant library. We then
transformed the pETM6-pnar-RBS (1-8)-mCherry plasmids into *E. coli*BL21(DE3) cells, respectively. Colony PCR identification showed
that we obtained the pETM6-pnar-RBS (1-5)-mCherry mutant library
(Figure 6).

![](https://static.igem.wiki/teams/4842/wiki/cimafen/figure-6.jpg)

**Figure
6** **Construction
results of pETM6-pnar-RBS (1-8)-mCherry mutant library****.**

**Test
2:**

We inoculated the positive transformants of the pETM6-pnar-RBS
(1-8)-mCherry mutant library, and then performed fluorescence
intensity tests to measure the results of the regulation of mCherry
expression by different RBS sequences. As shown in Figure 7, in
pETM6-pnar-RBS (1-8)-mCherry cells, there were three RBS sequences
(RBS 4, RBS 6, and RBS 1) that exhibited the strongest fluorescence.
These three RBS sequences are promising for regulating the expression
of (*S*)-equol pathway enzymes.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/figure-7.jpg)

**Figure
7** **Fluorescence
intensity assay results of RBS mutant library.**

**Learn
2:**

By
optimizing the RBS sequence, we achieved regulation of target protein
expression at the translational level. The three best-performing RBS
sequences have potential for application in producing (*S*)-equol
biosynthetic pathway enzymes. This method provides another useful
tool for regulating protein expression, facilitating the optimization
of biological processes in synthetic biology and metabolic
engineering.

**Reference**

[1] Legette L L, Prasain J, King J, et al. Pharmacokinetics of
equol, a soy isoflavone metabolite, changes with the form of equol
(dietary versus intestinal production) in ovariectomized rats [J].
Journal of agricultural and food chemistry, 2014, 62(6): 1294-1300.

[2] Mayo B, Vázquez L, Flórez A B. Equol: A Bacterial
Metabolite from The Daidzein Isoflavone and Its Presumed Beneficial
Health Effects [J]. Nutrients, 2019, 11(9): 2231.

© 2023 - Content on this site is licensed under a Creative Commons Attribution 4.0 International license.
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1. **Preparation of media**

1. **LB liquid medium:**

1. Weigh out 10 g tryptone, 5 g
   yeast extract, and 10 g NaCl, then add these components to an
   Erlenmeyer flask.

   - Dissolve the powder in 1 L
     distilled water in an Erlenmeyer flask.

     - Mix well and sterilize by
       autoclaving on liquid cycle at 121 °C for 15 min.

       - Allow medium to cool to room
         temperature after autoclaving before use.

         - Store prepared LB liquid
           medium at room temperature or 4 °C until use.

2. **LB solid medium**

   1. Weigh out 10 g tryptone, 5 g
      yeast extract, and 10 g NaCl, then add these components to an
      Erlenmeyer flask.

      - Weigh out 25 g of LB powder
        per liter of medium.

        - Dissolve the powder in
          distilled water in an Erlenmeyer flask.

          - Mix well and sterilize by
            autoclaving on a liquid cycle at 121 °C for 15 min.

            - Allow medium to cool to 50-60
              °C after autoclaving.

              - Aseptically pour plates in a
                laminar flow hood. Allow to solidify.

                - Store prepared LB agar plates
                  upside down at 4 °C until use.

2. **Construction of plasmid
   pETM6-pnar**

1. **Double enzyme digestion of
   fragments and vectors**

The double digestion system for
**pnar fragment**
is shown in the table below:

| **Reagent** | **Volume (μL)** |
| **Template****（****synthetic plasmid pCDM4-pnar****）** | 20 |
| ***Nde*I restriction enzyme** | 1 |
| ***Avr*Ⅱ restriction enzyme** | 1 |
| **10× CutSmart Buffer** | 5 |
| **ddH2O** | 23 |
| Total | 50 |

The double digestion system for
vector is shown in the table below:

| **Reagent** | **Volume (μL)** |
| **Template****（****pETM6****）** | 20 |
| ***Nde*I restriction enzyme** | 1 |
| ***Avr*Ⅱ restriction enzyme** | 1 |
| **10× CutSmart Buffer** | 5 |
| **ddH2O** | 23 |
| Total | 50 |

The enzyme digestion systems are
incubated at 37 ℃ for 3 h.

2. **Agarose gel electrophoresis**

1. Agarose gel preparation

1. Add 1 g agarose powder to 100 mL
   1× TAE buffer in a flask.

   - Heat in microwave on high for
     2-3 min, swirling occasionally until agarose dissolves completely.

     - Let agarose cool slightly and
       add 10 μL Ts-GelRed stain. Swirl to mix.

       - Pour agarose into a gel tray
         with a well comb inserted. Allow to solidify at room temperature for
         10 min.

         - Carefully remove the comb and
           place the gel in the electrophoresis chamber filled with 1×
           TAE buffer.

1. Electrophoresis

1. Pour about 400 mL TAE into the
   electrophoresis chamber.

   - Carefully take out the gel and
     replace it in the electrophoresis chamber.

     - Prepare
       DNA samples by mixing the 10× DNA loading buffer with the
       sample at a ratio of 1: 9.

       - Add 5 μL marker in the first
         well of the gel, then add 5 μL of each sample into the following
         wells.

         - Turn on the power of the
           electrophoresis chamber and wait for about 30-40 min.

1. Visualizing DNA bands

1. Take out the gel and place it
   under the Ultra Slim LED Illuminator

   - Open the Image Lab and scan the
     gel.

     - Comparison with markers to
       identify the target DNA bands

3. **Fragment extraction**

1. Cut the DNA gel:

1. Excise DNA bands of interest
   quickly using a clean scalpel under UV light.

   - Cut a minimal amount of agarose
     gel to minimize contaminants.

     - Transfer gel slices into
       pre-weighed microcentrifuge tubes and determine the weight of gel
       slices.

1. DNA gel extraction:

1. Add 3 times of sample volume of
   Buffer B2, and heat at 50 ℃ for 10 min until the gel is
   completely dissolved.

   - Transfer the solution into
     columns, centrifuge the tubes at 8,000 *×g*
     for 1 min, and discard the filtrate in the collection tube.

     - Add 500 µL Wash Solution,
       centrifuge the tubes at 9,000 *×g*
       for 1 min, and discard the filtrate in the collection tube.

       - Repeat the wash step.

         - Place the column in a clean 1.5
           mL tube. Add 30 μL Elution buffer to the center of the membrane.
           Incubate for 1 min at room temperature. Centrifuge at 9,000 *×g*for 1 min and keep
           the DNA solution.

4. **Ligation of pETM6-pnar**

The ligation system is shown in
the table below:

| **Reagent** | **Volume (μL)** |
| 10× T4 DNA Ligase buffer | 2 |
| **vector (pETM6)** | 6 |
| **fragment (pnar)** | 6 |
| T4 DNA Ligase | 1 |
| **ddH2O** | 1 |
| Total | 20 |

The ligation system is incubated
at 16 ℃ for 16 h, followed by 5 min at 65 ℃ to inactivate
the ligase.

5. **Transformation of recombinant
   plasmid to** ***E.
   coli*** **DH5α**

1. Add 10 μL of pETM6-pnar
   plasmid into 100 μL of *E.
   coli* DH5α
   competent cells.

   - Incubate the mixture
     on ice for 30 min.

     - Heat shock at 42 °C for 90
       sec, and put the mixture to the ice for 4-5
       min.

       - Add the LB culture into the
         tubes to 1 mL, and shake the tubes at 37 ℃, 220 rpm for 30-60
         min.

         - Centrifuge it at 9,000 ×*g*
           for 1 min, remove 800 μL
           of supernatant, and resuspend the cells in the remaining LB culture.

           - Spread 50 μL cells onto LB
             agar plates containing
             ampicillin and incubate overnight
             at 37 ℃.

6. **PCR identification of
   transformants**

1. Place 50 μL of Lysis Buffer
   for Microorganism to Direct PCR in a sterilized microtube. Pick a
   single colony with a sterilized toothpick or lance tip, and place it
   in the microtube.

   - After heat denaturation at 80 °C
     for 15 min, centrifuge at low speed and take 1~5 μL of
     supernatant after lysis as PCR template.

     - The PCR reaction system is shown
       in the table below:

| **Reagent** | **Volume (μL)** |
| **Lysis supernatant** | 1 |
| **Premix Taq** | 10 |
| **Forward Primer (10 μM)** | 1 |
| **Reverse Primer (10 μM)** | 1 |
| **sterilized water** | 7 |
| Total | 20 |

The PCR reaction condition is
shown in the table below:

| **Process** | **Temperature (˚C)** | **Time (sec)** |
| **Denaturation** | 94 | 30 |
| **Annealing** | 55 | 30 |
| **Extension** | 72 | 120 |
| **Back to step 1 (30 cycles)** | | |
| **Final elongation** | 72 | 300 |
|  | 4 | ∞ |

4. Inoculate the positive
   transformants in 3 mL LB culture medium containing 50 μg/mL
   ampicillin at a 37 ℃ shaker with 220 rpm overnight.

7. **Plasmid Extraction**

1. Column Equilibrium

1. Insert one spin column CP3 into
   the collection tube.

   - Add 500 μL buffer BL in the
     tube.

     - Centrifuge the tube at 12,000
       rpm for 1 min.

       - Discard the supernatant in the
         collection tube.

2. Bacteria Extraction

1. Harvest 1-5 mL overnight
   cultured bacteria into a centrifuge tube.

   - Centrifuge the tube at 12,000
     rpm for 1 min.

     - Discard the supernatant.

       - Invert the tube on the absorbent
         paper to dry.

3. Plasmid
   Extraction

1. Add 250 μL buffer P1 to the
   centrifuge tube.

   - Insert the tube into the vortex
     machine until the bacteria are suspended thoroughly.

     - Add 250 μL buffer P2 to the
       centrifuge tube.

       - Turn it upside down for 6-8
         times temperately.

         - Add 350 μL buffer P3 to the
           centrifuge tube.

           - Immediately turn it upside down
             6-8 times temperately until the solution is uniformly mixed.

             - Centrifuge the tube at 12,000
               rpm for 10 min.

               - Aspirate the supernatant and
                 transfer it to spin column CP3 in the collection tube.

                 - Centrifuge it at 12,000 rpm for
                   30-60 sec.

                   - Discard the supernatant in the
                     collection tube.

4. Wash

1. Add 600 μL buffer PW to the
   spin column CP3.

   - Centrifuge it at 12,000 rpm for
     30-60 sec.

     - Discord the supernatant in the
       collection tube.

       - Insert the spin column back into
         the collection tube.

         - Repeat the wash step.

           - Centrifuge the tube at 12,000
             rpm for 2 min.

5. Plasmid Elution

1. Insert the spin column CP3 into
   a clean microcentrifuge tube.

   - Add 50-100 μL buffer EB to
     the center of the spin membrane.

     - Place the tube at room
       temperature for 2 min.

       - Centrifuge it at 12,000 rpm for
         2 min.

         - Keep the plasmid solution in the
           centrifuge tube.

3. **Construction of plasmid
   pETM6-pnar-mCherry**

1. **Double enzyme digestion of
   fragments and vectors**

The double digestion system for
**mCherry fragment**
is shown in the table below:

| **Reagent** | **Volume (μL)** |
| **Template****（****pET28a-mCherry****）** | 20 |
| ***Xho*I restriction enzyme** | 1 |
| ***Avr*Ⅱ restriction enzyme** | 1 |
| **10× CutSmart Buffer** | 5 |
| **ddH2O** | 23 |
| Total | 50 |

The double digestion system for
vector is shown in the table below:

| **Reagent** | **Volume (μL)** |
| **Template****（****pETM6-pnar****）** | 20 |
| ***Xho*I restriction enzyme** | 1 |
| ***Avr*Ⅱ restriction enzyme** | 1 |
| **10× CutSmart Buffer** | 5 |
| **ddH2O** | 23 |
| Total | 50 |

The enzyme digestion systems are
incubated at 37 ℃ for 3 h.

2. **Agarose gel electrophoresis**

This step is as previously
described.

3. **Fragment extraction**

This step is as previously
described.

4. **Ligation of
   pETM6-pnar-mCherry**

The ligation system is shown in
the table below:

| **Reagent** | **Volume (μL)** |
| 10× T4 DNA Ligase buffer | 2 |
| **vector (pETM6-pnar)** | 6 |
| **fragment (mCherry)** | 6 |
| T4 DNA Ligase | 1 |
| **ddH2O** | 1 |
| Total | 20 |

The ligation systems are
incubated at 16 ℃ for 16 h, followed by 5 min at 65 ℃ to
inactivate the ligase.

5. **Transformation of recombinant
   plasmid to** ***E.
   coli*** **DH5α**

This step is as previously
described.

6. **PCR identification of
   transformants**

This step is as previously
described.

7. **Plasmid Extraction**

This step is as previously
described.

4. **Mutation library construction
   for the pETM6-pnar-RBSx-mCherry plasmid**

1. **RBS sequences design of the
   nar promoter**

Eight downstream RBS sequences of
nar promoter are designed by RBS calculator
(https://salislab.net/software/), and the sequences are shown in the
following table:

| **Name** | **Sequence (5’-3’)** |
| **RBS 1** | AGGTAGCCG |
| **RBS 2** | AGGCCCGAC |
| **RBS 3** | AGAAATGCA |
| **RBS 4** | AGAAACAAC |
| **RBS 5** | AGGACCAAA |
| RBS 6 | AGAGAACAC |
| RBS 7 | AGGCGGGGA |
| RBS 8 | AGAAACAGA |

2. **PCR**
   **Point mutations
   introduced by whole plasmid PCR**

For point mutation by whole
plasmid PCR, the following primers are designed:

| **Primer** | **Sequence (5’-3’)** |
| **Primer F1** | AGGTAGCCGTATACATATGGCAGATCTCAATTGGAT |
| **Primer F2** | AGGCCCGACTATACATATGGCAGATCTCAATTGGAT |
| **Primer F3** | AGAAATGCATATACATATGGCAGATCTCAATTGGAT |
| **Primer F4** | AGAAACAACTATACATATGGCAGATCTCAATTGGAT |
| **Primer F5** | AGGACCAAATATACATATGGCAGATCTCAATTGGAT |
| Primer F6 | AGAGAACACTATACATATGGCAGATCTCAATTGGAT |
| Primer F7 | AGGCGGGGATATACATATGGCAGATCTCAATTGGAT |
| Primer F8 | AGAAACAGATATACATATGGCAGATCTCAATTGGAT |
| Primer R | CTTTAATGAATTCTTTAAGGGCATTATACC |

The reaction system for PCR
amplification is shown in the table below:

| **Reagent** | **Volume (μL)** |
| **2× Hieff PCR Master Mix (With Dye)** | 25 |
| **Primer F (10 μM)** | 2.5 |
| **Primer R (10 μM)** | 2.5 |
| **DNA template** | 1 |
| **ddH2O** | 10 |
| **Total** | 50 |

The reaction procedure
for PCR amplification is shown in the table below:

| **Process** | **Temperature (˚C)** | **Time (sec)** |
| **Pre-denaturation** | 98 | 30 |
| **Denaturation** | 98 | 10 |
| **Annealing** | 60 | 20 |
| **Extension** | 72 | 20 |
| **Back to step 2 (30 cycles)** | | |
| **Final elongation** | 72 | 300 |
|  | 4 | ∞ |

3. **Degradation
   of PCR template with** ***Dpn*****Ⅰ**

The degradation
system is shown in the table below:

| **Reagent** | **Volume (μL)** |
| **PCR product** | 50 |
| ***Dpn*Ⅰ restriction enzyme** | 1 |
| **Green Buffer** | 5 |

The degradation
system is incubated at 37 ℃ for 30 min, followed by 5 min at 65
℃ to inactivate the ligase.

4. **Blunt-end
   treatment of PCR product and ligation**

1. The PCR products are end-blunted
   using the DNA Blunting Kit with the following reaction system:

| **Reagent** | **Volume (μL)** |
| **PCR product** | 1 |
| **10× Reaction Buffer** | 1 |
| **sterilized water** | 8 |

\* DNA fragments must be
precipitated by ethanol.

2. Keep at 70 ° C for 5 min,
   then transfer to 37 °C.

   - Add 1 μL T4 DNA Polymerase in
     the mixture. Mix gently.

     - Reaction at 37℃ for 5 min.

5. **Transformation
   of recombinant plasmids to** ***E.
   coli*** **BL21(DE3)**

(1) Add 5 μL of
pETM6-pnar-RBS(1-8)-mCherry plasmids into 50 μL of *E.
coli* BL21(DE3)
competent cells, respectively.

(2) Incubate the mixture on ice
for 30 min.

(3) Heat shock at 42 °C for
90 sec, and put the mixture to the ice for 4-5 min.

(4) Add the LB culture into the
tubes to 1 mL, and shake the tubes at 37 ℃, 220 rpm for 30-60
min.

(5) Centrifuge it at 9,000 *×g*
for 1 min, remove 900 μL of supernatant, and resuspend the cells
in the remaining LB culture.

(6) Spread 50 μL cells onto LB
agar plates containing ampicillin and incubate overnight at 37 ℃.

6. **PCR identification of
   transformants**

The PCR reaction system and
procedure are described previously.

5. **Fluorescence intensity
   detection of RBS mutant library.**

1. Inoculate the positive
   transformants in 3 mL LB culture medium containing 50 μg/mL
   ampicillin at a 37 ℃ shaker with 220 rpm overnight.

   - Inoculate overnight cultures at
     a ratio of 1:100 into 25 mL fresh LB culture medium containing 50
     μg/mL ampicillin.

     - Incubate at 37 °C, 200 rpm
       until OD600
       reaches 0.8.

       - Set the shaker speed to 50 rpm
         and 30 ℃ to induce protein expression.

         - After induction, mCherry
           expression is measured every half hour using a fluorescence enzyme
           marker (excitation light at 580 nm, emission light at 610 nm).

           - All samples are diluted with
             phosphate buffer solution (PBS) to the appropriate OD600
             for fluorescence determination.

             - The BL21 (DE3) strain
               transformed with the mutant promoter plasmid is assayed under the
               same conditions as the background and subtracted from the total
               fluorescence to obtain the actual red fluorescence intensity.

© 2023 - Content on this site is licensed under a Creative Commons Attribution 4.0 International license.
The repository used to create this website is available at gitlab.igem.org/2023/westcentral-hs.

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**Overview**

In
this study, we controlled the expression of (*S*)-equol
biosynthetic enzymes in engineered*E. coli*
using the *nar*promoter
induced by anaerobic conditions. This avoided the use of expensive
inducer IPTG during fermentation and provided a microaerobic
environment favorable for (*S*)-equol
biosynthesis. To regulate the expression levels of exogenous genes,
we adjusted the strength of
the ribosome-binding
site (RBS) by modifying its
sequence, thereby influencing the initiation and rate of protein
translation without constructing multiple variants. Using mCherry as
a model protein, we constructed a *nar*
promoter library with different RBS sequences and characterized the
mutant library by fluorescence intensity detection. We aimed to
obtain optimal RBS sequences to achieve regulated expression of
(*S*)-equol
pathway enzymes.

Our
experimental results mainly include:

(1)
Construction of plasmid pETM6-pnar,

(2)
Construction of plasmid pETM6-pnar-mCherry,

(3)
Mutation library construction for the plasmid
pETM6-pnar-RBSx-mCherry,

(4)
Fluorescence
intensity detection of RBS mutant library.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/figure-1.jpg)

**Figure
1 Schematic flow diagram of this experiment.**

(\*The
creation of engineering diagrams received guidance and assistance
from Associate Researcher Xia Xiudong, the Jiangsu Academy of
Agricultural Sciences.)

1. **Construction
   of plasmid pETM6-pnar**

To
construct the plasmid pETM6-pnar, we first double-digested the
synthetic pCDM4-pnar and pETM6 with *Nde*I
and *Avr*II
restriction enzymes, respectively, as shown in Figure 2A. After
recycling the target fragments, we ligated the *nar*fragment
with the pETM6 vector using T4 DNA ligase to obtain the complete
recombinant plasmid (Figure 2B).

We
then transformed the recombinant plasmid into *E.
coli*
DH5α competent cells. After overnight culture, positive
transformants grew on LB plates. We identified transformants with the
correct bands by colony PCR, and inoculated them for subsequent
plasmid extraction. The next day, we extracted plasmid from the
transformant for later construction of plasmid pETM6-pnar-mCherry.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/heart/2222.png)

**Figure
2 Construction result of the plasmid****pETM6-pnar****.**

2. **Construction
   of plasmid pETM6-pnar-mCherry**

To
construct plasmid pETM6-pnar-mCherry, we first double-digested
pET28a-mCherry and pETM6-pnar with *X**ho*I
and *Avr*II
restriction enzymes, respectively, as shown in Figure 3A.
After recycling the target fragments, we ligated the mCherry fragment
with the pETM6-pnar vector using T4 DNA ligase to obtain the complete
recombinant plasmid (Figure 3B).

We
then transformed the recombinant plasmid into *E.
coli*
DH5α competent cells. After overnight culture, positive
transformants grew on LB plates. We identified transformants with the
correct bands by colony PCR, and inoculated them for subsequent
plasmid extraction. The following day, we extracted the plasmid of
the transformant for the construction of pETM6-pnar-RBSx-mCherry
mutant library
containing different RBS sequences.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/figure-3.jpg)

**Figure
3 Construction result of the plasmid****pETM6-pnar-mCherry****.**

3. **Mutation
   library construction for the plasmids pETM6-pnar-RBSx-mCherry**

We
designed eight RBS sequences downstream of the *nar*promoter
using the RBS Calculator (https://salislab.net/software/), as shown
in the table below.

**Table
1 RBS sequences designed by RBS Calculator**

| **Name** | **Sequence (5’-3’)** |
| RBS 1 | AGGTAGCCG |
| RBS 2 | AGGCCCGAC |
| RBS 3 | AGAAATGCA |
| RBS 4 | AGAAACAAC |
| RBS 5 | AGGACCAAA |
| RBS 6 | AGAGAACAC |
| RBS 7 | AGGCGGGGA |
| RBS 8 | AGAAACAGA |

We
then introduced the RBS sequence mutations by whole plasmid PCR. As
shown in Figure 4, we successfully amplified
pETM6-pnar-RBS(1-8)-mCherry plasmids containing different RBS
sequences.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/heart/4444.png)

**Figure
4 Electrophoresis results of whole plasmid PCR.**

We
transformed pETM6-pnar-RBS (1-8)-mCherry plasmids into competent *E.
coli*
BL21(DE3) cells, respectively. After overnight culture, positive
transformants grew on LB plates. Colony PCR identification showed the
correct bands for pETM6-pnar-RBS (1-5)-mCherry transformants, while
no bands were observed for pETM6-pnar-RBS (6-8)-mCherry.
This
may be due to mistakes in picking colonies or omissions in adding
templates. Nevertheless, we inoculated all eight transformants for
subsequent fluorescence intensity testing.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/figure-5.jpg)

Drawing under the guidance of Mr. Wang

**Figure
5 Transformation and colony PCR identification results of plasmids
pETM6-pnar-RBSx-mCherry**

4. **Fluorescence
   intensity detection of RBS mutant library.**

We
measured mCherry expression every half hour after induction using a
fluorometer. Cell density (OD600)
was measured on a UV/VIS spectrophotometer. Total red fluorescence of
whole cells was determined by the fluorometer with excitation at 580
nm and emission at 610 nm. Background from transformants with mutant
promoter plasmids was subtracted under the same conditions to obtain
actual red fluorescence intensity.

As
shown in Figure 6A, as culture time increased, the fluorescence
intensity of pETM6-pnar-mCherry cells became stronger, while no
fluorescence was detected in pETM6-pnar cells. This indicates
successful mCherry expression induction by the *nar*
promoter. As shown in Figure 6B, there were three RBS sequences (RBS
4, RBS 6 and RBS 1) exhibited the strongest fluorescence in
pETM6-pnar-RBS (1-8)-mCherry cells. These three RBS sequences are
promising for regulating the expression of (*S*)-equol
pathway enzymes.

![](https://static.igem.wiki/teams/4842/wiki/cimafen/figure-6.jpg)

**Figure
6 Fluorescence intensity assay results of RBS mutant library.**

In
summary, using mCherry as a model protein, we successfully
constructed the pETM6-pnar-mCherry plasmid. Based on this, we
obtained a *nar*
promoter library with different RBS sequences. The mutant library was
characterized by fluorescence intensity detection, and the three
best-performing RBS sequences were identified. These RBS sequences
have the potential to achieve high-level regulated expression of
(*S*)-equol
pathway enzymes.

© 2023 - Content on this site is licensed under a Creative Commons Attribution 4.0 International license.
The repository used to create this website is available at gitlab.igem.org/2023/westcentral-hs.