iGEM 2022 — Team shanghai-city

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{% block title %}Notebook{% endblock %}
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Notebook

7/1

Ice breaking for the team
Introduction to the project
Discussion team name and logo design

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-notebook01.png)

7/2

PCR to amplify the target gene
Extract pET28(+) plasmid from Escherichia coli DH5-alpha

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-notebook02.png)

7/3

Liquid LB medium
Gel electrophoresis and gel extraction
Link vector and target gene by Gibson ligase
Vector transport target gene into competent cell

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-notebook03.png)

7/4

Pick bacteria after PCR
Gel electrophoresis test
Extraction of plasmid
Plasmid double digestion
Transport recombinant plasmid into BL21(DE3)
Plate on agar plate

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-notebook04.jpg)

7/5

Pick bacteria and PCR
Gel electrophoresis
Sample sequencing
Cultivate the bacteria
Prepare IPTG for inducing the protein expression
Prepare buffer A and buffer B for protein purification

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-notebook05.jpg)
![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-notebook06.jpg)

7/6

Secondary enlargement of bacteria
IPTG induce protein expression
Prepare the gel for SDS-PAGE

7/7

Protein purification
SDS-PAGE

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-notebook07.jpg)

7/8-7/9

Add different concentrations of protein to 96-well plate
Add bacteria E.coli into different holes
Constant temperature shake cultivate 12 hours
Put into the microplate reader to observe the results

7/10-7/11

The second time sterilization test
Add different concentrations of protein to 96-well plate
Add cultured mixed bacteria from water samples from the aquarium into different holes
Constant temperature shake cultivate 12 hours
Put into the microplate reader to observe the results

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-notebook08.jpg)

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Engineering

Introduction

In the process of fish farming, whether it is large-scale farming for fishery or family farming for ornamental
purposes, maintaining a clean growth environment is of great significance to the growth of fish. In particular, the
lack of a perfect water circulation system for fish farming can easily lead to poor water quality in the fish tank,
which will directly affect the growth of fish.

This project develops a product containing the natural antimicrobial peptide (AMP)
Hydramacin-1, which is discovered while investigating the epithelial defense of the ancient metazoan Hydra. It is
active both against Gram-positive and Gram-negative bacteria including multi-resistant human pathogenic strains. And
it has many advantages such as low side effects and not easy development of drug resistance. Our experimental data
indicated its significant effect on cleaning the water in the fish tank. Therefore, our product will provide a water
quality improvement plan for fish farming.

How we design our plasmid

Because the T7 promoter and T7 RNA polymerase have strong ability in translation and
usually be used as protein expression, we choose pET28a-vector and E.coli BL21(DE3), with T7 promoter and T7 RNA
polymerase respectively, to express our target protein Hydramacin-1. To achieve this, we designed the DNA sequences
of Hydramacin-1 to be inserted into the XbaI and XhoI sites of the pET-28a(+) vector (Figure 1.), transferred the
recombinant plasmid into E.coli BL21(DE3) for protein expression.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-engineering01.png)
Figure 1. The map of recombinant plasmid pET-28a(+)- Hydramacin-1.

How we build our plasmid

To build the plasmid, we let the synthetic company synthesize the DNA
fragment of Hydramacin-1 and integrate it
into the pUC57 vector. Then, we did is go for the PCR to amplify the DNA fragment of Hydramacin-1. At the same time,
we did the plasmid extraction to obtain the plasmid pET-28a(+). The second step was double-enzyme digestion. The
goal of digestion was to get the linearized pET-28a(+) vector and inserted DNA fragments of Hydramacin-1. The third
step was to ligate the inserts and linearized vector and transfer the ligation product into E.coli DH5α competent.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-engineering02.jpg)

Figure 2. The PCR gel electrophoresis picture of Hydramacin-1’s gene fragments.
Line M: DNA marker
Line 1: The gene fragment of Hydramacin-1, 252bp, correct
Line 2: The gene fragment of Sparamosin26-54, 84bp, correct
Line 3: The gene fragment of LL-37, 111bp, correct

We send the constructed recombinant plasmid to a sequencing company for
sequencing. The returned sequencing
comparison results showed that there were no mutations in the ORF region (Figure 3.), and the plasmid pET-28a(+)-
Hydramacin-1 was successfully constructed. And the last step was extracting the recombinant plasmid from E.coli DH5α
and transferring it into E.coli BL21(DE3) competent, so that can be used to express AMP proteins.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-engineering03.png)
Figure 3. The sequencing blast results of the recombinant plasmid pET-28a(+)-Hydramacin-1.

How we test our AMP

a) Protein expression and purification

In order to obtain the AMP protein, expanded the culture pET-28a(+)- Hydramacin-1/BL21(DE3) in the LB medium, and
added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and
culture, we
collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used
nickel column purification to purify the antibacterial peptide protein we wanted. The concentration of Hydramacin-1
protein was measured as 0.212mg/mL. At this point, we got the Hydramacin-1 protein solutions we wanted.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-contribution02.png)
Figure 4. The process of purification of protein by nickel column.

b) Functional test through inhibiting a single species of bacterial growth

To confirm the ability of our purified Hydramacin-1 protein to inhibit bacterial growth, we used E.coli DH5α as
bacteria for bacteriostatic test experiments. The positive control group is composed of 100μl of DH5α along with
100μl of liquid LB broth that contains Kanamycin and the negative control group contains 100μl of DH5α and 100μl of
the protein coded according to the empty pET-28a(+) plasmid.
As shown in the graph, the less diluted the Hydramacin-1 protein is, the more impact it has on bacterial growth.
Just like the experiments we ran on LL-37, Spheniscin-28, Sparamosin26-54, and Fusion. Although the
average
OD600
absorbance for the solution that contains 100μl of bacteria and 100μl of the Hydramacin-1 AMP that has been diluted
625, 125, and 25 times are relatively lower than the OD600 absorbance of the negative control group, the
error bars
of these variations overlap with each other. The overlap in their error bars hints that their OD600
absorbance is
not significantly different from each other, meaning that when diluted 625, 125, and 25 times, the AMP plays an
insignificant role in bacterial inhibition. However, there is a decrease in OD600 absorbance after
applying the AMP
that has been diluted 5 times. The drop in OD600 absorbance is further emphasized when we directly
applied the AMP
that has not been diluted to the DH5α bacteria. With the error bars of both of these variations not overlapping with
those of the negative control group, the data shows the feasibility of using Hydramacin-1 AMP as a way to inhibit
bacterial growth. While there is a drastic decrease in the OD600 absorbance where the Hydramacin–1 AMP
that has not
been diluted was applied, the graph shows that Kanamycin, the antibody that was used in the positive control group,
is still a better bacterial inhibitor. Nevertheless, we can continue to concentrate the peptide solution, making its
concentration higher than 0.212mg/mL. With a higher concentration, the Hydramacin-1 AMP may be a better bacterial
inhibitor compared to Kanamycin or other antibodies.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-engineering05.png)
Figure 5. Test results of protein Hydramacin-1 inhibiting bacterial growth.

c) Functional test through inhibiting real mixed bacterial growth

After confirming the feasibility of using AMP against E. coli DH5α which is cultured in the laboratory, we used the
same experimental set-up to test their effectiveness in inhibiting the growth of real mixed bacteria acquired from
Haichang Ocean Park. While the positive and negative control group are all composed of the same solutions, we
conducted this experiment with 11 different concentrations, each increasing with a ratio of 2. Through doing this,
we hope to find the best concentration at which our antimicrobial peptide works.
Similar to the trends presented in our results section, the less diluted the peptide, the more effective it is. Just
like the OD600 absorbance results when we tested the Hydramacin-1 AMP against DH5α, the OD600
absorbance is only
significantly lower than that of the negative control group when the peptide solution was diluted 8 times or less.
Specifically, we see a drastic decrease in the OD600 absorbance when the peptide was only diluted 4
times. Stopping
at an OD600 absorbance of 0.1296 when the peptide was undiluted at all, the OD600 absorbance
of the undiluted AMP is
slightly higher than that of the positive control group. Although the graph shows that Kanamycin, the antibody used
in the positive control group, was able to inhibit bacterial growth better, the Hydramacin-1 can plausibly be a
better bacterial inhibitor if we concentrate the protein more. With the current concentration being 0.212mg/mL,
increasing it to 0.4mg/mL or 0.5mg/mL may make it a better bacterial growth inhibitor when compared to existing
antibodies. In conclusion, our Hydramacin-1 products still have great potential in inhibiting real mixed bacteria
growth.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-engineering06.png)
Figure 6. Test results of protein Hydramacin-1 inhibiting real mixed bacterial growth.

d) Functional test with more specific concentrations

Based on the test results using the double dilution method to dilute the antimicrobial peptide and test on DH5α and
Oceanarium bacteria, we conducted another round of dilution and testing for more accurate data and effect analysis.
In this test, we uniformly diluted the antimicrobial peptide to micromolar concentrations and constructed at least
nine gradients for the peptide. Similar to the trend of our first experimental results, the lower the dilution of
the antimicrobial peptide, the more effective it was. When the concentration of Hydramacin-1 protein reached 8μM, it
could sterilize and inhibit bacteria to OD600 absorbance value 0.5 and below. When the concentration of
Hydramacin-1
protein reaches 15μM, it can sterilize and inhibit bacteria growth to the OD600 absorbance value of 0.25
or below,
and its antimicrobial effect is even better than that of kanamycin. This confirms our inference above. After the
concentration of Hydramacin-1 protein reaches a certain level (equal to or greater than 15μM), our peptide products
can achieve the effect of sterilization beyond antibiotics. This result is really encouraging.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-engineering07.png)
Figure 7. Test results of protein Hydramacin-1 inhibiting real mixed bacterial growth with more specific
concentrations.

How we learn from our project

We have already collected the figures from our experiments. Hydramacin-1 has the effect of controlling bacterial
proliferation. And we find out that the best dilution of this antimicrobial peptide is equal to or greater than
15μM. From the figure, we can find out that AMP has the perfect effect of antibacterial effect. After using AMP, we
can make the growth of the bacteria decrease by about 90%, so it can control bacterial proliferation efficiently. By
the way, when we compared these data with the positive control group, we surprisingly find that the effect of the
AMP is better than that.

Because of the great effect of the AMP, we believe that if we can use the AMP in the future and promote it in the
market, it will become a great power to fight against bacteria, and reduce the time period for changing the water in
large aquarium fish tanks, thereby achieving the purpose of protecting water resources and contributing to the
protection of the earth's ecological environment.

In the future, when the technology of AMPs becomes better, it may be applied to animal feeding, which can enhance
the ability of animals to resist bacterial infection without causing the production of “super bacteria”, making the
meat ingested by people more rest assured. In the future, AMPs products can even be used in the treatment of human
diseases to treat diseases such as inflammation caused by bacterial infection.

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Experiments

Bacterial culture

Making LB Medium

The growth of bacteria requires carbon(c), nitrogen(n), oxygen(O), hydrogen(H), and we also needed to keep the
osmosis at a balance, thus adding NaCl.

What we needed was 100mL of solid medium and 350mL of liquid medium.
According to the the formula, every 1L of medium requires 10g of tryptone which provides N, 5g of yeast extract
which provides C, 10g of NaCl, while O and H are from water. For the solid medium, a 1% concentration of agarose
powder is needed for solidification.

After adding the ingredients together, except the agarose, we used a
plastic sheet with filter paper in the middle to cover the flask and filter possible impurities during autoclaved
sterilization. We sterilized for about 20 minutes and let the medium cool down. For the solid medium, we added 50µL
of Kana antibiotic to kill the bacteria that didn’t contain the resistance gene. And for the final step, we poured
the melted solid medium into petri dish in the clean bench.

Construction of plasmids

Overview：

1. PCR
2. Plasmid extraction
3. PCR Gel Electrophoresis
4. PCR Gel Purification
5. Gibson Enzyme Connection
6. DNA transformation to plasmid
7. Apply to solid medium

PCR Gel Electrophoresis

The sizes of our DNA fragments are:
Hydramacin-1: 252bp
Sparamosin26-54: 84bp
LL-37: 111bp

Since the DNA contains negative electric charge, we placed the gel with
wells toward the negative charge of the buffer tank, as the other side is positively charge, the DNB fragments will
run toward that side and separate due to different sizes. The larger the size, the slower the DNA moves. Keep in
mind that the TAE buffer needs to fully cover the gel. And also never forget to add marker or you won’t be able to
compare the sample with the right band. Run the electrophoresis at constant 150V for 20 minutes.

Before running the electrophoresis, we added DNA loading buffer to the PCR
product, according to its concentration.

After the electrophoresis, we put the gel under ultra slim LED illuminator
to see the DNA and cut out the gel with DNA fragments for the next step.

THE RESULTS ARE AS FOLLOWS

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments01.jpg)

PCR Gel Purification

We wanted the purified DNA/Gel to insert it into the plasmid---the process
is similar to the plasmid extraction.

1. Add 500µL B2 buffer to the gel
2. Water bath at 50℃for 5-10 minutes to make the gel melt in buffer
3. Add around 140µL isopropanol (for the DNA fragments that are less than 500 bp)
4. Move all the liquid into the absorption column with membrane, centrifuge for 30 seconds, 8000Xg
5. Add 500µL Wash Solution, 9000Xg centrifuge for 30 seconds, repeat this step again
6. Add nothing and centrifuge again for 1 minute, 9000Xg
7. Put the absorption column into a clean 1.5mL centrifuge tube, add 15-40µL Elution Buffer, put in room temperature
for 1 minute and centrifuge for 1 minute to keep the DNA solution.
8. Test the DNA concentration using Nanodrop machine
    a) Hydramacin-1: 48.6ng/µL
    b) Sparamosin26-54: 29.9ng/µL
    c) LL-37: 28.95ng/µL

Lab design:

1. Compare group

○ Positive control --- Kana antibacteria
○ Negative control --- empty pET28a plasmid

2. Experiment group

○ Hydromasin-1
○ Spheniscin-2
○ LL-37
○ Sparamosin 26-54
○ LL-37-spa

Hydromasin-1 was in puc47 plasmid and we needed to do PCR to take it out
and move into pET28a.

We use NcoI and XhoI to cut the pET28a plasmid and connect the DNA fragments in.
We didn’t use T4 connecting enzyme but Gibson since T4 requires restriction sites while Gibson can process
homologous recombination.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments02.png)

Gibson

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments03.png)
The sequences here need to be the same (designed PCR primer)

\*\*\*Put the two strands into Gibson master mix and incubate for 15 minutes to 1 hours at 50℃
![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments04.png)

Gibson Enzyme Connection

Materials: 20µL
1. pET28a plasmid 8µL
2. Target gene
3. Gibson 2µL
4. Buffer (5X) 4µL
5. ddH2O
30fmol of pET28a：60fmol of DNA

Plasmid: 30fmol=0.02\*n(ng)
5.4kb=5400ng
0.02\*5400ng=108ng
108ng | plasmid concentration (ng/µL) --- measured when extracting plasmid = 8µL

Target DNA --- 60fmol=0.04\*n(ng)
According to different bp

Kb=kilobase
Bp=base pair

Steps:

1) Add 0.1µL of DNA
2) According to DNA bp \* 0.04/concentration

After adding all the ingredients together(buffer and enzyme last as they are stored in refrigerator)
PCR 37℃ 30min --- let target DNA connect into the plasmid.

DNA transformation to plasmid

1. Precool 1.5ml tube, pET28a plasmid is really sensitive to temperature, easy to die
2. Add 35µL of pET28a into the tube, and 5µL of plasmid, put the 1.5ml tube on the ice. We stored the leftover
plasmid at 4℃ in case we need them in the future
3. Put the mix in ice for 30 minutes to fully mix them
4. Water bath at 42℃ for exactly 90 seconds, or else the cell will keep expanding and die, to make competent cell
expand, allowing plasmid to enter.
5. Put the tube into ice for 2-3 minutes to shrink the membrane
6. Add 700µL non-kana medium to activate the plasmid, prepare the E.coli for kana selection
7. Put in shaker for 1 hour, 37℃, 220RPM
8. Centrifuge 1000RPM 1 min, precipitate the E.coli, get rid of 650µL of supernatant
9. Resuspend the e-coli and apply to the solid medium

Apply to solid medium

1) Put 50µL of e-coli solution to the solid LB medium petri dish
2) Use apply stick to distribute the solution evenly
3) Raise the e.coli over night in the incubator
4) Get the strain and put into liquid LB with kana
5) Then run colony PCR to see if the DNA is right

Identification of recombinant plasmids

Overview：

1. Take colony and do colony PCR
2. Plasmid Extraction
3. Double Digestion
4. Transforming and applying to the petri dish
5. Run Gel electrophoresis and test sequencing

Take Colony

◇ We added 50µ LB medium with no bacteria and antibiotics into each 1.5mL EP tube
◇ Used tips to take the E.coli colony and put them into EP tubes
◇ Do Colony PCR

Colony PCR

System:

1. We added 5µL ddH2O
2. Added 10µL Primer S. Mixer (which includes dNTP, DNA polymerase, and buffer[contains salt to adjust pH])
3. Added 3µL of DNA template
4. Added 1µL of forward and reverse primer

Condition:

1) 95℃, 5min, pre-degenerate
2) 95℃, 5-10sec, DNA degeneration
3) Mininum Tm value -5℃, 10-15sec, annealing
4) 72℃, time depends on the speed of replication of the enzyme, elongation
5) 72℃, 10min, fully elongate
6) 4℃, infinite time, preserve

DNA Gel Electrophoresis

After doing the Colony PCR, we wanted to check whether the BL21 was
successfully transfected.

Since we used the Gibson enzyme, there are three possibilities for the BL21 on the petri dish to survive (Resistant
gene is in the plasmid):
1. No target DNA in BL21, only the plasmid
2. Two plasmids connected with each other
3. Plasmid with target DNA inside, which is what we wanted.

We check the bands after gel electrophoresis to see whether the survived BL21s (another selection) were the plasmid
we wanted

We picked three colonies that are supposed to have our target DNA H: A, B, and C
We made TAE solution: 1L in total=980mL ddH2O with 50X TAE buffer

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments05.png)

According to the PCR result, A and C had the target DNA we wanted and the according
colony can be put into liquid LB to culture

Plasmid Extraction

We cultured E.coli with pET28a the previous night and used that to do plasmid extraction
The plasmid concentration tested were:

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments06.png)

Double Digestion

Materials:
For every 50µL system:
1) Plasmid: 1.5µg
2) NcoI: 1µL
3) XHoI: 1µL
4) Cut Smart(50X): 5µL (Buffer)
5) ddH2O: Fill to 50µL
37℃ water bath for 2 minutes

Transforming and applying to the petri dish

We transformed the Spheniscin-2, Fusion, and DH5a colonies
30µL BL21, target DNA being 1/10 the amount of BL21

Take colony and do colony PCR

Needed to be done in clean bench

10µL system:
1. 1.5µL ddH2O
2. 5µL Primer S. Mixer
3. 2.5µL template DNA
4. 0.5µL each forward and reverse primer

Gel electrophoresis and test sequencing

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments07.jpg)

We picked the best ones for sequencing, to verify whether the DNA sequence had the
correct DNA

Protein expression

Overview：

1. Small shake, Test OD600, turn to big shake, add IPTG to induce protein expression
2. Protein purification
3. SDS-PAGE Gel Electrophoresis

Small Shake

To amplify the colony by 100 times, and the big shake the next day would amplify it again for 100 times
1. We added 20µL of Kana antibiotics to 20mL of non-antibiotic LB medium
2. Mixed 3.5mL of Kana LB medium (liquid) with 10µL of E.coli solution
3. 220RPM, 37℃ shaker overnight

Petri Dish

We poured the melted solid LB medium with Kana antibiotic into the petri
dish in the
clean bench

Test OD600, turn to big shake, add IPTG to induce protein expression

We turned the overnight small shake to big shake for around 2 hours

Used OD 600 to check whether the big shake is good for IPTG or not,
normally 0.4-0.6 is ideal

Added 350µL IPTG to 350mL of shaked solution with E.coli and LB medium

Mechanism of IPTG:

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments08.jpg)

We needed 20mM of IPTG:
IPTG=238.31g/L=1mol
We needed 10mL of solution thus: 238/31g/L/100=2/3831g --- 1mol
1mol= 50\*20mM
So: 2.3831/50=0.047 IPTG

Protein purification

Make buffer A and B for protein purification

Protein Purification:
Buffer A: Make His tag bind with Ni2
\*Low Imidazole 500mL
1. 20mM Na2HPO4 1.42g
2. 500mM NaCl 14.5g
3. 20mM Imidazole 0.68g
4. Adjust the pH to 7.4 by using HCl
5. Add ddH2O to total volume: 500mL

Sample Calculation:
NaHPO4:1L 142g=1mol
500ml 71g=1mol
20mMol=1/50 mol
Thus, 500mL 71/50g=20mMol

Buffer B: Washes off His tag and Ni2 so we can get the antibacterial peptide
\* High Imidazole 250mL
1. 20mM Na2HPO4 0.71g
2. 500mM NaCl 7.3g
3. 500mM Imidazole 8.5g
4. Adjust the pH to 7.4 by using HCl
5. Add ddH2O to total volume: 0.25L

Preparation:

We used two 50mL centrifuge tubes to centrifuge the bacteria solution after
big shake, repeated the step until all bacteria solution is centrifuges and the E.coli were precipitated to the
bottom. (5000 RPM for 10 minutes each time, tube’s precipitate side face up)

We discarded the supernatant, added 5mL of buffer A to tube 1 and vortexed
them to resuspend the E.coli, poured the solution to the other tube with E.coli, then added another 5mL of buffer A
to tube 1 to fully wash the tube, and added them to the other tube, and resuspended all the E.coli.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments09.png)

We put the tube with buffer A and bacteria in ice and do ultrasonic cell
wall breaking. (The ice is to prevent overheating) 40% power, 25℃ temperature, 30 minutes (3 seconds working and 4
seconds resting as a loop)

\*Extracted 50µL solution after breaking for future electrophoresis.

We centrifuged the tube for 30 minutes, 12000RPM to separate the cell
fragments and solution

Put the tube with supernatant on ice, take 50µL for electrophoresis

Added 100µL of ddH2O to precipitate and vortex to resuspend, take 50µL for
electrophoresis

Protein Purification

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments10.png)

**1. Prepare Ni Column** --- we used 1mL Ni Beads (Buffer A and B added need to be 10
times
of Ni Beads)

Note that never let the solution in the column fully go out or the Ni
Column would go dry and unable to be used again.

Column wash: Open both the plugs to let the 20% ethanol solution to drip,
then add 10mL ddH2O to drip, and 10mL buffer A to prepare for purification to drip, plug in the plugs to prevent dry
out.

2. Protein purification

1. Add all the supernatant we had into the Ni Column and wait for 10
minutes to let protein and the Ni beads bind. Do not let it drip, prepare a new tube to catch the drip later.

2. Take 50µL of binded solution for electrophoresis

3. Let the solution drip, and then add 10mL of buffer A to wash, collect
them in the same tube, and take 50µL of solution from that tube for electrophoresis

4. Plug the bottom plug and add 10mL of buffer B for elution, to elute off
the antibacterialpeptide, wait for 3-5 minutes and let it drip. Take a new 15mL tube for collection.

5. Take 50µL for electrophoresis.

6. Store all the solutions on ice, or in the fridge

Remember to rewash the Ni Column for future usage:
1) 5mL buffer A to drip
2) 10mL ddH2O to drip
3) 10mL 20% ethanol and plug both plugs

SDS-PAGE Gel Electrophoresis

Make 1mm SDS-PAGE Gel for Electrophoresis

This Gel is used for running antibacterial peptide samples to see if the
thing we purified is the right ones or not

We made 15% lower gel since we had 230 base pairs, aka 76.6 amino acids.
Every 20 amino acids have an average of relative molecular weight of 128 (0.128kDa)

Thus, our samples should have about 9.813kDa. And according to the
instruction, a 15% lower gel fits the best

Make lower gel:
1. We added 2.7mL of lower gel solution
2. Added 2.7mL of lower gel buffer
3. Added 60µL of coagulant
4. Added the mix to gel making glass plate and add some ddH2O to make the get flat
5. Wait until the gel solidify, pour out the water

Make upper gel
1) Mixed 0.75mL upper gel solution with 0,75mL of colored upper gel buffer
2) Added 15µL of coagulant
3) Plugged in the well comb
4) Waited until the gel solidify and plug out the comb, used paper towel to wrap around the glass plate and added
ddH2O to keep the gel moistured

We vertically assembled the gel cassette with the chamber
and the buffer tank, added buffer into the chamber to check whether it leaks or not, then added recycle buffer into
the tank to let electricity able to pass through.

For the wells on the gel, we added the samples in the sequence as follows
for all 5 genes and empty pET28a:

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments11.png)

1. Marker
2. Crude extract (Solution after ultrasonic cell wall breaking)
3. Supernatant (Top liquid after centrifuge)
4. Precipitation (Precipitate after centrifuge and re-suspended)
5. Mixture solution (After supernatant combine with nickel column)
6. Washing liquid (After washed by buffer A)
7. Eluent (After washed by buffer B)
Run 60V for 30min and then 120V for 1 hour

For sample 2-6, we mixed 1µL of sample with 7µL buffer B and 2µL 5X loading
buffer since the concentration of the samples were high and needed to be diluted by buffer B.
For sample 7, we simply mixed 8µL of solution with 2µL loading buffer.

Then we put the 2-7 PCR tube into PCR machine, 95℃ for 5 minutes to
denature the protein and able to run the SDS-PAGE Gel Electrophoresis

After the electrophoresis:

1. We poured the running buffer back to the recycle bottle
2. Took out the get, cut off and discarded the top gel with wells, used coomassie brilliant blue stain to cover the
gel
3. Put the gel on the shaker overnight (if time is limited, head to 60℃ and shake for 2 hours) to color the protein
with stain, anything that is not protein would not show color, or at least we cannot see
4. Used detergent to wash off the stain
    a) Detergent: 100µL of methanol, 100µL acetic acid, and 800 µL ddH2O

Bacteriostatic functional test

Serial Dilution of Protein

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-experiments12.png)

Original protein solution
We dilute from original to (time of dilution): 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024
300µL of LB medium in each tube to dilute

Make bacteria solution

We had three bottles of water with bacteria from different fish tanks in
total, so we mixed them together to 2mL and added 18mL of LB solution to dilute them for 10 times

Add sample to 96-well plates

Except for the compare groups, each well we added 100µL of bacteria
solution and 100µL of protein we got

For the four compare groups, the systems are as follows:

· Blank control: 200µL of LB medium
· Environmental Control: 100µL of LB medium and 100µL of bacteria solution
· Negative control: 100µL of LB medium and 100µL of empty pET28a protein
· Positive control: 100µL of bacteria solution and 100µL of 10X kana antibiotic

Shake at 37℃, 220 RPM for 12 hours and then use microplate reader to test
absorbance. The less the number, the less the bacteria growth.

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Results

1. Construction of the antimicrobial peptide expression plasmids

We design five plasmids: The DNA sequences of the four antimicrobial peptides Hydramacin-1, Spheniscin-2, LL-37, and
Sparamosin26-54 were inserted into the XbaI and XhoI sites of the pET-28a(+) vector, respectively. The fifth one was
a fusion antimicrobial peptide that connects LL-37 and Sparamosin26-54 through a linker, which was also inserted
into the pET-28a(+) vector for protein expression, as shown in Figure1.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results01.png)
Figure 1. Antimicrobial peptide expression plasmids in this project

In order to build our plasmids, we let the synthetic company synthesize
three target
gene fragments, Hydramacin-1 integrate it into the pUC57 vector, Spheniscin-2,
LL-37-Linker-Sparamosin26-54,
integrate them into the pET-28a(+) vector respectively. The certificate of synthesize analysis are as follows.

Certificate of Analysis of Hydramacin-1-pUC57

Certificate of Analysis of Spheniscin-2- pET-28a(+)

Certificate of Analysis of LL-37-Linker-Sparamosin26-54-
pET-28a(+)

Then, we amplify Hydramacin-1, LL-37, and Sparamosin26-54 by PCR (Figure2.),
double-enzyme digestion, and ligase to pET28a(+) carrier, respectively, to obtain the other three plasmids
Hydramacin-1- pET28a(+), LL-37- pET28a(+), and Sparamosin26-54- pET28a(+).

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results02.png)

Figure 2. Gel electrophoresis results of target gene fragments.
M: DNA Marker.
L: The gene fragment of LL-37, 111bp, correct
S: The gene fragment of Sparamosin26-54, 84bp, correct
H: The gene fragment of Hydramacin-1, 252bp, correct

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing
comparison results showed that there were no mutations in the ORF region (Figure3-7.), and the plasmid was
successfully constructed. So far, we have successfully obtained five recombinant plasmids, which were respectively
on the pET28a(+) vector, which can be used to express antimicrobial peptide proteins.

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results03.png)
Figure 3. The sequencing blast results of the recombinant plasmid Hydramacin-1-pET28a(+).

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results04.png)
Figure 4. The sequencing blast results of the recombinant plasmid Spheniscin-2-pET28a(+).

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results05.png)
Figure 5. The sequencing blast results of the recombinant plasmid LL-37-pET28a(+).

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results06.png)
Figure 6. The sequencing blast results of the recombinant plasmid Sparamosin26-54-pET28a(+).

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results07.png)
Figure 7. The sequencing blast results of the recombinant plasmid Fusion-pET28a(+).

2. Protein expression and purification

In order to obtain the five antimicrobial peptide proteins, we transferred the recombinant plasmids into E.coli
BL21(DE3), expanded the culture in the LB medium, and added IPTG to induce protein expression when the
OD600 reached
0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release
the intracellular proteins. Next, we used nickel column purification to purify the antibacterial peptide protein we
wanted. The concentration of each protein was measured as:
0.212mg/L Hydramacin-1, 0.208mg/mL Sparamosin26-54, 0.42mg/mL LL-37, 0.74mg/mL Spheniscin-2, and
0.431mg/mL Fusion.
At this point, we got the five antimicrobial peptide protein solutions we wanted.

3. Antibacterial ability test

Overview

To confirm the ability of the our purified antimicrobial peptide to inhibit bacterial growth, we used E.coli
DH5-alpha as bacteria, and antibiotics as a positive control for bacteriostatic test experiments.
To better show the relationship between the concentration of antimicrobial peptides and the inhibition of bacterial
growth, we added 100 μL of DH5α and 100 μL of different concentrations of the antimicrobial peptide to each of the
five test tubes. Our five test tubes were filled with the antimicrobial peptide stock solution and diluted 1, 5, 25,
125, and 625 times solution, and repeated three times for each concentration to form the average data graph with
error bars.
The results showed that the five antimicrobial peptide proteins had significant antibacterial effects at double
dilution concentrations. A single antimicrobial peptide protein can be seen to inhibit the growth of about 60% of
bacteria in the range of 5 to 25 times dilution. The antibacterial effect of the Fusion antimicrobial peptide is the
best, with about 60% when diluted 625 times. It shows that our antimicrobial peptide products do have a significant
antibacterial function, and the antibacterial function of the fusion antimicrobial peptide is the best. A detailed
analysis of the antibacterial effect of each antimicrobial peptide is given below.

a) Functional test of Hydramacin-1

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results08.png)
 Figure 8. Test results of protein Hydramacin-1 inhibiting bacterial growth.

We confirmed the ability to use the Hydramacin-1 antimicrobial peptide to inhibit bacterial growth by first testing
it against DH5α. As shown in the graph, the less diluted the antimicrobial peptide is, the more impact it has on
bacterial growth. Just like the experiments we ran on Fusion, LL-37, Spheniscin-28, and Sparamosin-26, the positive
control group is composed of 100μl of DH5α along with 100μl of liquid LB broth that contains Kanamycin and the
negative control group contains 100μl of DH5α and 100μl of the peptide coded according to the empty pET-28a(+)
plasmid. Although the average OD600 absorbance for the solution that contains 100μl of bacteria and 100μl
of the
Hydramacin-1 antimicrobial peptide that has been diluted 625, 125, and 25 times are relatively lower than the
OD600
absorbance of the negative control group, the error bars of these variations overlap with each other. The overlap in
their error bars hints that their OD600 absorbance is not significantly different from each other,
meaning that when
diluted 625, 125, and 25 times, the antimicrobial peptide plays an insignificant role in bacterial inhibition.
However, there is a decrease in OD600 absorbance after applying the antimicrobial peptide that has been
diluted 5
times. The drop in OD600 absorbance is further emphasized when we directly applied the antimicrobial
peptide that
has not been diluted to the DH5α bacteria. With the error bars of both of these variations not overlapping with
those of the negative control group, the data shows the feasibility of using Hydramacin-1 antimicrobial peptide as a
way to inhibit bacterial growth. While there is a drastic decrease in the OD600 absorbance where the
Hydramacin–1
antimicrobial peptide that has not been diluted was applied, the graph shows that Kanamycin, the antibody that was
used in the positive control group, is still a better bacterial inhibitor. Nevertheless, we can continue to
concentrate the peptide solution, making its concentration higher than 0.212mg/ml. With a higher concentration, the
Hydramacin-1 antimicrobial peptide may be a better bacterial inhibitor compared to Kanamycin or other antibodies.

b) Functional test of Spheniscin-2

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results09.png)
 Figure 9. Test results of protein Spheniscin-2 inhibiting bacterial growth.

The graph above indicates that the less diluted the protein Spheniscin-2 solution is, the more bactericidal it is.
Compared to other bars in the chart, the one with 1:1 dilution has the most significant effect on sterilization,
which can eliminate almost 77.79% of bacteria. However, the histograms of Spheniscin-2 antimicrobial peptides
diluted by 625, 125, and 25-fold LB medium were virtually identical to the graph of the negative control group,
indicating that they had a little sterilizing effect. And the antimicrobial peptide diluted by five times had some
antibacterial ability, but it was not as significant as the sterilization effect of Spheniscin-2 antimicrobial
peptide stock solution.

c) Functional test of LL-37

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results10.png)
 Figure 10. Test results of protein LL-37 inhibiting bacterial growth.

We also used the same operation to dilute an antimicrobial peptide called LL37. In this histogram, the heights of
antimicrobial peptides that have been diluted 625x and 125x with LB medium are almost the same as that of the
negative control group, indicating that the sterilization ability of these two samples is practically absent. In
contrast, the sample diluted 25 times LB had a significant sterilizing effect but was not as good as the one with
five times. The best sterilization effect was the LL37 stock solution, which could sterilize up to 75.74%.

d) Functional test of Sparamosin 26-54

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results11.png)
 Figure 11. Test results of protein Sparamosin 26-54 inhibiting bacterial growth.

We performed dilution of the antimicrobial peptide Sparamosin26-54 using the same procedure and operation. By
comparing the height of the histograms and the margin of error, we learned that the antimicrobial peptide
sterilization after 625-fold LB solution dilution was not competent because its height was higher than the image
height of the negative control. The samples diluted by 125x and 5x were similar in sterilization ability, but the
25x was a bit worse than both. Among all models, the Sparamosin26-54 stock solution had the best antimicrobial
effect, which could reach 76.20%.

e) Functional test of Fusion

![](https://static.igem.wiki/teams/4248/wiki/t-shanghai-city-results12.png)
 Figure 12. Test results of protein Fusion inhibiting bacterial growth.

As the graph shows, the less the Fusion antimicrobial peptide was diluted, the stronger its ability to inhibit
bacterial growth. However, even considering the error bars, the 625 times dilution had a better ability to inhibit
bacterial growth than the 125-fold dilution. Therefore, we compared the experimental results of the Fusion
antimicrobial peptides with the crude extracts in the preliminary experiment and found that these antimicrobial
peptides have a slightly better ability to inhibit bacterial growth at lower concentrations than at concentrations
around the median. The OD600 absorbance was not significantly different from that of the negative control
group when
it was located at the median concentration, i.e., 125-fold and 25-fold diluted solutions, which shows that the
antimicrobial peptides did not work efficiently at this concentration. However, with the decrease in dilution and
the increase of antimicrobial peptide concentration, the OD600 absorbance decreased significantly and
reached an
average of 0.1602 at double dilution. Although this value is still somewhat high compared to the positive control
with kanamycin, it can be inferred from the above table that at higher concentrations of the antimicrobial peptide,
Fusion antimicrobial peptide is likely to replace kanamycin as a more effective bacterial inhibitor.

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