iGEM 2023 — Team dku

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RNA Optimization

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Phage Platform

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Protocols

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AMP Optimization

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{% block title %}Engineering Success{% endblock %}
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# **Cycle 1**

![](https://static.igem.wiki/teams/4587/wiki/cycle-all.png)

**Cycle 1**

# Cycle 1

**Cycle1: LdtR Expression Strain Construction**

In order to research LdtR and the functions of our genetic
circuit, E. coli instead of L. crescens was used as the model
organism because of its short reproduction time, convenient
transformation, and known expression system. Therefore, in the
first engineering cycle, we attempted to introduce the
transcriptional factor of interest, LdtR, into E. coli BL21
(DE3)
to construct the Lcr-mimicing strain for further research.

![](https://static.igem.wiki/teams/4587/wiki/cycle-design.png)

**Cycle 1**

## Design

To yield a stable and massive expression, E. coli strain BL21
(DE3) was selected as the chassis organism, while vector
pRSFDuet-1 was selected as the expression vector, the T7
promoter of which is expected to be activated vigorously by T7
RNApol of the strain. Retrieved from open Lcr genome dataset,
LdtR gene sequence was codon-optimized for success expression in
E. coli. Also, HA tag was fused to its N-terminal for easier
detection.

![](https://static.igem.wiki/teams/4587/wiki/cycle-build.png)

**Cycle 1**

## Build

The optimized LdtR gene was inserted between NcoI and BamHI
sites of pRSFDuet-1 vector to become pRSF-LdtR, which was
confirmed by DNA sequencing data. Kanamycin selection
yielded colonies with success transformation of pRSF-LdtR.

![](https://static.igem.wiki/teams/4587/wiki/cycle-test.png)

**Cycle 1**

## Test

In order to investigate whether LdtR introduction can stably
produce wanted LdtR protein, western blot assay was employed to
detect the LdtR-HA fusion protein with the target of HA tag.
After induction at 25°C with IPTG, strong signal was detected
with HA-tag primary antibodies.

![](https://static.igem.wiki/teams/4587/wiki/cycle-learn.png)

**Cycle 1**

## Learn

The constant expression in transformed strains indicated
constitutive, stable, and massive expression of LdtR in our
strain BL21-LdtR, which can be used for further studies.

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# **Cycle 2**

![](https://static.igem.wiki/teams/4587/wiki/cycle-all.png)

**Cycle 2**

# Cycle 2

**Cycle2: Promoter Sensor Construction**

We attempted to obtain an accurate and efficient element that
specifically react to LdtR so that the engineered phage only
release its effects in CLas bacteria. Previous studies showed
that promoters of LdtP and ZnuA2 have regulatory roles under
LdtR. Therefore, we obtained the two promoters as sensor
candidates. After analysis of published whole transcriptome
sequencing dataset, we identified a gene of unknown functions,
the ID of which was B488\_05770, that was down-regulated
vigorously when LdtR was inhibited. This suggests that the
promoter of B488\_05770 might be exclusively activated by LdtR.
Therefore, P\_B488\_05770 was also selected as the sensor
candidate of the overall genetic circuit.

The genome of CLasMV1 is a double stranded circular DNA
containing 8 genes. Despite functions of most genes are unknown,
the gene 1 was identified to code for its capsid protein, which
is essential for phage assembly, recognition, and invasion. Due
to the limit of whole phage genome materials, we attempted to
selected the capsid protein gene as a represent and cloned it
into a phage-derived plasmid pBluescript II to mimic the
engineered phage DNA. By placing our genetic circuit under the
control of candidate sensors, we could optimize the promoter
choice for an accurate and effective sensor.

![](https://static.igem.wiki/teams/4587/wiki/cycle-design.png)

**Cycle 2**

## Design

After obtained from open datasets, the CLasMV1 capsid protein
sequence was codon-optimized for success expression in E. coli.
Capsid protein gene was fused with FLAG tag for easier
detection, which was placed after the candidate promoters
(P\_LdtP, P\_ZnuA2, P\_B488\_05770). The whole cassette was placed
into vector p\_Bluescript II KS(+) to yield a engineered phage
model, which would be further transformed into BL21-LdtR for
detection.

![](https://static.igem.wiki/teams/4587/wiki/cycle-build.png)

**Cycle 2**

## Build

The sensor-capsid protein expression cassettes were cloned
between PciI and HindIII sites of pBluescript II KS(+) vector to
become p\_BA, pLP, and p\_ZA, which were confirmed by DNA
sequencing data. Ampicillin and kanamycin double
selection yielded colonies with success transformation of the
phagemids into BL21-LdtR.

![](https://static.igem.wiki/teams/4587/wiki/cycle-test.png)

**Cycle 2**

## Test

The sensor-capsid protein expression cassettes were cloned
between PciI and HindIII sites of pBluescript II KS(+) vector to
become p\_BA, pLP, and p\_ZA, which were confirmed by DNA
sequencing data. Ampicillin and kanamycin double
selection yielded colonies with success transformation of the
phagemids into BL21-LdtR.

![](https://static.igem.wiki/teams/4587/wiki/cycle-learn.png)

**Cycle 2**

## Learn

The sensor-capsid protein expression cassettes were cloned
between PciI and HindIII sites of pBluescript II KS(+) vector to
become p\_BA, pLP, and p\_ZA, which were confirmed by DNA
sequencing data. Ampicillin and kanamycin double
selection yielded colonies with success transformation of the
phagemids into BL21-LdtR.

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# **Cycle 3**

![](https://static.igem.wiki/teams/4587/wiki/cycle-all.png)

**Cycle 3**

# Cycle 3

**Cycle3: Signal Amplifier Construction**

After picking the potential sensors for LdtR recognition of
CLas, we attempted to amplify the signal for more efficient
production of downstream genes. RinA was identified as an strong
activator of its target promoter and has been applied in genetic
engineering as an amplifier in previous works, which was
selected as the potential amplifier for construction of the
sensor-amplifier-downstream gene expression cassette.

![](https://static.igem.wiki/teams/4587/wiki/cycle-design.png)

**Cycle 3**

## Design

Based on the phagemid in the previous cycle, we cloned the RinA
gene under the control of selected sensors (P\_LdtP,
P\_B488\_05770). To quantify the amplification efficiency, we
placed AcGFP as reporter after the target promoter region of
RinA.

![](https://static.igem.wiki/teams/4587/wiki/cycle-build.png)

**Cycle 3**

## Build

ThThe sensor-amplifier-GFP expression cassettes were cloned
between PciI and BamHI sites of pBluescript II KS(+) vector to
become pBAG and pPAG, which were confirmed by DNA sequencing
data. Expression cassette of sensor-GFP was also
constructed for control. Ampicillin and kanamycin double
selection yielded colonies with success transformation of the
phagemids into BL21-LdtR.

![](https://static.igem.wiki/teams/4587/wiki/cycle-test.png)

**Cycle 3**

## Test

In order to quantify the amplification efficiency, we measured
fluorescence intensity for GFP expression index with or without
amplifier. After 24 hours induction, the expression under the
sensor P\_B488\_05770 was dramatically elevated by RinA amplifier,
with an almost 100-fold increase in fluorescence intensity.
However, only a little enhancement was observed by RinA
amplification system under the sensor P\_LdtP.

![](https://static.igem.wiki/teams/4587/wiki/cycle-learn.png)

**Cycle 3**

## Learn

In order to quantify the amplification efficiency, we measured
fluorescence intensity for GFP expression index with or without
amplifier. After 24 hours induction, the expression under the
sensor P\_B488\_05770 was dramatically elevated by RinA amplifier,
with an almost 100-fold increase in fluorescence intensity.
However, only a little enhancement was observed by RinA
amplification system under the sensor P\_LdtP.

Previous

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# **Cycle 4**

![](https://static.igem.wiki/teams/4587/wiki/cycle-all.png)

**Cycle 4**

# Cycle 4

**Cycle4: MaSAMP Expression Strain Construction**

Research conducted by Huang et. al in 2021 examined the
effectiveness of MaSAMP on different types of bacteria, showing
the most toxicity on α-proteobacteria, especially L. crescens
that they used as model organism in place of Candidatus
Liberibacter asiaticus (CLas). E. coli, which is
γ-proteobacteria, were tested to show less sensitivity to MaSAMP
. Therefore, we decided to use E. coli BL21 (DE3) to express
MaSAMP.

![](https://static.igem.wiki/teams/4587/wiki/cycle-design.png)

**Cycle 4**

## Design

Research conducted by Huang et. al in 2021 examined the
effectiveness of MaSAMP on different types of bacteria, showing
the most toxicity on α-proteobacteria, especially L. crescens
that they used as model organism in place of Candidatus
Liberibacter asiaticus (CLas). E. coli, which is
γ-proteobacteria, were tested to show less sensitivity to MaSAMP
. Therefore, we decided to use E. coli BL21 (DE3) to express
MaSAMP.

![](https://static.igem.wiki/teams/4587/wiki/cycle-build.png)

**Cycle 4**

## Build

We constructed our recombinant plasmid by inserting our
designed sequence into pET-30a (+) at the Ndel & Xhol
restriction enzymes site. It was then transformed into E. coli
BL21 (DE3), amplified and induced by IPTG for massive
expression.

![](https://static.igem.wiki/teams/4587/wiki/cycle-test.png)

**Cycle 4**

## Test

After collecting our constructed strain, we both observed it
directly under fluorescent microscope and did western blot after
ultrasonication to confirm the expression our fusion protein.

![](https://static.igem.wiki/teams/4587/wiki/cycle-learn.png)

**Cycle 4**

## Learn

MaSAMP along with fusion protein can be expressed in E. coli
BL21 (DE3) in a high level. The SUMO protein was confirmed to be
an effective tag to hide MaSAMP toxicity.

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# **Cycle 5**

![](https://static.igem.wiki/teams/4587/wiki/cycle-all.png)

**Cycle 5**

# Cycle 5

**Cycle5: Adjust 6xHis-tag position in recombinant plasmid for
better MaSAMP biosynthesis**

Adjust 6xHis-tag position in recombinant plasmid for better
MaSAMP biosynthesis
6xHis-tag is an important element often added at the N- or
C-terminus of the protein for the affinity purification for
recombinant protein. In engineering cycle 1, which we used the
recombinant plasmid of AMP group, we added the 6xHis-tag at the
C-terminus of our fusion protein, right after MaSAMP. However,
this 6xHis-tag could not be removed from MaSAMP after SUMO
protease cleavage, and it might have the potential to influence
the sterilizing effect of MaSAMP. Therefore, designed a new
plasmid (named FAMP) by adjusting the 6xHis-tag position. We
would then compare the purification process of AMP and FAMP
group and also the sterilizing effect of two peptides (with and
without 6xHis-tag) released.

![](https://static.igem.wiki/teams/4587/wiki/cycle-design.png)

**Cycle 5**

## Design

6xHis-tag was moved from the C-terminus of MaSAMP to the
position between GFP and SUMO tag since it would be removed
along with SUMO tag after SUMO protease cleavage.

![](https://static.igem.wiki/teams/4587/wiki/cycle-build.png)

**Cycle 5**

## Build

The modified sequence based on the design was inserted into
pET-30a (+) at the Ndel & Xhol restriction enzymes site, and
then transformed into E. coli BL21 (DE3), amplified and induced
by IPTG for massive expression.

![](https://static.igem.wiki/teams/4587/wiki/cycle-test.png)

**Cycle 5**

## Test

After conducting fusion protein extraction and purification, we
did SUMO protease cleavage to release our MaSAMP in both groups.
However, the concentration of fusion protein is very low in the
supernatant of lysate after centrifuging, making the SUMO
protease cleavage hard.

![](https://static.igem.wiki/teams/4587/wiki/cycle-learn.png)

**Cycle 5**

## Learn

After conducting fusion protein extraction and purification, we
did SUMO protease cleavage to release our MaSAMP in both groups.
However, the concentration of fusion protein is very low in the
supernatant of lysate after centrifuging, making the SUMO
protease cleavage hard.

Previous

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# **Cycle 6**

![](https://static.igem.wiki/teams/4587/wiki/cycle-all.png)

**Cycle 6**

# Cycle 6

**Cycle6: Improve MaSAMP for better sterilizing effect**

The net positive charge and the amphiphilic (hydrophilic and
hydrophobic) α-helical are two typical characteristics of
antimicrobial peptides (AMP). This is a general conclusion
of AMP, but the net positive charge does not fit our AMP. In
addition, AMP normally has only one amphiphilic (hydrophilic and
hydrophobic) α-helical. The common method of increasing
sterilizing effect of AMP is to use lysine to substitute every
amino acid. Considering our MaSAMP consists of 67 amino
acids including two alpha helixes, it may not be suitable for us
to screen every mutant. By referring to the improvement of
stapled amino acids, we found the tool for calculating
lyticity and the conclusion that substituting amino acids with
lysine at the hydrophilic face will increase the sterilizing
effect. We came up with the idea of developing a tool that can
help us automatically improve amino acid sequence and
calculating biophysical parameters, such as total
hydrophobicity, net charge and 3D hydrophobic moment vector.

![](https://static.igem.wiki/teams/4587/wiki/cycle-design.png)

**Cycle 6**

## Design

The principle of improving is to substitute amino acids in the
range at the hydrophilic face with lysine. To avoid excessive
calculation, we set the substituting range from the 5 amino
acids before the alpha helix to the 5 amino acids after the
alpha helix. Biophysical parameters, such as total
hydrophobicity, net charge and 3D hydrophobic moment vector, are
calculated for reference.

![](https://static.igem.wiki/teams/4587/wiki/cycle-build.png)

**cycle 6**

## Build

Briefly, we used alphafold 2 to predict the protein structure,
saving the pdb file and output the position of alpha helix. For
the downstream, we used python packages or designed tools to
calculate biophysical parameters. Connections between each part
were made.

![](https://static.igem.wiki/teams/4587/wiki/cycle-test.png)

**Cycle 6**

## Test

6 mutants of the main function part of MaSAMP were chemically
synthesized to test the potentially increased effect. We will
use fluorescent staining to visualize the results.

![](https://static.igem.wiki/teams/4587/wiki/cycle-learn.png)

**Cycle 6**

## Learn

The calculated biophysical parameters can only be used for
reference. To fully prove the accuracy of our one-step assembled
toolbox, we will further use it to create a mutant library to
adapt the principle of our improvement and then narrow the
substituting range.

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# **Cycle 7**

![](https://static.igem.wiki/teams/4587/wiki/cycle-all.png)

**Cycle 7**

# Cycle 7

**Cycle7: Primer construction for gene amplification from D.
citri DNA sample**

To obtain gene silencing materials, it is necessary to clone
native genes from D. citri, which requires construction of
corresponding primers. In this cycle, the common genome of D.
citri in NCBI was used as reference.

![](https://static.igem.wiki/teams/4587/wiki/cycle-design.png)

**Cycle 7**

## Design

To design the primers, we searched for our target gene
sequences (DCR, EFE4, CP450) in the National Center for
Biotechnology Information (NCBI) system. We designed the primers
based on the mRNA sequence of the genes without the restriction
enzyme sites to confirm the amplification of the gene. Then, we
designed the primers with restriction enzyme sites to amplify
both the antisense and sense direction of target genes.

![](https://static.igem.wiki/teams/4587/wiki/cycle-build.png)

**Cycle 7**

## Build

For the sense fragment, we utilized two different restriction
enzyme sites for each of the forward and reverse primers. The
same strategy is utilized for building the antisense fragment.
As a whole in the L4440 vector system, there will be a linker in
between the sense and antisense fragments, so that when it is
transcribed, it will form a hairpin RNA.

![](https://static.igem.wiki/teams/4587/wiki/cycle-test.png)

**Cycle 7**

## Test

We tested the primers by using polymerase chain reaction (PCR)
using the extracted D. citri DNA as the DNA template. Then, we
ran gel electrophoresis to confirm the amplification. The gel
result shows that the three target genes were successfully
amplified. We then ran the PCR using the primers with the
restriction enzyme sites. The result showed successful
amplification of the CP450 gene with restriction enzyme sites
for sense and antisense directions. However, the EFE4 gene was
not amplified. We moved on to cloning with the CP450 gene.

![](https://static.igem.wiki/teams/4587/wiki/cycle-learn.png)

**Cycle 7**

## Learn

From the gel result, we learned that the genes matched the
expected fragment length (405 bp for EFE4 and 412 bp for CP450).
DCR gene had introns between the forward and reverse primers,
resulting in a longer fragment (800 bp) than expected (275 bp).
Therefore, we extracted RNA from D. citri, converted it into a
cDNA, and ran the PCR with the cDNA template, which identified
the correct amplification of the DCR gene in both sense and
antisense directions. We tried to troubleshoot for amplification
of EFE4 with primers with restriction enzyme sites by trying
different annealing temperatures and using, but it was not
successfully amplified, which may be due to the long length of
the primer.

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# **Cycle 8**

![](https://static.igem.wiki/teams/4587/wiki/cycle-all.png)

**Cycle 8**

# Cycle 8

**Cycle8: Cloning of CP450 gene onto the L4440 plasmid**

In our next cycle, we tried to clone one of our target genes
onto the L4440 plasmid, which was confirmed of a successful
amplification by engineering cycle 1

![](https://static.igem.wiki/teams/4587/wiki/cycle-design.png)

**Cycle 8**

## Design

To utilize the HT115-L4440 RNAi production method, we tried to
clone the amplified fragment of sense and antisense strand CP450
onto the L4440 plasmid vector.

![](https://static.igem.wiki/teams/4587/wiki/cycle-build.png)

**Cycle 8**

## Build

We first tried to digest the CP450 antisense PCR fragment and
the L4440 plasmid with antisense restriction enzymes, XbaI and
SacI. To confirm successful digestion, we planned to confirm
with a gel by running the digested PCR product and plasmid. Then
we tried to ligate the digested PCR product and plasmid
together. We transformed the ligated product into the E.coli
DH5a strain to screen for the vector with successful ligation
and an empty vector. After miniprep, we confirmed successfully
ligated vector with PCR using the ligated plasmid vector as the
template and using CP450 gene primers.

![](https://static.igem.wiki/teams/4587/wiki/cycle-test.png)

**Cycle 8**

## Test

According to the gel result, it showed that the digested vector
bands had a larger band size than the undigested vector. For the
PCR digestion, we did not see a clear band on the gel. After
ligation, digestion, miniprep, and PCR, the gel result showed an
amplification of the CP450 gene for one of the plasmids we
tested, indicating successful ligation of the CP450 antisense
gene onto the L4440 vector.

![](https://static.igem.wiki/teams/4587/wiki/cycle-learn.png)

**Cycle 8**

## Learn

The first gel result shows that both restriction enzymes work
well on our vector, since we can see that the band of the uncut
vector shows a smaller band size than the digested vector. This
indicates that the digested vector is a linear form of the
plasmid rather than a circular, undigested one. The vector
cloned with CP450 antisense could be used for further cloning of
CP450 sense fragment to make a complete hpRNA.

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# **Cycle 9**

![](https://static.igem.wiki/teams/4587/wiki/cycle-all.png)

**Cycle 9**

# Cycle 9

**Cycle9: Increasing the production of hpRNA using additional T7
promoters**

In this engineering cycle, we tried to test if having an
additional T7 promoter in our vector L4440 would increase the
hpRNA production, using three different target genes of D. citri
(MP20, CP64, and DM2).

![](https://static.igem.wiki/teams/4587/wiki/cycle-design.png)

**Cycle 9**

## Design

In the vector L4440 which already has a T7 promoter, we added
an additional T7 promoter sequence to the target gene sequence.
The T7 and three target gene sequences were successfully cloned
into the plasmid via a commercial biotech company. We then
planned to test the efficiency of the three genes without
additional T7 promoters and those with additional T7 promoters
utilizing the HT115(DE3) E. coli bacterial strain and IPTG
induction.

![](https://static.igem.wiki/teams/4587/wiki/cycle-build.png)

**Cycle 9**

## Build

We transformed the cloned plasmids into HT115(DE3) E. coli
bacteria, and successful transformation was screened with
tetracycline and ampicillin antibiotics. Then we cultured the
colonies and added IPTG to induce hpRNA transcription. We
utilized CTAB miniprep to extract hpRNA and ran the final
product on the gel with the treatment with RNase A to leave only
the hpRNA produced.

![](https://static.igem.wiki/teams/4587/wiki/cycle-test.png)

**Cycle 9**

## Test

We tested the final product of RNAi production on the gel. The
gel result shows successful induction of hpRNA for samples that
were treated with IPTG and RNase A

![](https://static.igem.wiki/teams/4587/wiki/cycle-learn.png)

**Cycle 9**

## Learn

We learned from the gel that it is difficult to observe if the
system with additional T7 promoters produced more hpRNA.
Therefore, to meet our ultimate goal of testing the efficiency
of hpRNA production under an additional T7 promoter system, we
need to measure the RNA quantity using the Nanodrop method.

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{% endblock %}

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

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM
project. These should be detailed enough for another team to repeat your
experiments.

If you made Parts this year, please remember to put all information,
characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU
  Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020
  Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020
  NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

#### Results

##### Promoter of B488\_05770 was deduced to possess specificity to LdtR

LdtR is the only MarR family member transcriptional factor in *Liberibacter asiaticus*[1], the panthogen that caused citrus greening. LdtR is specific to CLas and its close family members. In an attempt to obtain a biosensor that specifically and efficiently respond to LdtR, we proposed to find promoters specifically activated by LdtR. Previous research have revealed regulatory relationships between LdtP promoter, ZnuA2 promoter and LdtR [1,2,3]. P\_LdtP (B488\_10900) and P\_ZnuA2 (B488\_07960) were subsequently selected as candidates for the sensor. Previous research sequenced whole transcriptome of *Liberibectar crescens* (Lcr) with functional LdtR or inhibited LdtR [1]. By analyzing the published dataset and comparing differentially expressed genes (DEGs) with the threashold of foldchange < 0.9 or > 1.1, adjust p value < 0.05, we identified 121 down-regulated genes and 131 up-regulated genes (Figure 1). Under the condition of LdtR inhibition, down-regulation of expression might indicate potential positive regulatory roles of LdtR on the gene's promoter. Interestingly, the gene with unknown function B488\_05770 was dramatically down regulated with a fold change of 0.123 when LdtR was inhibited (Figure 1). This suggested strong posibility that P\_B488\_05770 might positively and specifically respond to LdtR. Therefore, B\_488\_05770 promoter was selected as one of the sensor candidates for further investigation.

![alt Figure 1](https://static.igem.wiki/teams/4587/wiki/wiki-image/figure-1.png)

**Figure 1. DEGs under conditions with and without intact LdtR.** DEGs were identified with the threashold of foldchange < 0.9 or > 1.1, adjust p value < 0.05. Volcanal plot was generated by ggplot2. The arrow annotated gene B488\_05770.

##### P\_B488\_05770 and P\_LdtR mediated sensor specifically detected LdtR

*E. coli* is a classic substitution of unculturable CLas bacteria for molecualar research, such as the study of viral factor OptA [4]. To construct a substitutive model organism our LdtR-centered research, we introduced the LdtR transcriptional factor into *E. coli* BL21 (DE3). The protein coding sequences of LdtR (HA tagged) was retrieved from Genbank (Gene ID: 8223535), which were codon-optimized for *E. coli* expression using Genscript's Gensmart codon optimization services (Genscript, China), which were cloned into kanamycin resistant pRSF plasmid backbone. The recombinant plasmid pRSF-LdtR were subsequently transformed into *E. coli* BL21 (DE3) and selected by kanamycin. To check the expression status of LdtR, western blot targetting HA tag on LdtR and Commassie Blue staining assays were performed, the results of which suggested constitutive expression of the LdtR transcriptional factor in *E. coli* BL21 (DE3), condirming the success construction of LdtR expressing strain for further studies (Figure 2).

CLasMV1 is a natural phage that lives on CLas, which obtained a symbiosis relationship for unknown reasons [5,6]. The capsid protein of CLasMV1 constructs the outer layer of CLasMV1, which is essential for CLas recognition and invasion. In order to conquer the problem of lacking phage genome material, we only selected the essential part of CLasMV1, which is capsid protein, to create a phagemid expression system for testing. CLasMV1 capsid protein sequence (FLAG tagged) was acquired from Genbank (GenBank: CP045566.1), cloned into Ampicillin resistant pBluescript backbone. Promoters of LdtP, ZnuA2, and B488\_05770 were cloned to control CLasMV1 capsid protein (BBa\_K4587198, BBa\_K4587197, BBa\_K4587196) (Figure 2). Recombinant phagemids was either transformed into previously constructed LdtR-expressing *E. coli* strains or wild type *E. coli* BL21 strains containing no plasmid. Capsid protein expression was detected and quantified with Western Blot (targeting FLAG tag, Figure 2). P\_ZnuA2 showed greatest expression efficiency under the activation of LdtR, however, the considerable expression quantity without LdtR suggested non-specificity of this candidate sensor. The specificity of P\_LdtP was obviously higher with almost no expression without LdtR, but its efficiency was relatively low. Interestingly, P\_B488\_05770 showed specificity to LdtR, and a moderate efficiency in expression exhibition with the activation of LdtR (Figure 2). The severe expression leakage of P\_ZnuA2 controlled expression cassette suggested unspecificity of P\_ZnuA2 as a sensor. In contrast, P\_LdtP and P\_B488\_05770, especially P\_B488\_05770 seemed to be specifically activated by LdtR, which were selected as sensors of CLas to be further optimized.

![alt Figure 2](https://static.igem.wiki/teams/4587/wiki/wiki-image/figure-2.png)

**Figure 2. LdtR expressing strain and sensor selection.** **a.** schematic view of LdtR expression cassette that was cloned in pRSF; **b.** schematic view of sensor-capsid expression cassette that was cloned in pBluescript; **c.** Coomassie blue staining results. Bacteria were induced with or without IPTG for 12 hours under 25°C. LdtR-HA has the size of ~26kDa. LdtR-OE: LdtR expressing *E. coli* strain; WT: Wild type *E. coli* BL21 (DE3); **d.** Western blot result with rabbit anti-HA antibody as the primary antibody; Bacteria were induced with or without IPTG for 12 hours under 25°C. LdtR-HA has the size of ~26kDa. LdtR-OE: LdtR expressing *E. coli* strain; WT: Wild type *E. coli* BL21 (DE3); **e.** Western blot results with rabbit anti-FLAG antibody as primary antibody. Bacteria were induced with IPTG for 12 hours under 25°C. Capsid-FLAG has the size of ~55kDa. LdtR: the LdtR expressing *E. coli* strain background; P\_LdtP: Bacteria with phagemids featuring LdtP promoter; P\_ZnuA2: Bacteria with phagemids featuring ZnuA2 promoter; P\_B488\_05770: Bacteria with phagemids featuring B488\_05770 promoter.

##### RinA amplification system significantly magnified signal conferred by P\_B488\_05770 mediated sensor

To magnify the expression of our downstream genes including AMP and dsRNA, we simplified and adopted a RinA amlifier system from iGEM 2019 BEAS China to better cater to our needs [7]. Sequences of RinA amplifier and RinA-speciific promoter were acquired from Genbank (NCBI Reference Sequence: YP\_001285353.1) and cloned into ampicillin resistant pBluescript phagemid backbone containing AcGFP reporter under control of P\_LdtP or P\_B488\_05770 (BBa\_K4587195, BBa\_K4587194) (Figure 3). The recombinant phagemids were transformed into previously constructed LdtR-expressing *E. coli* BL21 strains. Expression level of signal GFP was detected by fluorescence intensity at 489nm. RinA has proven to amplify the signal around 20-fold in P\_B488\_05770 sensor system and 2-fold in P\_LdtP sensor system (Figure 3). Given varying performances of RinA amplifier in P\_LdtP mediated and P\_B488\_05770 mediated sensors, P\_B488\_05770 mediated sensor amplified by RinA system was obviously the optimal system to specifically sense the presence of LdtR and efficiently activate downstream genes.

![alt Figure 3](https://static.igem.wiki/teams/4587/wiki/wiki-image/figure-3.png)

**Figure 3. RinA amplifier amplifies signal conferred by sensor.** **a.** Schematic view of sensor-RinA amplifier-GFP cassette that was cloned into pBluescript; **b.** Fluorescence intensity at 488nm of sensor-RinA amplifier-GFP system after 24h in LdtR expressing *E.coli* strain.

##### Sequence and structural optimization of MaSAMP by *ProteinOpti*

The amino acid sequence of MaSAMP was found in the appendix of Huang et al., 2021 [7]. Codon optimization was done using VectorBuilder to reach rational GC% and high Codon Adaption Index (CAI) for the E. coli expression system (Figure 4). The amino acid sequence of MaSAMP was found in the appendix of Huang et al., 2021[7]. Amino acid optimization was conducted via substituting amino acids in the hydrophilic face with single of multiple lysine. From the result of single and double substitution, the lyticity hardly changes, which indicates that our substitution will not enhance lysing human cells via software calculating. Total hydrophobicity slightly increases after substitution while net charge increases immensely. The total positive charges promote MaSAMP to bind with the negative cell membrane. As a result, we arranged the mutants based on the net charge from low to high (top to bottom) and selected 6 candidates with highest net charge for testing. For efficiency, we chemically synthesized the second alpha helix of the six candidates considering the second alpha helix is the main function domain. The 3D hydrophobic Moment vector will be further used in figuring out the interaction between MaSAMP mutants and bacteria membrane.

![alt Figure 4](https://static.igem.wiki/teams/4587/wiki/wiki-image/figure-4.png)

**Figure 4. Obtain MaSAMP and Optimization.** **a.** The pasted sequence is the amino acid sequence of MaSAMP. The improved DNA has 47.55% GC content and 0.98 Codon Adaption Index (CAI); **b.** Red characters point out the position substituted. Blue characters are the main function domain (second alpha helix). The following biophysical parameters are calculated via our assembled toolkit – ProteinOpti.

##### AMPs could be biosynthesized for massive tests

Since chemical peptide synthesis is expensive, time consuming, and not practical for massive testing assays, we proposed to biologically synthesize AMPs for our usages. Detailed description of the sythesis genetic module can be found in [Enginnering Success]. In brief, AMP gene sequences were marked by GFP as reporter, as well as SUMO tag for toxicity concealment. After massively synthesis by high efficient T7 RNApol-promoter *E. coli* system, AMP could be purified after protease cleavage of SUMO. Recombinant plasmids were constructed by cloning designed sequences BBa\_K4587209 (FAMP), BBa\_K4587210 (AMP), and BBa\_K4587215 (BAMP) separately into pET-30a(+) with Kanamycin-resistant gene at the Ndel & Xhol restriction enzyme sites. The recombinant plasmids were subsequently transformed into BL21 (DE3) selected by kanamycin (Figure 5). The size of plasmid FAMP, AMP, and BAMP are 6513 bp, 6477 bp, and 6498 bp respectively, while the size of empty plasmid pET-30a(+) is 5422 bp. Clear band size differences between recombinant plasmids and the pET-30a(+) empty plasmid were found in the gel electrophoresis results after plasmid extraction (Figure 5). Two or more bands in one lane were prone to be various conformations of plasmids such as supercoiled, nicked circular, and linear conformation. These results indicated successful construction of bacteria for AMP bio-manufacturing.

After induction with 1mM IPTG at 37°C for 12 hours, green fluorescence signal from GFP were visualized by fluorescent microscope. Strong green fluorescence signal was exhibited in both the FAMP and AMP group, indicating the successful expression of GFP in the corresponding recombinant plasmids (Figure 5). The AMP group showed a strong green fluorescence signal even without IPTG induction. The green fluorescence signal was relatively weak compared to the FAMP and AMP group. To further confirm the expression of our fusion protein with the exact size, Tris-SDS-PAGE and multiple western blots were performed for each group (Figure 5). According to our design, the size of the fusion protein for FAMP, AMP, and BAMP group were 47.4 kDa, 47.4 kDa, and 48.2 kDa respectively. Large amount of expression was observed in both supernatant and pellet of AMP group after ultrasonication compared to the control groups (Figure 5). Much thicker bands were observed in FAMP and AMP groups, indicating higher levels of fusion protein expression, which were confirmed by Tris-SDS-PAGE results (Figure 5). Therefore, we proceeded our experiment with the FAMP and AMP.

![alt Figure 5](https://static.igem.wiki/teams/4587/wiki/wiki-image/figure-5.png)

**Figure 5. Consturction of AMP biosynthesis system.** **a.** Transformation success of recombinant plasmids into E. coli BL21 (DE3). 50uM Kanamycin was used for selection; **b.** Gel electrophoresis of recombinant plasmids extracted from transformed E. coli that were amplified overnight; **c.** Fluorescence microscopic imaging of engineered E. coli BL21.FAMP; **d.** Western blot of both supernatant and pellet of E. coli BL21 with recombinant plasmid (AMP) after ultrasonication using 6X His tag antibodies, while E. coli having pET-30a(+) plasmid without our designed sequence (Empty) and without plasmid were tested as controls. Expression under both IPTG induction and without induction was examined; **e.** Western blot of supernatant of E. coli BL21 with recombinant plasmids FAMP, AMP and BAMP. Expression under both IPTG induction and without induction was examined; **f.** Tris-SDS-PAGE and Coomassie brilliant blue staining results of recombinant protein expression in E. coli BL21 with recombinant plasmids AMP and FAMP. pET-30a(+) was tested as a control; **g.** Western blot result of recombinant protein expression in E. coli BL21 with recombinant plasmids AMP and FAMP using Anti-6X His tag antibodies. The red rectangle indicates the recombinant protein size and expression.

##### Purified AMP (showed high lethality to Lcr)

Large-scale extraction and purification of our fusion protein were performed after successful small amount extraction. Recombinant E. coli strains were diluted to OD600=0.1 in 100 mL LB respectively in 400 mL conical flask and cultured at 37°C to reach the OD600=0.4~0.6. IPTG induction was followed for another 6~8 hrs. After induction with 1mM IPTG at 37°C for 12 hours, whole protein were extracted with High-Pressure Homogenizer. For protein purification, we used the His-tag Protein Purification Kit produced by Beyotime to purify the fusion protein in the FAMP group. The bands in lane 5 of Figure 6 were purified fusion protein. However, after SUMO protease cleavage, no significant band size change and no band near 7kD was observed, which means the MaSAMP was not successfully released from our fusion protein (Figure 6). After troubleshooting, we found that most of our fusion protein was in the pellet of *E. coli* lysate. For the AMP group, we did not find any fusion protein in the supernatant of E. coli lysate, while the pellet had protein of the correct size (Figure 6). For the FAMP group, we can also see abundant fusion protein in the pellet of lysate compared to the supernatant, and no thick band for purification product (Figure 6). Therefore, we adjusted our protocol to conduct inclusion body purification to purify our fusion protein.

![alt Figure 6](https://static.igem.wiki/teams/4587/wiki/wiki-image/figure-6.png)

**Figure 6. Bio-manipulation of AMP.** **a.** 10% Tris-SDS-PAGE gel. Lane 1: Supernatant after ultrasonication and centrifugation; Lane 2: Supernatant after chelation; Lane 3: Supernatant after first wash; Lane 4: Supernatant after second wash; Lane 5: Supernatant after elution; Lane 6: SUMO protease digestion at 4°C; Lane 7: SUMO protease digestion at 30°C; **b.** 18% Tris-SDS-PAGE gel. Lane 1: Supernatant after ultrasonication and centrifugation; Lane 2: Supernatant after chelation; Lane 3: Supernatant after first wash; Lane 4: Supernatant after second wash; Lane 5: Supernatant after elution; Lane 6: SUMO protease digestion at 4°C, +salt; Lane 7: SUMO protease digestion at 4°C, -salt; **c.** 10% Tris-SDS-PAGE gel of recombinant E. coli lysate in AMP group. Lane 1: Mixture of supernatant and pellet after using High-Pressure Homogenizer; Lane 2: supernatant of lysate; Lane 3: pellet of lysate; **d.** 10% Tris-SDS-PAGE gel of recombinant E. coli lysate and purified product after washing and eluting using gradient imidazole solution. Lane 1: Pellet after lysing cells and centrifuging; Lane 2: Beads after final elution; Lane 3: First elution (0.3M imidazole solution); Lane 4: 0.03M imidazole solution Washing; Lane 5: Second elution using 0.3M imidazole solution; Lane 6: Supernatant after extraction; Lane 7: 0.06M imidazole solution; Lane 8: Lysis solution washing.

##### Optimized AMP 6 showed high lethality to Lcr

SAMP\_5: BBa\_K4587222, SAMP\_6: BBa\_K4587224) of the MaSAMP second alpha-helix generated by the ProteinOpti software (developed by DKU iGEM team) were synthesized by GeneScript and dissolved in DMSO to make 30 µM SAMP solutions. Equal volumes of DMSO and 30 µM SAMP solutions (including unmutated SAMP and 6 mutants) were added to the Lcr culture at OD600=0.4, and 3 replicates for each group were made. After 5 hours, the OD600 of each culture was measured using Microplate Reader (Figure 7). The Abs OD600 of SAMP\_4 and SAMP\_6 was lower than that of SAMP, exhibiting better sterilizing effect on Lcr compared to unmutated SAMP. Other mutants have a variation of sterilizing effects, but they are not as effective as unmutated

![alt Figure 7](https://static.igem.wiki/teams/4587/wiki/wiki-image/figure-7.png)

**Figure 7. The measurement of the OD600 absorption for SAMP mutants.** Mean (±1 SD) OD600 of Lcr culture in different SAMP solutions after 5 hours of culturing (n=3). SAMP showed a significant sterilizing effect compared to the control group DMSO (p=0.003215<0.05), while our mutants SAMP\_4 and SAMP\_6 had more significant sterilizing effect (p=0.0019, p=0.002947) than SAMP.

##### dsRNA can be biosynthesized for massive tests

We designed our primers for our targeted genes- BBa\_K4587003 (DCR), BBa\_K4587006 (CP450), BBa\_K4587009 (MP20), BBa\_K4587012 (CP64), BBa\_K4587015 (DM2). The lengths of these genes are respectively 399bp, 1836bp, 757bp, and 480bp. The mRNA sequences of these primers (both forward and reverse) are BBa\_K4587004 (DCR forward), BBa\_K4587005 (DCR reverse), BBa\_K4587007 (CP450 forward), BBa\_K4587008 (CP450 reverse), BBa\_K4587010 (MP20 forward), BBa\_K4587011 (MP20 reverse), BBa\_K4587013 (CP64 forward), BBa\_K4587014 (CP64 reverse), BBa\_K4587016 (DM2 forward), BBa\_K4587017 (DM2 reverse). Figure 8a shown below demonstrates the successful amplification of CP450 sense and antisense sequence

We constructed our plasmids by inserting our target genes (BBa\_K4587006, BBa\_K4587009, BBa\_K4587012, BBa\_K4587015) respectively into L4440 plasmid with T7 promoter and Ampicillin resistance gene at the KpnI & HindIII (for sense sequence) and SacI & XbaI (for antisense sequence) restriction enzyme sites. The gel results as shown in Figure proves that both restriction enzymes work well since we can see that the band of the uncut vector shows a smaller band than the +RE ones, meaning that the RE ones are linear instead of circular, though concentration problems exist. The size of the undigested L4440 plasmid is 2790bp. A hairpin structure is formed since there’s a linker between the sense and antisense sequence.

We successfully transformed different L4440 plasmids (DM2, MP20, CP64, CP450 respectively) into the HT115 E. coli strain following the Transformation protocol and cultured them. Small colonies can be seen in the following figures (Figure 8b), which are the successfully cultured HT115 E. coli that can produce hairpin RNA with IPTG induction. However, for Figure 8b, it demonstrates the successfully constructed L4440 plasmid with the CP450 antisense in DH5α bacteria.

We successfully produced hpRNA for MP20, CP64, and DM2 genes by transforming the HT115 E. coli strain and utilizing the CTAB miniprep protocol (Figure 8c).

![alt Figure 8](https://static.igem.wiki/teams/4587/wiki/wiki-image/figure-8.png)

**Figure 8. Success biosynthesis of dsRNA.** *E. coli* HT115 strains with dsRNA synthesis functions were constructed and efficiently produce dsRNA. a. Gel-electrophoresis of testing the function of restriction enzymes; b. Success transformation of L4440 + CP450, DM2, MP20 with T7, DM2 with T7, CP64, CP64 with T7, and MP20 (left to right, top to down); c. Gel electrophoresis result of hpRNA induction from plasmids extracted from HT115(DE3) bacteria, which were induced with IPTG and treated with RNase A.

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