iGEM 2023 — Team fshs-gd

{% 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 page\_content %}

![](https://static.igem.wiki/teams/4846/wiki/notebook.png)

**Notebook**

**Day****1 [****19/07****]**

•
 Learned how to safely use pipettes.

•
 Extraction of target plasmids.

•
 Amplification of fragments by PCR.

•
 Agarose gel electrophoresis of fragments.

![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook177.png) ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook179.png)

**Day****2 [27/07]**

•
 Cutting of the agarose gel and the extraction of the gel.

•
 Double enzyme digestion

•
 Formation of recombinant plasmids

 ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook324.png)

**Day 3**

•
 Preparation of LB solid culture medium from the liquid medium.

•
 Transformed

 the recombinant plasmids.

 ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook444.png) ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook446.png)

**Day****4**

•
 PCR identification of transformants

•
 Inoculate positive transformant seed solutions to LB culture medium
plates.

•
 Processed the resulting pictures for the experiments (gel, petri dish
with bacteria etc.)

 ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook668.png) ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook670.png)

**Day****5-6**

•
 Expression of positive transformant containing plasmid A and C.

•
 Prepared competent cell (BL21(DE3) containing plasmid B).

•
 Transformed plasmid D to competent cell BL21 containing plasmid B.

•
 Prepared SDS-PAGE gel.

 ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook911.png) ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook913.png)

**Day****7**

•
 Obtain

cell
lysate
 by ultrasonic crushing machine.

•
 Used Ni2+ column purification method to purify the cell lysate.

•
 Used SDS-PAGE gel electrophoresis to identify protein samples.

•
 Stained the gel after electrophoresis for observation.

 ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook1174.png)

**Day 8**

•
 Isolated bacteria from *E.coli* and inoculated them to petri dishes.

•
 Destained the SDS-PAGE gel.

•
 *In vitro* cleavage of purified proteins.

•
 Proceeded with agar diffusion growth inhibition assay.

![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook1388.png) ![](https://static.igem.wiki/teams/4846/wiki/notebook/notebook1390.png)

**Day 9**

•
 Analyzed the results of the whole experiment

•
 Uploaded basic and composite parts to the iGEM website.

**Day 10-11**

•
 Proceeded with agar diffusion growth inhibition assay.

**Day 12**

•
 Prepare samples and sent to SubCat for agar diffusion growth
inhibition assay.

{% 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 page\_content %}

![](https://static.igem.wiki/teams/4846/wiki/engineering/engineering.png)

**Engineering Success**

**Overview:**

The goal of this project was to enable *E.coli* capable of co-express
ing
 the nisin (or bicereucin) and darobactin. Nisin,
bicereucin, and darobactin require precursor peptides and post-translational modifying (PTM) enzymes to
gain antimicrobial activity. We utilized two approaches to produce bsjA-darA and nisA-darA fusion
peptides, respectively.

In Cycle 1, the precursor proteins and PTM enzymes
were expressed separately on two plasmids, *i.e.*, pRSFduet-DarL-BsjL-His-DarA-BsjA and
pETduet-DarE-BsjM.

In Cycle 2, the precursor proteins and PTM enzymes
were co-expressed on the same plasmid, *i.e.*, pRSFduet-DarL-NisL-His-DarA-NisA-DarE and
pETduet-NisB-NisC.

![](https://static.igem.wiki/teams/4846/wiki/engineering/figure-1.jpg)

Figure
 1. Schematic diagram of cycle 1 and cycle
2.

**Cycle 1:**

**Design:**

To achieve separate expression of the precursor
proteins and PTM enzymes, we designed the expression frame of DarL-BsjL-His-DarA-BsjA and DarE-BsjM,
where DarL and BsjL are leaders of the core peptides (DarA and BsjA) for recognition and binding by the
PTM enzymes (DarE and BsjM). Based on the literature, *E. coli* endogenous peptidases can cleave the darobactin
leader, so a His-tag was included between the leader and core peptide, to enable purification of the mature
product after leader removal.

![](https://static.igem.wiki/teams/4846/wiki/engineering/figure-2.jpg)

Figure
2.
 Expression frame of the plasmids constructed
in
cycle
1
.

**Build:**

We used the restriction enzymes to create
complementary sticky ends on the vector (pRSFduet and pETduet) and fragments (DarL-BsjL-His-DarA-BsjA
and DarE-BsjM). T4 DNA ligase was then used to ligate the vector with the fragments, generating plasmid
A (pRSFduet-DarL-BsjL-His-DarA-BsjA) and plasmid C (pETduet-DarE-BsjM).

![](https://static.igem.wiki/teams/4846/wiki/engineering/figure-3.jpg)

Figure
 3. The generation of the plasmids constructed
in
cycle
1
.

After constructing the recombinant plasmids A and C,
we needed to co-transform them into the same bacterial strain. Plasmid A was first transformed, and
positive transformants were selected to prepare competent cells containing plasmid A, and plasmid C was
then transformed into it. We performed gel electrophoresis and sequencing to verify the successful
transformation
o
f two plasmids into *E.coli*.

![](https://static.igem.wiki/teams/4846/wiki/engineering/figure-4.jpg)

Figure
 4. Colony PCR and sequencing results of transformants containing plasmids
A and C.

**Test:**

We inoculated positive clones and induced protein
expression with IPTG. After induction, bacterial cells were lysed by sonication to release cellular
contents and proteins. Nickel column purification was then performed on the lysate supernatant to obtain
the target fusion peptides at higher purity (Figure 5).

![](https://static.igem.wiki/teams/4846/wiki/engineering/figure-5.jpg)

Figure 5. SDS-PAGE result of the protein expression and
purification.

The purified proteins were subjected to in vitro cleavage with lysyl endopeptidase to release the core peptides. Finally, agar diffusion growth inhibition assays demonstrated that the cleaved DarA-BsjA fusion peptide have little inhibitory effect on Escherichia coli (Gram-negative), while they have antibacterial effect on Bacillus subtilis (Gram-positive), but the antibacterial effect is not very significant (Figure 6).

![](https://static.igem.wiki/teams/4846/wiki/engineering/6a.png)![](https://static.igem.wiki/teams/4846/wiki/engineering/6b.png)

Figure 6. Antibacterial effect of DarA-BsjA fusion peptide antimicrobial peptides on Escherichia coli (top) and Bacillus subtilis (below)

**Learn:**

Perhaps it is because after expressing the fusion peptide, in vitro cleavage experiments are required, and the cleavage results need to be detected by mass spectrometry. The fusion peptide was not completely cleaved, resulting in weak activity and no antibacterial effect. In the future, we will improve the experiment to ensure the cutting effect and the antibacterial activity of the antibacterial peptide.

**Cycle 2:**

**Design:**

To achieve co-expression of the precursors and PTM
enzymes, we designed two expression frames. One was T7-DarL-NisL-His-DarA-NisA-T7-DarE, where
the
first
 MCS contained DarL-NisL-His-DarA-NisA, and
the
second
 MCS contained the darobactin PTM enzyme DarE.
DarL and NisL are leaders of DarA and NisA for recognition and binding by the PTM enzymes. The second
expression frame was NisB-NisC, the PTM enzyme
of
 NisA.

![](https://static.igem.wiki/teams/4846/wiki/engineering/figure-7.jpg)

Figure 7. Expression frame of the plasmids constructed
in
cycle
2.

**Build:**

Through enzymatic digestion and ligation, we ligated
the vector backbones pRSFduet and pETduet with the fragments DarL-NisL-His-DarA-NisA-DarE and NisB-NisC
to construct plasmid B (pRSFduet-DarL-NisL-His-DarA-NisA-DarE) and D
(pETduet-NisB-NisC).

![](https://static.igem.wiki/teams/4846/wiki/engineering/figure-3.jpg)

Figure 8. The generation of the plasmids constructed
in

cycle
 2.

After constructing the recombinant plasmids B and D,
we needed to co-transform them into the same bacterial strain. Plasmid B was first transformed, and
positive transformants were selected to prepare competent cells containing plasmid B, and plasmid D was
then transformed into it. We performed gel electrophoresis and sequencing to verify the successful
transformation
o
f two plasmids into *E.coli*.

![](https://static.igem.wiki/teams/4846/wiki/engineering/figure-9.jpg)

Figure

9. Colony PCR and sequencing results of transformants containing plasmids
B and D.

**Test:**

We inoculated the positive transformant and induced
protein expression with IPTG. The bacterial cells were then lysed by sonication, and nickel purification
was performed to obtain the fusion peptides at higher purity (Figure 10).

![](https://static.igem.wiki/teams/4846/wiki/engineering/figure-10.jpg)

Figure 10. SDS-PAGE result of the protein expression and
purification.

![](https://static.igem.wiki/teams/4846/wiki/engineering/11.png)

Figure 11. Antibacterial effect of DarA-NisA fusion antimicrobial peptide on Bacillus subtilis

In vitro
lysyl endopeptidase cleavage released the core peptides from the purified proteins.
Finally, agar diffusion assays showed the cleaved DarA-NisA fusion peptide inhibited
Bacillus subtilis
(Gram-positive), but the antibacterial effect is not very significant (Figure 11).

**Learn:**

Co-expressing the precursors and PTM enzymes on the same plasmid enabled the modification of
DarA and NisA to confer antimicrobial activity. At the molecular level, we fused DarA and NisA to generate a peptide with complementary
anti-Gram-positive and anti-Gram-negative effects.Perhaps it is because after expressing the fusion peptide, in vitro cleavage experiments
are required, and the fusion peptide is not completely cleaved, resulting in weak activity and no antibacterial effect. In the future,
we will use mass spectrometry to detect the cutting effect and ensure the formation of fusion peptides for antibacterial purposes.
In addition, in the future, we will also conduct antibacterial tests on other common Gram negative and Gram positive bacteria.

{% endblock %}

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

![](https://static.igem.wiki/teams/4846/wiki/experiments.png)

**Experiments/Protocols**

1
**Preparation of the experiment**

1.1
**Preparation of LB** **liquid****culture medium**

(1)
Mix 1% NaCl, 1% peptone, 0.5% yeast extraction, and ddH₂O into in
glass bottle or flask.

(2)
Mix well and autoclave on liquid cycle at 121°C for
20
 min to sterilize.

(3)
Allow medium to cool after autoclaving.

(4)
Store the LB
liquid
 culture medium at room temperature or 4°C.

1.2
**Preparation of LB solid culture
medium**

(1)
Mix 1% NaCl, 1% peptone, 0.5% yeast extraction,
1.5%

agar
, and ddH₂O into in glass bottle or flask.

(2)
Mix well and autoclave on liquid cycle at 121 °C for
20
 min to sterilize.

(3)
Allow medium to cool to 50-60 °C after autoclaving.

(4)
Aseptically pour plates in a laminar flow hood. Allow to
solidify.

(5)
Store prepared LB agar plates at 4 °C.

1.3
**Strain inoculation**

(1)
Inoculate the strains synthesized by the company, which were
transformed into the plasmids (containing target fragments) into 5 mL of liquid LB culture medium,
respectively.

(2)
Overnight incubation at 37°C, 220 rpm.

2
**Plasmid construction and
transformation**

2.1
**Preparation of LB** **liquid****culture medium** **Plasmid extraction**

**In this section, we used the DiaSpin Plasmid
Mini-Preps Kit to extract four plasmids:**

A.
pUC57-DarL-BsjL-His-DarA-BsjA,

B.
pUC57-DarL-NisL-His-DarA-NisA-DarE,

C.
pUC57-DarE-BsjM and

D.
pUC57-NisB-NisC-DarE

**The procedure is as follows****：**

(1)
Equilibrate column: Add 500 μL Buffer S to balance the Miniprep
column, and centrifuge at 12,000 ×*g* for 1 min.

(2)
Collect 5 mL of overnight overnight LB bacteria culture. Centrifuge at
8,000 ×*g* for 2 min to pellet the bacteria and discard the
supernatant.

(3)
Add 250 μL Buffer SP1 (containing RNaseA) to suspend the bacteria
completely by vortexing.

(4)
Add 250 μL Buffer SP2, and gently invert the tube 5-10
times.

(5)
Add 350 μL Buffer SP3 and immediately gently invert the tube 5-10
times. Centrifuge at 12,000 ×*g* for 5-10 min.

(6)
Transfer the supernatant to the column, centrifuge at 8,000
×*g* for 30 sec, and discard the filtrate in the collection tube.

(7)
Add 500 μL Wash Solution, centrifuge at 9,000 ×*g* for 30 sec, and repeat this step.

(8)
Place the column in a clean 1.5 mL tube. Add 50 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.

2.2
**Amplification of target fragments by
PCR**

The reaction system and procedure for PCR amplification of fragments
are shown in the table below:

|  |  |  |
| --- | --- | --- |
| Reagent | Volume (μL) | Procedure |
| PrimeStar Max Premix (2×) | 25 | Denaturation: 98 ℃ for 30 sec |
| Primer F (10 μM) | 1 | Annealing: Tm for 15 sec |
| Primer R (10 μM) | 1 | Extension: 72 ℃, 30 sec/kb |
| DNA template | 1 | Back to step 1 (30 times) |
| ddH2O | 22 | Final elongation: 72 ℃ for 2 min |
| Total | 50 | 10 ℃ hold |

The PCR amplified fragment information is listed below:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Fragment | Amplified  fragment | Length (bp) | Annealing time (sec) | Tm (℃) |
| F1 | BsjA | 399 | 60 | 63 |
| F2 | DarL-DarE | 1715 | 60 | 64 |
| F3 | DarE | 1293 | 60 | 65 |
| F4 | NisB-NisC | 2988 | 90 | 62 |

2.3
**Agarose gel electrophoresis**

(1)
**Preparation of 1× TAE solution**

Dilute 50× TAE concentrates to a working concentration (1×),
*i.e.*,add 20 mL of the 50× TAE concentrates mix to 980 mL of ddH2O.

(2)
**Preparation of 1% DNA agarose gel**

a)
Weigh 0.6 g agarose powder using a balance.

b)
Pour 60 mL 1× TAE solution using a measuring cylinder.

c)
Add the 1× TAE solution and agarose powder into a conical flask and mix
them well.

d)
Cover the conical flask and heat it in a microwave until all powder is
dissolved completely.

e)
Cool agarose solution to about 60 °C and add YeaRed™ nucleic acid
gel stain to a final concentration of 0.1 μL/mL.

f)
Pour gel solution into gel tray and insert well comb. Wait for 20-30
min until the gel is solidified.

(3)
**Electrophoresis conditions**

a)
Remove the comb and place the solidified agarose gel into the
horizontal electrophoresis Geltank.

b)
Pour the TAE solution into the Geltank, and make sure the solution
covers the surface of the agarose gel.

c)
Prepare DNA samples by mixing the 10× DNA loading buffer with PCR
products at a ratio of 1: 9.

d)
Load 5 μL DNA marker and 3.5 μL PCR products into wells of
solidified agarose gel.

e)
Run gel electrophoresis at 180 V in 1× TAE buffer until sufficient
migration (at about 25-30 min).

f)
Visualize DNA bands under UV light.

2.4
**Fragment extraction**

(1)
Cut the DNA gel:

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

b)
Cut a minimal amount of agarose gel to minimize
contaminants.

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

(2)
DNA gel Extraction:

In this section, we used the DiaSpin DNA Gel Extraction Kit to extract
the DNA fragments, the procedure is as follows
：

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

b)
Transfer the solution into columns, centrifuge the tubes at 8000
*×g* for 1 min, and discard the filtrate in the collection tube.

c)
Add 500 µL Wash Solution centrifuge the tubes at 9000
*×g* for 1 min, and discard the filtrate in the collection tube.

d)
Repeat the wash step.

e)
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.

2.5
**Double enzyme digestion of fragments and
vectors**

The digestive sites of the fragments and vectors are shown in the table
below:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Fragment | | Vector | | Digestive site | |
| F1 | BsjA | V1 | pRSFduet | *Nco*I | *Not*I |
| F2 | DarL-DarE | V2 | pRSFduet | *BamH*I | *Xho*I |
| F3 | DarE | V3 | pETduet | *Nco*I | *Not*I |
| F4 | NisB-NisC | V4 | pETduet | *Nco*I | *Hind*III |

The reaction system and procedure for double enzyme digestion are shown
in the table below:

|  |  |  |
| --- | --- | --- |
| Reagent | Volume (μL) | Procedure |
| 10× rCutsmart buffer | 2 | Incubate at 37 ℃ for 3 h |
| fragments or vectors | 15 |
| Enzyme 1 | 0.5 |
| Enzyme 2 | 0.5 |
| ddH2O | 2 |

2.6
**Agarose gel electrophoresis of digest product of
vectors**

This procedure is described previously.

2.7
**Ligation of recombinant plasmids**

The reaction system and procedure for ligation of vector and fragment are shown in the table below:

|  |  |  |
| --- | --- | --- |
| Reagent | Volume (μL) | Procedure |
| 10× T4 DNA Ligase buffer | 2 | Incubate at 16 ℃ for 16 h |
| vector | 4 |
| fragment | 12 |
| T4 DNA Ligase | 1 |
| ddH2O | 1 |

After the enzyme ligation reaction, we will get the following four
recombinant plasmids:

Plasmid A
：
pRSFduet-DarL-BsjL-His-DarA-BsjA

Plasmid B
：
pRSFduet-DarL-NisL-His-DarA-NisA-DarE

Plasmid C
：
pETduet-DarE-BsjM

Plasmid D
：
pETduet-NisB-NisC-DarE

2.8
**Transformation of recombinant
plasmids**

In this step, we transformed the plasmids A and B into BL21(DE3)
competent cells, respectively. The procedure is as follows:

(1)
Thaw BL21(DE3) competent cells on ice.

(2)
Add 5
μL
 recombinant plasmids to the 50
μL
 BL21(DE3) competent cells and gently mix. Do not
vortex.

(3)
Incubate the mixture of plasmids and competent cells on ice for 30
min.

(4)
Heat shock the cells at 42 °C for 90 sec.

(5)
Immediately return the mixture to the ice for 2 min.

(6)
Add 950
μL
 LB culture medium without antibiotics into the mixture.

(7)
Centrifuge the tubes at 4000 *×g* for 2 min to pellet the cells.

(8)
Remove 600 μL of supernatant and resuspend the cells in the
remaining LB culture medium.

(9)
Spread 150 μL cells onto LB agar plates containing appropriate
antibiotics.

(10)
Incubate the solution at 220 rpm, 37 ℃ for 60 min.

(11)
Culture in a petri dish overnight at 37 ℃.

2.9
**PCR identification of transformants**

(1)
Pick 8 well-isolated colonies from the transformation plate onto a new
LB agar plate, and incubate at 37 ℃ for 6 h.

(2)
Pick the colonies, resuspend them in 20 μL of sterile water, and the resuspension is lysed at 98 °C for 10
min.

(3)
Centrifuge the tubes for 5 min to pellet the cell debris.

(4)
Add 1 μL of the supernatant as a DNA template and followed by PCR
amplification according to the following table
:

|  |  |  |
| --- | --- | --- |
| Reagent | Volume (μL) | Procedure |
| PrimeStar Max Premix (2×) | 10 | Denaturation: 98 ℃ for 30 sec |
| Primer F (10 μM) | 0.5 | Annealing: Tm for 15 sec |
| Primer R (10 μM) | 0.5 | Extension: 72 ℃, 30 sec/kb |
| DNA template | 1 | Back to step 1 (30 times) |
| ddH2O | 8 | Final elongation: 72 ℃ for 2 min |
| Total | 20 | 10 ℃ hold |

(5)
After amplification, analyze 3.5 μL of the PCR reaction by agarose
gel electrophoresis. The procedure is as previously described.

(6)
If the target band appears, it indicates that the plasmid has been
successfully transformed into the BL21(DE3) competent cells.

(7)
Inoculate the positive transformants in an LB culture medium containing
antibiotics at 220 rpm, 37 ℃ overnight.

2.10
**Preparation of Competent Cells**

In this step, we need to prepare competent cells containing A (or B)
plasmid, and then transform the C (or D) plasmid into them. The procedure is as follows:

(1)
Inoculate overnight cultures 1: 100 into 50 mL fresh LB culture medium
in conical flasks. Incubate at 37°C, 220 rpm until OD600 reaches 0.3-0.5.

(2)
Transfer the culture solution into the centrifuge tube and place it on
the ice for 20 min.

(3)
Centrifuge at 5,000 rpm at 4 ℃ for 5 min and discard the supernatant.

(4)
Resuspend the pellet gently in 10 mL of pre-cooling 0.1 M
CaCl2 and ice bath for 30 min. Centrifuge as before and discard the
supernatant.

(5)
Resuspend the pellet gently in 2 mL of pre-cooling 0.1 M
CaCl2 containing 10% glycerol.

(6)
Aliquot competent cells into pre-chilled tubes, 100 μL per
tube.

(7)
Flash freeze in dry ice and transfer to -80°C for long-term
storage.

2.11
**Transformation of another plasmids and PCR
identification**

In this step, we transform the C (or D) plasmid into previously
prepared competent cells containing A (or B) plasmid, respectively. The transformation steps described
previously.

PCR identification of the transformants required the use of two pairs
of primers specifically paired with different plasmids for fragment amplification, and only
transformants with two observed bands contain both plasmids successfully. The PCR reaction system and
procedure are described previously.

3
**Protein expression and purification**

3.1
**Protein expression**

(1)
Inoculate the positive transformants in an LB culture medium containing antibiotics at 220 rpm, 37 ℃ overnight.

(2)
Inoculate overnight cultures 1: 100 into 200 mL fresh LB culture medium containing antibiotics in conical flasks.

(3)
Incubate at 37°C, 220 rpm until OD600 reaches 0.8.

(4)
After the temperature of the LB bacterial medium is reduced to 18 ℃,
add IPTG to a final concentration of 0.8 mM.

(5)
Induction at 18 ℃, 200 rpm for 20 hours.

(6)
Harvest cells by centrifugation at 4,000 rpm for 10 min at 4 °C.
Discard supernatant.

3.2
**Protein purification by affinity chromatography on
Ni-NTA resin**

(1)
Resuspend the pellet in lysis buffer by adding 10 mL of lysis buffer
(containing 8 M urea) to 1 g of wet-weight bacteria.

(2)
Sonication of the bacterial resuspension is performed using φ6
probes at 30% power for 10 min with 3 sec on and 3 sec off.

(3)
Centrifuge at 12,000 rpm at 4 ℃ for 20 min to remove
debris.

(4)
Resuspend 50% His-tag purification resin gently, take 1 mL resin, and
centrifuge at 1,000 rpm, 4 °C for 30 sec to discard the storage solution.

(5)
Equilibrate the resin by adding 1-5 bed volumes of denaturing lysis
buffer and mixing well, centrifuge again, and discard the supernatant.

(6)
Mix the resin with lysate supernatant and incubate for 60 min with
gentle agitation on a side or horizontal shaker at 4
℃
.

(7)
Load the mixture of lysate and resin into an empty chromatography
column.

(8)
Remove the snap-off cap at the bottom of the column, and allow the
liquid to flow out of the column by gravity.

(9)
Wash the resin with 1-2 bed volumes of denaturing lysis buffer
(containing 8 M urea) and allow the liquid to flow out by gravity. Repeat washing 5
times.

(10)
Elute protein 5-10 times with denaturing elution buffer (containing 8 M urea), with one-bed volume of denaturing elution buffer each
time.

(11)
All flow-out components need to be identified by SDS-PAGE
analysis.

3.3
**Preparation of SDS-PAGE gel**

In this section, we used the SDS-PAGE Color Preparation Kit to prepare
the SDS-PAGE gel. Take the example of preparing a piece of gel, and the procedure is as
follows
:

(1)
Assemble the gel casting stand.

(2)
Prepare to resolve gel by adding 2.7 mL of 2× gel solution and 2.7 mL
of 2× resolving gel buffer, and mix evenly.

(3)
Add 55 μL modified polymerization accelerator to the mixture. Mix
by gently swirling to avoid bubbles.

(4)
Pour the resolving gel solution into the casting stand 1.5 cm from the
top. Layer with water to ensure the

 surface is without bubbles.

(5)
Allow to polymerize at 25 °C for 10 min until distinct interface
forms.

(6)
Prepare stacking gel by adding 0.75 mL of 2× stacking gel solution and
0.75 mL of 2× colored stacking buffer, and mix evenly.

(7)
Add 15 μL modified polymerization accelerator. Mix by gently
swirling to avoid bubbles.

(8)
Pour stacking gel on top of resolving gel to the top of glass plates.

(9)
Insert comb slowly to avoid bubbles.

(10)
Allow to polymerize for 10-15 min. Carefully remove comb for the
further SDS-PAGE analysis.

3.4
**SDS-PAGE analysis of protein**

(1)
Install the SDS-PAGE gel into the vertical electrophoresis
holder.

(2)
Prepare protein samples by mixing with 5× SDS sample loading buffer at
a ratio of 1: 4, and boiling at 98
℃
for 5 min.

(3)
Load 6 μL of protein sample per well and 4 μL of protein
ladder in a separate well.

(4)
Assemble the SDS-PAGE apparatus and fill it with 1× Tris-glycine SDS
running Buffer.

(5)
Run at 90 V through stacking gel then increase to 120 V.

(6)
Stop when the dye front reaches the bottom of the gel.

(7)
Carefully remove gel and stain with Coomassie blue R-250 for 60 min
with gentle agitation.

(8)
Destain gel with destain solution (containing 40% ethanol and 10% acetic acid) until protein bands are visible and the
background is clear.

4
**Structure and activity verifications of
proteins**

4.1
**Detecting the structures of purified
protein**

(1)
Filter the collected eluate through a 0.45 μm filter membrane.

(2)
Purify by RP-HPLC using a C4 column, with mobile phase A: water with
0.1% trifluoroacetic acid (TFA) and mobile phase B: 80% acetonitrile with 0.1% TFA.

(3)
Lyophilize the precursor peptides then dissolve them in deionized
water.

(4)
Filter through a 0.22 μm filter membrane.

(5)
Detect the molecular weight of each precursor peptide using
Q-TOF/MS.

(6)
Transfer 30 μL of the peptide solution into a glass vial lined
with a plastic insert, avoiding the formation of bubbles.

(7)
Select the ESI ion source and positive ion mode on the mass
spectrometer.

(8)
Inject a 5 μL sample volume, with mobile phase A: water with 0.1%
TFA and mobile phase B: 80% acetonitrile with 0.1% TFA.

4.2
***In vitro*****cleavage on proteins**

In this step, the purified protein needs to be cleaved by protease
(lysyl endopeptidase) and the released core peptide is the final mature product. The procedure is as
follows
：

(1)
Equilibrate the powder of lysyl endopeptidase in a drying oven for at
least 30 min.

(2)
Dissolve powder of lysyl endopeptidase in a buffer (20 mM Tris-HCl, pH
8.5) to a concentration of 200 AU/mL.

(3)
Add lysyl endopeptidase to the purified protein in a 100 AU: 1 g ratio.

(4)
Incubate at 37 °C for 4 h.

4.3
**Agar diffusion growth inhibition
assay**

(1)
The indicator strain B. subtilis 168 is cultured overnight at 37
°C in a liquid LB culture medium.

(2)
Agar plates are prepared by mixing 20 mL melted agar (ca. 42 °C)
with the cell cultures (1%, v/v) and poured into a sterile petri dish immediately.

(3)
All core peptides are dissolved in sterile ddH2O to a stock concentration of 100 μM.

(4)
10 μL of the stock solutions are separately spotted for the agar
diffusion growth inhibition assay. The sterile ddH2O is used as the negative control.

(5)
Plates are incubated at 37 °C for 12 h after being
spotted.

(6)
The antimicrobial activity is determined by the presence/size of the
growth inhibition zones.

{% endblock %}

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4846/wiki/results.png)

**Results**

**Overview**

In this project, we genetically fused nisin (or bsjA) with
darobactin
via molecular cloning and heterologously expressed the fusion peptides in *E. coli*. Two strategies were also used to modify nisin, bsjA, and
darobactin,
one was the separate expression of the precursor peptide and the post-translational modifying (PTM)
enzyme, and the other was the co-expression of the precursor peptide and the PTM enzyme. After obtaining
the fusion proteins, we performed *in vitro* cleavage assays to obtain the core peptides and verified
their
activity.

Our experimental results are mainly composed of parts, which are
(1)
plasmid construction and transformation, (2) expression and purification of the fusion peptide, and (3)
validation of the structure and activity of the fusion peptide.

**Plasmid construction and
transformation**

For the construction of the following four plasmids, gene
sequences:
*nisB*, *nisC*, *bsjM*, *bsjA1-L*, *darL*, *darA-core*, and *bsjA1-core* were handed over to the company for synthesis and then
cloned
directly into the pUC57 cloning vector. The fragments were ligated to the vector and by enzymatic
ligation, resulting in four recombinant plasmids:

Plasmid A：
pRSFduet-DarL-BsjL-His-DarA-BsjA

Plasmid B：
pRSFduet-DarL-NisL-His-DarA-NisA-DarE

Plasmid C：
pETduet-DarE-BsjM

Plasmid D：pETduet-NisB-NisC

Plasmid A and plasmid C will be transferred into the same strain,
while
plasmid B and plasmid D will be transferred into the same strain

![](https://static.igem.wiki/teams/4846/wiki/results/results1461.png)

Figure 1 T
he
 plasmids constructed in the project

We first extracted four plasmids containing the target genes
synthesized by the company and amplified the target fragments using PCR. As shown in Figure 2, DNA
electrophoresis results proved that we successfully amplified four fragments. After obtaining the target fragments, we processed the target
fragment
and the vector (pRSFduet and pETduet) using double enzyme digestion and formed the recombinant plasmids
by enzymatic ligation.

![](https://static.igem.wiki/teams/4846/wiki/results/results2091.png)

|  |  |  |
| --- | --- | --- |
|  | Amplified fragment | Length  ( bp) |
| F1 | DarL-BsjL-His-DarA-BsjA | 426 |
| F2 | DarL-NisL-His-DarA-NisA-DarE | 1716 |
| F3 | DarE-BsjM | 1304 |
| F4 | NisB-NisC | 3018 |

Figure 2 DNA electrophoresis results of PCR amplified
target
fragments

Subsequently, the plasmids A and B were transferred into the
BL21(DE3)
competent cell, respectively. As shown in Figure 3, the transformants were successfully grown after
overnight culture. The results of colony PCR showed that both transformants amplified the target bands,
indicating that both plasmids A and B were successfully transformed into the BL21(DE3).

![](https://static.igem.wiki/teams/4846/wiki/results/results2528.png)

Figure 3 PCR results of colonies of transformants

On the premise that both A and B plasmids are successfully
transformed,
these two plasmids were transformed into competent cells, and then C or D plasmid was transformed into
the competent cells containing A or B plasmid, respectively. As shown in Figure 4, for the transformants
containing the A and C plasmids, all transformants could amplify two target bands, indicating that the A
and C plasmids were successfully transferred into BL21(DE3). For the transformants containing the B and
D plasmids, transformants 3, 4, 5, 6, and 7 were able to amplify two target bands, indicating that the
strain
was successfully constructed.

![](https://static.igem.wiki/teams/4846/wiki/results/results3211.png)

Figure 4 Colony PCR and sequencing results of transformants
containing
dual plasmids

**3. Expression and purification of
proteins**

We inoculated the positive transformants and when the
OD600 value of the bacterial solution was about 0.8, IPTG was
added to
induce expression. After centrifugation to obtain the bacterium, the bacterium was resuspended with a
buffer containing 8 M urea and sonicated. As shown in Figure 5, we did not observe target bands on
SDS-PAGE, likely due to the small sizes of the final products (His-DarA-BsjA: 13.4 kDa, His-DarA-NisA: 8
kDa). And the nickel column purification was not effective. This may be because we did not elute the
impurities sufficiently, which resulted in the low purities of the final target proteins.

![](https://static.igem.wiki/teams/4846/wiki/results/results3964.png)

Figure 5 SDS-PAGE result of the protein expression and
purification

4.
**Agar diffusion growth inhibition assay**

After obtaining the purified proteins, we performed in vitro cleavage experiments using lysyl endopeptidase to obtain the core peptide for subsequent testing in inhibition experiments.

![](https://static.igem.wiki/teams/4846/wiki/results/results4260.png)

Figure 6. Antibacterial effect of DarA-BsjA fusion peptide antimicrobial peptides on Escherichia coli

![](https://static.igem.wiki/teams/4846/wiki/gd-results-update.png)

Figure 7. Antibacterial effect of two antimicrobial peptides on Bacillus subtilis

We used Gram negative bacteria Escherichia coli and Gram positive bacteria Bacillus subtilis as indicator bacteria to verify the antibacterial effect of antimicrobial peptides. From the Figure 6 and 7, it can be seen that antimicrobial peptides have little inhibitory effect on Escherichia coli, while they have antibacterial effect on Bacillus subtilis, but the antibacterial effect is not very significant. This result is not very ideal, possibly because after expressing the fusion peptide, in vitro cleavage experiments are required, and the cleavage results need to be detected by mass spectrometry. The fusion peptide was not completely cleaved, resulting in weak activity and no antibacterial effect. In the future, we will use mass spectrometry to detect the cutting effect and ensure the formation of fusion peptides for antibacterial purposes. In addition, we will also conduct antibacterial tests on other common Gram negative and Gram positive bacteria in the future.

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