iGEM 2023 — Team bnds-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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![cover-back](https://static.igem.wiki/teams/4593/wiki/cover-back/notebook.png)

## Menu

[5.26-7.23](#5.26-7.23)
[7.23-8.14](#7.23-8.14)
[8.14-8.21](#8.14-8.21)
[8.21-10.4](#8.21-10.4)

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{% 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 %}

![cover-back](https://static.igem.wiki/teams/4593/wiki/cover-back/es-cover.jpg)

## Menu

[Lysing part](#1. Lysing part)
[QS part](#2. QS part)
[References](#3. References)

## Engineering success - Lysing part

---

### *#1st cycle: Verification of LysDZ25's bactericidal ability against S. aureus*

---

#### *Design*

---

Endolysin, the bacteria-originated enzyme that specifically lyse *S. aureus*, is the most important component of our in vivo elimination module. Although 3 types of endolysins will be present in our final product, we choose LysDZ25 which has the widest salt tolerance to be integrated into our testing device. [(Chang et al., 2023)](#References_1) In the first engineering cycle, we plan to extract LysDZ25 and test its bactericidal ability in vitro under its optimal reaction condition. To achieve this goal, we designed plasmid pET28a(+)\_DZ25 (Fig.1), which allows N terminal his-tagged LysDZ25 to be expressed when IPTG is present.

![Plasmid map of pET28a(+)_DZ25](https://static.igem.wiki/teams/4593/wiki/es/figure-1.png)

*Figure 1. Plasmid map of pET28a(+)\_DZ25*

#### *Build*

---

To get purified LysDZ25, plasmid pET28a(+)\_DZ25 (assembled by Genscript) is transformed into *E. coli* BL21 (DE3).

Purification of LysDZ25 is done through nickel bead columns. Its length and purity are affirmed using SDS-PAGE, which is subsequently stained with Coomassie Brilliant Blue for visualization (Fig.2).

![SDS-PAGE Result of DZ25 Purification](https://static.igem.wiki/teams/4593/wiki/es/figure-2.png)

*Figure 2. SDS-PAGE Result of DZ25 Purification*

Endolysin LysDZ25, possessing a molecular weight of 58kDa, presented a distinct band in lane 2, closely aligning with the 55kDa marker on the ladder, pointing towards the successful purification of DZ25. However, it appears that the final wash did not completely remove all contaminant proteins, potentially introducing some degree of uncertainty to the findings.

The purity of DZ25, as evident from its lane on the gel, is lower than expected, implying the presence of approximately 50% other proteins in the mixture. Nonetheless, this deviation did not impede the subsequent applications of DZ25, which still exhibited a promising capability in sterilizing *S. aureus*.

#### *Test*

---

The bactericidal activity of endolysin DZ25 against *S. aureus* was evaluated under various concentrations. An overnight culture of *S. aureus* was diluted 500-fold into fresh TSB medium. The cells were then centrifuged once the OD600 reached a value of 2.0. The pelleted cells were then re-suspended in a reaction buffer (20 mM Tris, 300 mM NaCl, pH 8.0). The initial two groups received 1ml of water and the elution buffer of DZ25, respectively, while the subsequent two were treated with DZ25 to achieve concentrations of 0.05mg/mL and 0.01mg/mL. The OD600 for each group was recorded just after the endolysin was added, and at intervals of 5, 10, 15, 20, 30, and 60 minutes after the DZ25 addition, with each time point being repeated three times.

![OD 600 of S. aureus under different concentrations of endolysin DZ25 with respect to time (some error bars are too small to be visible)](https://static.igem.wiki/teams/4593/wiki/es/figure-3.png)

*Figure 3. OD 600 of S. aureus under different concentrations of endolysin DZ25 with respect to time (some error bars are too small to be visible)*

The data underscore the formidable bactericidal properties of endolysin DZ25. As time progressed, we observed a marked decline in the OD600 values, indicating a reduction in the *S. aureus* colony density. Also, the rate of bacterial elimination was faster at a concentration of 0.01mg/ml compared to 0.05mg/ml, especially in the initial 5 and 20-minute intervals. This unexpected result could not be explained - the salt concentration difference between the two groups should be the same, and even though there is a minor difference, it should not affect the activity of DZ25 due to its wide optimal salt concentration. However, by the one-hour mark, both concentrations had effectively reduced the bacterial count, exhibiting only minor differences in their overall efficacy.

To offer a more comprehensive perspective, cultures from each group were diluted 500-fold and plated on *S. aureus* chromogenic agar post 120 minutes of reaction. The colonies' density after an overnight incubation corroborated the insights derived from the OD600 assessments.

![Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin DZ25 concentrations for 120 min.](https://static.igem.wiki/teams/4593/wiki/es/figure-4.png)

*Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin DZ25 concentrations for 120 min.*

*A) 0.05mg/ml LysDZ25 B) 0.01mg/ml LysDZ25*

*C) water D) Elution buffer*

#### *Learn*

---

Although the concentration-efficiency relationship is not as expected, the result we get is sufficient to conclude that LysDZ25 has significant bactericidal ability against *S. aureus*. Also, although counterintuitive, our result reveals that LysDZ25 has greater lytic efficiency at a relatively smaller concentration (0.01mg/ml) than at a higher concentration (0.05mg/ml). Further experiments are required to explain this effect and get a broader view of the concentration-lytic activity curve. However, for the purpose of our project, this engineering cycle makes us confident that LysDZ25 is a suitable endolysin to be applied in the next cycles.

### *# 2nd cycle: Verification of Spn1s\_LysRZ's lysing ability in E. coli*

---

#### *Design*

---

In our project, *E. coli* has to lyse itself to release the *S. aureus* targeting endolysin (such as LysDZ25). Therefore, our team chose endolysin complex Spn1s\_LysRZ, a complex of holin, endolysin, and two Rz/Rz1-like spanin that specifically lyse *E. coli* and *S. Typhimurium* [(Lim et al., 2012)](#References_2), to assist the release.

In the second engineering cycle parallel to the first one, we plan to examine the lytic efficiency of Spn1s\_LysRZ by letting *E. coli* express it under controlled conditions. To achieve this goal, we designed plasmid pET28a(+)\_Spn1s\_LysRZ (Fig.5), which allows Spn1s\_LysRZ to be expressed when IPTG is present.

![Plasmid map of pET28a(+)_Spn1s_LysRZ](https://static.igem.wiki/teams/4593/wiki/es/figure-5.png)

*Figure 5. Plasmid map of pET28a(+)\_Spn1s\_LysRZ*

#### *Build*

---

To prepare the strain, plasmid pET28a(+)\_Spn1s\_LysRZ (assembled by Genscript) is transformed into *E. coli* BL21 (DE3).

#### *Test*

---

To get a view of the self-lytic process at different expression levels over time, *E. coli* BL21 (DE3) with Spn1s\_LysRZ-pET28a was shaken overnight and then diluted 500-fold into fresh LB medium (K+). The culture is separated into 4 groups of different sterilized tubes, 5 ml each after its OD 600 reaches 1.1. Various amount of 1 M IPTG is added to each tube subsequently, resulting designated final concentration (0mM, 0.01mM, 0.1mM, 1mM). The OD 600 of each group is recorded (with 3 repeats each time) at 15, 30, 60, 120, and 240 minutes after IPTG is added (Fig. 6).

![OD 600 of E. coli with plasmid Spn1s_LysRZ-pET28a under different IPTG concentrations with respect to time. (Error bars are too small to be visible)](https://static.igem.wiki/teams/4593/wiki/es/figure-6.png)

*Figure 6. OD 600 of E. coli with plasmid Spn1s\_LysRZ-pET28a under different IPTG concentrations with respect to time. (Error bars are too small to be visible)*

As the increase in protein concentration is not instant, all 4 groups show an increase in OD 600 during the first 15-minute interval. However, the groups with inducer show a significant decrease in growth speed and even a decrease in OD600 after 30 minutes or longer of induction compared with the blank group. Although protein overexpression could result in slower growth of bacteria, it could not lead to such a significant decrease in bacteria density in a short amount of time. Therefore, the result reveals that Spn1s\_LysRZ is actively lysing E. coli.

To better visualize the result, after 240 min of induction, each group of culture is diluted 300-fold and 200 ul of each diluted bacteria culture is spread on K+ LB plates. The colony density on each plate after growing overnight is consistent with the relationship generated from OD 600 measurement. Overall, the result reveals a positive correlation between Spn1s\_LysRZ expression level and lysing speed of *E. coli*, proving that this part is working successfully.

![Overnight LB agar plate of E. coli with plasmid Spn1s_LysRZ-pET28a induced with different IPTG concentrations for 240 min.](https://static.igem.wiki/teams/4593/wiki/es/figure-7.png)

*Figure 7. Overnight LB agar plate of E. coli with plasmid Spn1s\_LysRZ-pET28a induced with different IPTG concentrations for 240 min.*

*A) 0.01mM IPTG. B) 0.1 mM IPTG. C) 1 mM IPTG. D) 0 mM IPTG*

#### *Learn*

---

Spn1s\_LysRZ works as expected in this engineering cycle. Considering that although IPTG could slow the growth of *E. coli* but not eliminate it under the concentration we used, the conclusion that Spn1s\_LysRZ has a significant lysing ability to *E. coli* when expressed by *E. coli* itself could be drawn. On vector pET28a(+), there are no significant differences in the lytic efficiency when the concentration of IPTG is between 0.1M and 1M. However, for practical usage on other vectors, the strength of the promoter and RBS should be further tuned to achieve the optimal expression level.

### *# 3rd cycle: Integration of endolysin production module and self-lysing release module*

---

#### *Design*

---

In the two previous engineering cycles, we successfully demonstrated that the endolysin LysDZ25 is capable of lysing *S. aureus*, and the self-lysing endolysin SPN1S\_LysRZ can effectively lyse *E. coli*, as originally designed.

As per our initial design, the release of LysDZ25 can occur after SPN1S\_LysRZ has lysed *E. coli*, enabling DZ25 endolysin to target and eliminate *S. aureus*.

To find out deeper into this concept, we conducted an investigation to assess the bactericidal and lysing effects of combining these two endolysins. The genetic circuits utilized in both cycles have been integrated into a single plasmid. The gene circuit of this integrated design is illustrated in Figure 8.

![The design of LysDZ25_SPN1S_LysRz. Created by biorender.com](https://static.igem.wiki/teams/4593/wiki/es/figure-8.png)

*Figure 8. The design of LysDZ25\_SPN1S\_LysRz. Created by biorender.com*

The plasmid contains three ORFs that code for the LysDZ25, the SPN1S, and the Rz-like spanin respectively. The inducible xylose promoter (PxylA) is introduced to the upstream of LysDZ25 for the constant expression, and LacO had been placed upstream of Spn1s\_LysRZ to regulate the self-lysing expression. This plasmid allows for the induction of LysDZ25 expression via xylose and the simultaneous induction of SPN1S and the Rz-like spanin through IPTG.

#### *Build*

---

LysDZ25\_SPN1S\_LysRz-pET28a (Figure 2) was designed as depicted in the gene circuit. The xylose promoter was sourced from addgene (plasmid pXylA-agrCA-I), while the SPN1S\_LysRZ and LysDZ25 components were obtained from previously synthesized plasmids. All these parts were integrated into the pET28a vector using the Goldengate Assembly.

![The AGE result of the PCR product for the construction of plasmid LysDZ25_SPN1S_LysRz-pET28a. The bands indicate the correctness of each component's length.](https://static.igem.wiki/teams/4593/wiki/pcr/dz-25-crp.png)

*Figure 9. The AGE result of the PCR product for the construction of plasmid LysDZ25\_SPN1S\_LysRz-pET28a. The bands indicate the correctness of each component's length.*

![Plasmid map of LysDZ25_SPN1S_LysRz-PET28a.](https://static.igem.wiki/teams/4593/wiki/es/figure-10.png)

*Figure 10. Plasmid map of LysDZ25\_SPN1S\_LysRz-PET28a.*

#### *Test*

---

The constructed plasmid was transformed into the *E. coli* TOP10 strain and subsequently extracted for sequencing confirmation. Once confirmed, the *E. coli* transformed with the plasmid was inoculated into LB medium containing K+ and 0.5% (w/v) xylose and incubated for 4 hours at 37 degrees Celsius, 200rpm.

Following this, 0.5mM IPTG was added to the medium, and the culture was shaken for another 4 hours at 37 degrees Celsius, 200rpm. The resulting culture was then centrifuged at 3000g for 10 minutes, and the supernatant was collected. The control group - the K+ LB medium without the transformed *E. coli*, underwent the same procedures as the experimental group.

In parallel, *S. aureus* was inoculated in 5mL TSB medium for 8 hours, and the culture was subsequently centrifuged at 3000 rpm for 10 minutes. The *S. aureus* cultures were gently spread across an *S. aureus* chromogenic media plate. Next, two 5mm x 5mm square wells were created along the plate's diameter at approximately 1/3 and 2/3 of the diameter.

To these wells, 20 μL of the *E. coli* supernatant collected as mentioned above, and 20 μL of LB K+ media without *E. coli* culture were added onto the *S. aureus* chromogenic media plate coated with *S. aureus* immediately after creating the wells. The plate was then incubated overnight, and the observations and results are depicted in Figure 3.

![The S. aureus chromogenic media plate coated with S. aureus. The experimental group is on the left side, while the control group is on the right side.](https://static.igem.wiki/teams/4593/wiki/es/figure-11.png)

*Figure 11. The S. aureus chromogenic media plate coated with S. aureus. The experimental group is on the left side, while the control group is on the right side.*

Observations show an obvious inhibition zone surrounding the left hole, while the surroundings of the right hole do not show observable differences compared to the rest of the plate. This indicates that the engineered *E. coli* with the plasmid LysDZ25\_SPN1S\_LysRz-PET28a is capable of lysing *S. aureus* after our proposed endolysin expression-self lysing procedure.

#### *Learn*

---

The setup of the control groups is less than ideal, and regrettably, we do not have sufficient time to develop a fully prepared control group experiment. To finalize the experimental settings, we need to include only the SPN1S and DZ25 groups as controls, and these will be further investigated in our future study.

After the test, we preliminarily concluded that the integration of endolysin production module and self-lysing release module in one plasmid LysDZ25\_SPN1S\_LysRz-PET28a are successful and the transformed post-induction *E. coli* culture supernatant are capable of lysing bacteria *S. aureus*. However, the conclusion that the lysis effect results from LysDZ25 endolysin production cannot be given since the control used is clear LB K+ media making us unable to rule out the possibility that other contents originate from *E. coli* cell lysate have bactericidal ability against *S. aureus*. But overall, this engineering cycle provides verification that the engineered *E. coli* with the production and release of endolysin integrated could possibly lysis the bacteria *S. aureus*, which provides direction for further plans of engineering *E. coli* that can sense and lysis *S. aureus* automatically.

## Engineering Success—QS part

---

### *#1st cycle: QS characterization in E. coli*

---

#### *Design*

---

The QS System is a mechanism employed by specific bacteria to sense their population density and subsequently regulate gene expression accordingly. In the case of *S. aureus*, it produces a signaling molecule known as autoinducing peptide (AIP) during its growth. AIPs are recognized by a membrane receptor called AgrC. AgrC, in turn, phosphorylates AgrA, leading to the activation of the downstream promoter P2. [(Marchand & Collins, 2013)](#References_3) For further details, please refer to the Design page. During this engineering cycle, our primary objective is to verify the proper functioning of the P2 promoter.

Initially, our objective is to confirm the inducibility of the P2 promoter by AIPs. In our plasmid design, sfGFP serves as a reporter to indicate whether AgrA activated the transcription of genes downstream P2 promoter in the presence of AIPs. Consequently, the presence of observable fluorescence would signify the effective activation of the P2 promoter, validating its utility in our project. The plasmid design is depicted below.

![The gene circuit of p2 characterization in E. coli.](https://static.igem.wiki/teams/4593/wiki/es/figure-12.png)

*Figure 12. The gene circuit of p2 characterization in E. coli.*

#### *Build*

---

The PCR is employed to amplifiy our plasmid, the AgrC and AgrA was directly amplified from the plasmid pXylA-agrCA-I obtained from Addgene. [1]. P2 promoter was synthesized according to previous exist sequence (Part:BBa\_K1960900) that was designed by iGEM 16\_XMU-China. The sfGFP was obtained from 2002p derived from BNDS-China 2021. All the fragments were added to the pET28a (+) backbone through Goldengate Assembly.
The plasmid is transformed into TOP 10 competent cells, and the cells are spread on the LB agar plate with K+. After individual clones are selected and allowed to shake overnight, the plasmid is extracted and sent for sequencing, with the outcome being confirmed as correct.

![The AGE result of the PCR product for the construction of the characterization plasmid. The bands indicate the correctness of each component's length.](https://static.igem.wiki/teams/4593/wiki/pcr/qs-1-con.jpg)

![The AGE result of the PCR product for the construction of the characterization plasmid. The bands indicate the correctness of each component's length.](https://static.igem.wiki/teams/4593/wiki/pcr/pcr-qs-pcr-22.png)

*Figure 13. The AGE result of the PCR product for the construction of the characterization plasmid. The bands indicate the correctness of each component's length.*

The RBS directly synthesized according to previous existing sequence (Part:BBa\_B0034), and PCR is employed to amplify the plasmid parts(P1 and P2), to add RBS downstream of the promoter. The plasmid undergone the same procedure, transformed into TOP 10 competent cells and was sent for sequencing, with the outcome being confirmed as correct.

![The AGE result of the PCR product for the construction of the characterization plasmid. The bands indicate the correctness of each component's length.](https://static.igem.wiki/teams/4593/wiki/pcr/qs-rbs-final.jpg)

 *Figure 14. The AGE result of the PCR product for constructing the characterization plasmid. The bands indicate the correctness of each component's length. Lane 1: 2428bp; Lane 2: 4373bp.*

#### *Test*

---

The extracted plasmid is introduced into BL21 to enable the expression of sfGFP. After an overnight incubation period, IPTG is introduced to induce expression. In conditions where *S. aureus* is not present, we observed the expression of sfGFP. This observation suggests that P2 is not induced but rather expressed constitutively. As a result, it becomes evident that P2 is not functioning as originally intended in our experiment, and a redesign of the gene circuit is required.

![The picture of the induced BL21. Fluorescence can be observed in the absence of AIPs.](https://static.igem.wiki/teams/4593/wiki/es/figure-14.png)

*Figure 15. The picture of the induced BL21. Fluorescence can be observed in the absence of AIPs.*

To determine the cause of the failure, the dry lab component was employed, and the transcriptional rates of the P2 promoter in *E. coli* were assessed using the De novo DNA calculator [(LaFleur et al., 2022)](#References_4). The result is shown below.

![The predicted transcription rates of p2 specifically in E. coli.](https://static.igem.wiki/teams/4593/wiki/es/figure-15.png)

*Figure 16. The predicted transcription rates of p2 specifically in E. coli.*

The calculated result reveals that it is evident that the transcription rate at the beginning of the sequence is exceptionally high even without an activator, which is consistent with the wet lab result.

#### *Learn*

---

Two main reasons contribute to the failure of our design. First, the structure of RNA polymerase and the microenvironment for transcription is very different in *E. coli* and *S. aureus*, causing the transcription activity of P2 to be exceptionally high. Also, as *E. coli* is a gram-negative bacteria and *S. aureus* is a gram-positive bacteria, the membrane protein AgrC may not locate itself successfully on the cell membrane of *E. coli*, causing the signal transduction to fail. For those reasons, we decided to move the QS detection module to a gram-positive bacteria, Bacillus subtilis, which has a more shared feature with *S. aureus*.

### *#2nd cycle: QS characterization in B. subtilis*

---

#### *Design*

---

Improving upon the previous plasmid construction within *E. coli*, our team has chosen to construct the plasmid in Bacillus subtilis, a gram-positive bacterium. We changed the Ori of the plasmid to suit plasmid replication in *B. subtilis*. Additionally, we use xylose inducible promoter pXylA to control the expression of AgrA and AgrC to suit the gene expression in *B. subtilis* and to reduce the expression burden, which is consistent with the design of the original backbone.

![The plasmid map of p2 characterization in B. Subtillus](https://static.igem.wiki/teams/4593/wiki/es/figure-16.png)

*Figure 17. The plasmid map of p2 characterization in B. Subtillus*

#### *Build*

---

The characterization plasmid is built upon pXylA-agrCA-I, obtained from Addgene. [(Addgene: PXylA-AgrCA-I Sequences, n.d.)](#References_5) First, PCR is employed to amplify our plasmid parts as shown in Figure 17. The fragment lengths are 3531bp, 4691bp, and 189bp, respectively. Gel extraction was successful for the first two groups, with concentrations of 30 ng/ul and 137 ng/ul, respectively. However, the gel extraction for the "62-1" group failed.

![The PCR result for three different groups of templates and primers. (Bands from left to right are: DNA marker, BN23_0055+58 (lane 1), BN23_0056+61 (lane 2-6), two groups of BN23_0059+62 (lane 7-8)(failed), DNA marker)](https://static.igem.wiki/teams/4593/wiki/es/figure-17.png)

*Figure 18. The AGE result of the PCR product for consrtucting the characterization plasmid. The PCR result for three different groups of templates and primers. (Bands from left to right are: DNA marker, BN23\_0055+58 (lane 1), BN23\_0056+61 (lane 2-6), two groups of BN23\_0059+62 (lane 7-8)(failed), DNA marker)*

![The DNA concentration of the band from the annealing temperature of 62 °C and 63 °C was measured at 36ng/µl, and the band from the annealing temperature of 67°C was measured at 137ng/36ng/µl.](https://static.igem.wiki/teams/4593/wiki/pcr/rqs-pcr-1.jpg)

*Figure 19. The AGE result of the PCR product for consrtucting the characterization plasmid. The DNA concentration of the band from the annealing temperature of 62 °C and 63 °C was measured at 36ng/µl, and the band from the annealing temperature of 67°C was measured at 137ng/µl.*

While the gel extraction did not meet our expectations, we used DNA purification to extract PCR products. Also, the primer's annealing temperature has a great difference. Hence, the primers need to be fixed.

![Gel electrophoresis for pxyl-Lane4: pxyl-agr+BN0086+87;Lane5: pxyl-agr](https://static.igem.wiki/teams/4593/wiki/pcr/rqs-pcr-2.jpg)

*Figure 20. The AGE result of the PCR product for constructing the characterization plasmid. The bands did not indicate the correctness of the component.*

#### *Learn*

---

The plasmid assembly for characterizing the p2 promoter was unsuccessful, and we don't have time for further debug. It is suspected that this failure may be attributed to our lack of familiarity with the pXyl vector, which is not native to *E. coli*. However, during the process, we have gained valuable experience in plasmid construction.

## References

---

Chang, Y., Li, Q., Zhang, S., Zhang, Q., Liu, Y., Qi, Q., & Lu, X. (2023). Identification and Molecular Modification of Staphylococcus aureus Bacteriophage Lysin LysDZ25. *ACS Infectious Diseases, 9*(3), 497-506. <https://doi.org/10.1021/acsinfecdis.2c00493>

Lim, J.-S., Shin, H., Kang, D.-H., & Ryu, S. (2012). Characterization of endolysin from a Salmonella Typhimurium-infecting bacteriophage SPN1S. *Research in Microbiology, 163*(3), 233-241. <https://doi.org/10.1016/j.resmic.2012.01.002>

Marchand, Nicholas, and Cynthia H. Collins. “Peptide-Based Communication System Enables Escherichia Coli to Bacillus Megaterium Interspecies Signaling: Peptide-Based Interspecies Communication System.” *Biotechnology and Bioengineering 110*, no. 11 (November 2013): 3003-12. <https://doi.org/10.1002/bit.24975.>

LaFleur, T. L., Hossain, A., & Salis, H. M. (2022). Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria. *Nature Communications, 13*(1). <https://doi.org/10.1038/s41467-022-32829-5>

*Addgene: pXylA-agrCA-I Sequences*. (n.d.). Www.addgene.org. Retrieved October 11, 2023, from <https://www.addgene.org/53437/sequences/>

{% 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 %}

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

![cover-back](https://static.igem.wiki/teams/4593/wiki/cover-back/result-cover.jpg)

## Menu

[The “Quorum sensing detect” Module](#The “Quorum sensing detect” Module)
[The "Elimination" Module](#The “Elimination” Module)
[The "Aptamer detect" Module](#The “Aptamer detect” Module)

## The “Quorum sensing detect” Module

---

#### *Characterization of P2 in E. coli*

---

Initially, our objective is to confirm the inducibility of the P2 promoter by AIPs. In our plasmid design, sfGFP serves as a reporter to indicate whether AgrA activated the transcription of genes downstream P2 promoter in the presence of AIPs. Consequently, the presence of observable fluorescence would signify the effective activation of the P2 promoter, validating its utility in our project. The plasmid design is depicted below.

![The gene circuit of p2 characterization in E. coli.](https://static.igem.wiki/teams/4593/wiki/es/figure-12.png)

*Figure 1. The gene circuit of p2 characterization in E. coli.*

#### *Construction of the plasmid*

---

The PCR is employed to amplifiy our plasmid, the AgrC and AgrA was directly amplified from the plasmid pXylA-agrCA-I obtained from Addgene. [1]. P2 promoter was synthesized according to previous exist sequence (Part:BBa\_K1960900) that was designed by iGEM 16\_XMU-China. The sfGFP was obtained from 2002p derived from BNDS-China 2021. All the fragments were added to the pET28a (+) backbone through Goldengate Assembly.
The plasmid is transformed into TOP 10 competent cells, and the cells are spread on the LB agar plate with K+. After individual clones are selected and allowed to shake overnight, the plasmid is extracted and sent for sequencing, with the outcome being confirmed as correct.

![The AGE result of the PCR product for the construction of the characterization plasmid. The bands indicate the correctness of each component's length.](https://static.igem.wiki/teams/4593/wiki/pcr/qs-1-con.jpg)

![The AGE result of the PCR product for the construction of the characterization plasmid. The bands indicate the correctness of each component's length.](https://static.igem.wiki/teams/4593/wiki/pcr/pcr-qs-pcr-22.png)

*Figure 2. The AGE result of the PCR product for the construction of the characterization plasmid. The bands indicate the correctness of each component's length.*

The RBS directly synthesized according to previous existing sequence (Part:BBa\_B0034), and PCR is employed to amplify the plasmid parts(P1 and P2), to add RBS downstream of the promoter. The plasmid undergone the same procedure, transformed into TOP 10 competent cells and was sent for sequencing, with the outcome being confirmed as correct.

![The AGE result of the PCR product for the construction of the characterization plasmid. The bands indicate the correctness of each component's length.](https://static.igem.wiki/teams/4593/wiki/pcr/qs-rbs-final.jpg)

 *Figure 3. The AGE result of the PCR product for constructing the characterization plasmid. The bands indicate the correctness of each component's length. Lane 1: 2428bp; Lane 2: 4373bp.*

#### *First time induction*

---

The extracted plasmid is introduced into BL21 to enable the expression of sfGFP. After an overnight incubation period, IPTG is introduced to induce expression. In conditions where *S. aureus* is not present, we observed the expression of sfGFP. This observation suggests that P2 is not induced but rather expressed constitutively. As a result, it becomes evident that P2 is not functioning as originally intended in our experiment, and a redesign of the gene circuit is required.

![The picture of the induced BL21. Fluorescence can be observed in the absence of AIPs.](https://static.igem.wiki/teams/4593/wiki/es/figure-14.png)

*Figure 4. The picture of the induced BL21. Fluorescence can be observed in the absence of AIPs.*

See more information in the Engineering page.

#### *Charaterization of P2 in Bacillus Subtillus*

---

Improving upon the previous plasmid construction within *E. coli*, our team has chosen to construct the plasmid in Bacillus subtilis, a gram-positive bacterium. We changed the Ori of the plasmid to suit plasmid replication in *B. subtilis*. Additionally, we use xylose inducible promoter pXylA to control the expression of AgrA and AgrC to suit the gene expression in *B. subtilis* and to reduce the expression burden, which is consistent with the design of the original backbone.

![The plasmid map of p2 characterization in B. Subtillus](https://static.igem.wiki/teams/4593/wiki/es/figure-16.png)

*Figure 5. The plasmid map of p2 characterization in B. Subtillus*

The characterization plasmid is built upon pXylA-agrCA-I, obtained from Addgene. [(Addgene: PXylA-AgrCA-I Sequences, n.d.)](#References_5) First, PCR is employed to amplify our plasmid parts as shown in Figure 17. The fragment lengths are 3531bp, 4691bp, and 189bp, respectively. Gel extraction was successful for the first two groups, with concentrations of 30 ng/ul and 137 ng/ul, respectively. However, the gel extraction for the "62-1" group failed.

![The PCR result for three different groups of templates and primers. (Bands from left to right are: DNA marker, BN23_0055+58 (lane 1), BN23_0056+61 (lane 2-6), two groups of BN23_0059+62 (lane 7-8)(failed), DNA marker)](https://static.igem.wiki/teams/4593/wiki/es/figure-17.png)

*Figure 6. The AGE result of the PCR product for consrtucting the characterization plasmid. The PCR result for three different groups of templates and primers. (Bands from left to right are: DNA marker, BN23\_0055+58 (lane 1), BN23\_0056+61 (lane 2-6), two groups of BN23\_0059+62 (lane 7-8)(failed), DNA marker)*

![The DNA concentration of the band from the annealing temperature of 62 °C and 63 °C was measured at 36ng/µl, and the band from the annealing temperature of 67°C was measured at 137ng/36ng/µl.](https://static.igem.wiki/teams/4593/wiki/pcr/rqs-pcr-1.jpg)

*Figure 7. The AGE result of the PCR product for consrtucting the characterization plasmid. The DNA concentration of the band from the annealing temperature of 62 °C and 63 °C was measured at 36ng/µl, and the band from the annealing temperature of 67°C was measured at 137ng/36ng/µl.*

While the gel extraction did not meet our expectations, we used DNA purification to extract PCR products. Also, the primer's annealing temperature has a great difference. Hence, the primers need to be fixed.

![Gel electrophoresis for pxyl-Lane4: pxyl-agr+BN0086+87;Lane5: pxyl-agr](https://static.igem.wiki/teams/4593/wiki/pcr/rqs-pcr-2.jpg)

*Figure 8. The AGE result of the PCR product for constructing the characterization plasmid. The bands did not indicate the correctness of the component.*

However, since the lack of experience in transforming plasmid into *B. subtillus*, we failed to confirm the function of this plamid.

## The “Elimination” Module

---

#### *LL-37*

---

#### Cell spreading under different concentrations of LL-37

---

The LL-37 was synthesized by Genescript company, with the sequence shown below,
LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

![Figure 2. The report of the synthesized protein was provided by Jinsirui](https://static.igem.wiki/teams/4593/wiki/es/jinsirui.png)

*Figure 9. The report of the synthesized protein was provided by Genescript*

The protein's 0.8mg white lyophilized powder was dissolved by 800μL of Glacial Acetic Acid to 1mg/mL. Dissolved LL-37 was diluted into different concentrations (1.0mg/ml, 0.5mg/ml), and the S.aureus solution which OD600=0.48 was resuspended with the prepared LL-37 solution, water, and Glacial Acetic Acid with each of 200 μl (as controls), spreading to the four agar plates, respectively. The plates were cultivated under 37 degrees Celsius overnight.

![Figure 2. Overnight S.aureus chromogenic plate of S.aureus treated with different dissolved LL-37 concentrations A)Glacial Acetic Acid   B)water   C) 0.5mg/ml LL-37   D)1.0mg/ml LL-37](https://static.igem.wiki/teams/4593/wiki/contribution/contribution/ll37-2.png)

*Figure 10. Overnight S.aureus chromogenic plate of S.aureus treated with different dissolved LL-37 concentrations*

*(A)Glacial Acetic Acid (B)water (C) 0.5mg/ml LL-37 (D)1.0mg/ml LL-37*

The result shows a success proof of LL-37's bactericidal effect.

#### LL-37 lysing effect towards *S. aureus* under different concentration

---

To gain a detailed view of the sterilization effect of LL-37 against *S. aureus* at different concentrations, overnight S. aureus culture was inoculated to fresh TSB medium their OD 600 value was tested. After the OD 600 value of cultures reaches 1.0, they are separated into 4 different sterilized tubes, 5 ml each. Then 100μl of Glacial Acetic Acid and dissolved LL-37(1mg/ml) are added to 4 tubes respectively, achieving different final concentrations (0.1mg/ml, 0.05mg/ml, 0.01mg/ml). The OD 600 values over time of all 4 tubes are recorded after 0.5min, 5min, 10min, 20min, and 30min being treated; each group repeated three times.

![Figure 3. OD600 of  S. aureus under different concentrations of LL-37with respect to time (error bars are too small to be visible)](https://static.igem.wiki/teams/4593/wiki/contribution/contribution/ll37-3.png)

*Figure 11. The OD600 value of S. aureus under different concentrations of LL-37 with respect to time*

*(error bars are too small to be visible)*

The data clearly indicates the sterilization ability of LL-37. As the time increases, there is an obvious decrease in OD 600 values of *S. aureus*. At the beginning of the experiment, OD 600 values for all groups are close to 1.0, while the number drops to 0.8 for most groups and even 0.3 for the 0.1mg/ml group after 30 minutes. The effect of ll-37 at a concentration of 0.01mg/mL is not obvious since its concentration is too low for LL-37 to work normally.

## Concluison

BNDS-China gains a different perspective on the sterilization effects of LL-37 targets S. aureus.

#### *Endolysins*

---

#### *Protein expression, extraction, purification, and verification.*

---

The ORFs of LysDZ25 and ClyC are being synthesized and cloned to vector pET-28a, and the ORF of LysGH15 are being synthesized and cloned to vector pET-15b (finished by Genscript), resulting in the 3 plasmids shown below.

![](https://static.igem.wiki/teams/4593/wiki/result/9-10.png)
 *Figure 12. The plasmid maps of endolysins*

The plasmids are transformed separately to *E. coli* BL21 (ED3) and are induced with IPTG by proper procedures. After induction, the cell is harvested and broken by a High-Pressure Homogenizer. Proteins are purified through affinity chromatography using nickel bead columns. For more detailed procedures, please refer to our protocol and Lab notebook.

A series of SDS-PAGE are applied to verify the length of the extracted protein, which the proteins would be pushed and separated by an electrical field through a gel that contains small pores. The gel was stained with Coomassie Brilliant Blue (CBB) and then decolorated.

![SDS-PAGE Result of ClyC Purification](https://static.igem.wiki/teams/4593/wiki/result/figure-10.png)

*Figure 13. SDS-PAGE Result of ClyC Purification.*

The protein eluted displays prominent bands at about 35 kDa in both lanes, showing consistency with the calculated molecular mass (34.6 kDa). Interestingly, the final wash also shows a high purity of ClyC, suggesting that the amount of ClyC expressed might exceed the binding capacity of the column.

Although lane 2 does contain some contaminant bands, these are unlikely to impact the final application of ClyC. The increased prominence of both the target protein and contaminants in the elution fraction likely results from the high imidazole concentration used in elution, which eluted undesired protein with higher affinity from the beads.

![SDS-PAGE Result of GH15 Purification](https://static.igem.wiki/teams/4593/wiki/result/figure-11.png)

*Figure 14. SDS-PAGE Result of GH15 Purification.*

Endolysin LysGH15, with a molecular weight of 58kDa, displayed a prominent band in lanes 3, 4, and 5 that's just behind the 55 kDa marker on the ladder, suggesting the correctness in the length of the GH15 protein. The empty lane 2 indicates that the quantity of washing is enough, and a higher concentration of desired protein in the eluate must be obtained through a higher than current (20mM) concentration of imidazole in the wash buffer. While this is a concern, the contamination's impact appears minimal, especially when considering the high purity of GH15 observed in lane 4, even if in a reduced yield.

![SDS-PAGE Result of DZ25 Purification](https://static.igem.wiki/teams/4593/wiki/result/figure-12.png)

*Figure 15. SDS-PAGE Result of DZ25 Purification.*

Endolysin LysDZ25, possessing a molecular weight of 58kDa, presented a distinct band in lane 2, closely aligning with the 55kDa marker on the ladder, pointing towards the successful purification of DZ25. However, it appears that the final wash did not completely remove all contaminant proteins, potentially introducing some degree of uncertainty to the findings.

In contrast to GH15, the purity of DZ25, as evident from its lane on the gel, was markedly lower, implying the presence of approximately 50% other proteins in the mixture. Nonetheless, this deviation did not impede the subsequent applications of DZ25, which still exhibited a promising capability in sterilizing *S. aureus*.

#### *ClyC*

---

To get a broader view of the sterilizing process at different ClyC endolysin concentration levels over time, *S. aureus* is shaken overnight and then diluted 300-fold into fresh Tryptic Soy Broth (TSB) medium. After the medium's OD600 reaches 1.0, the cell is collected by centrifuge and then resuspended in reaction buffer with the same volume (50 mM Tris-Cl buffer, pH 6.8, no salt) and then separated into 5 groups of different sterilized tubes, 1ml *S.aureus* cell pellets for each. Equivolum water/elution buffer/diluted ClyC solution is added to each tube, resulting in the final concentration shown in the figure. The OD600 for each group was recorded just after the endolysin was added, and at intervals of 0, 15, 35, and 60 minutes post ClyC addition, with each time point being repeated three times.

![OD 600 of S. aureus under different concentrations of ClyC with respect to time](https://static.igem.wiki/teams/4593/wiki/result/figure-13.png)

*Figure 16. OD 600 of S. aureus under different concentrations of ClyC with respect to time.*

*(some error bars are too small to be visible)*

The data highlights the potent bactericidal capability of endolysin ClyC. Over time, there's a noticeable decrease in the OD600 readings, signaling the dwindling of the *S. aureus* population. Interestingly, the bacterial reduction was more rapid at the 0.8uM concentration than at the 2uM and 0.2uM concentrations. This intriguing outcome could be attributed to the zero-salt environment required by the endolysin [1]. Also, the OD 600 of the mixture exhibits a significant growth just after the endolysin is added even though the resuspended bacteria in each tube are almost homogeneous, which is not explainable. Nonetheless, by the 60-minute mark, all three concentrations substantially curtailed the bacterial numbers, which successfully proved in the lytic activity.

To offer a more comprehensive perspective, 4 groups of *S. aureus* treated as same as above were diluted 500 folds and spread on *S. aureus* chromogenic agar post 120 minutes of lysing. Due to limitations in the availability of chromogenic plates, we were unable to analyze both the 0.8uM and 0.2uM concentration groups simultaneously. To address this, we selected a 0.5uM concentration as a compromise, considering it to be a more representative concentration level for evaluating the efficiency of ClyC endolysin. The colonies' density after an overnight incubation shows consistency with the conclusion derived from the OD600 assessments. Altogether, the findings confirm the successful execution of this part of the study.

![Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin ClyC concentrations for 120 min.](https://static.igem.wiki/teams/4593/wiki/result/figure-14.png)

*Figure 17. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin ClyC concentrations for 120 min.*

*A) water B) elution buffer C) 2uM ClyC D) 0.5uM ClyC*

#### *DZ25*

---

The bactericidal activity of endolysin DZ25 against *S. aureus* was evaluated under various concentrations. An overnight culture of *S. aureus* was diluted 500-fold into fresh TSB medium. The cells were then centrifuged once the OD600 reached a value of 2.0. The pelleted cells were then re-suspended in a reaction buffer (20 mM Tris, 300 mM NaCl, pH 8.0). The initial two groups received 1ml of water and the elution buffer of DZ25, respectively, while the subsequent two were treated with DZ25 to achieve concentrations of 0.05mg/mL and 0.01mg/mL. The OD600 for each group was recorded just after the endolysin was added, and at intervals of 5, 10, 15, 20, 30, and 60 minutes after the DZ25 addition, with each time point being repeated three times

![OD 600 of S. aureus under different concentrations of ClyC with respect to time](https://static.igem.wiki/teams/4593/wiki/result/figure-15.png)

*Figure 18. OD 600 of S. aureus under different concentrations of endolysin DZ25 with respect to time.*

*(some error bars are too small to be visible)*

The data underscore the formidable bactericidal properties of endolysin DZ25. As time progressed, we observed a marked decline in the OD600 values, indicating a reduction in the *S. aureus* colony density. Also, the rate of bacterial elimination was faster at a concentration of 0.01mg/ml compared to 0.05mg/ml, especially in the initial 5 and 20-minute intervals. This unexpected result could not be explained - the salt concentration difference between the two groups should be the same, and even though there is a minor difference, it should not affect the activity of DZ25 due to its wide optimal salt concentration [2]. However, by the one-hour mark, both concentrations had effectively reduced the bacterial count, exhibiting only minor differences in their overall efficacy.

To offer a more comprehensive perspective, cultures from each group were diluted 500-fold and plated on *S. aureus* chromogenic agar post 120 minutes of reaction. The colonies' density after an overnight incubation corroborated the insights derived from the OD600 assessments. Altogether, the findings confirm the successful execution of this part of the study.

![Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin DZ25 concentrations for 120 min.](https://static.igem.wiki/teams/4593/wiki/result/figure-16.png)

*Figure 19. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin DZ25 concentrations for 120 min.*

*A) 0.05mg/ml LysDZ25 B) 0.01mg/ml LysDZ25*

*C) water D) Elution buffer*

#### *GH15*

---

For assessing the effectiveness of endolysin GH15 in lysing *S. aureus*, a similar overnight culture technique was used. Cells were centrifuged upon reaching an OD600 of 1.0 and resuspended in a specialized reaction buffer (20 mM PBS, 500 mM NaCl, pH 8.0). The initial two groups received 1ml of water and the elution buffer of GH15, respectively, while the subsequent three were treated with GH15 to achieve concentrations (0.25uM, 0.05uM, and 1uM). The OD600 for each group was recorded just after the endolysin was added, and at intervals of 5, 10, 15, 20, 30, and 60 minutes post GH15 addition, with each time point being repeated three times.

![OD 600 of S. aureus under different concentrations of endolysin GH15 with respect to time](https://static.igem.wiki/teams/4593/wiki/result/figure-17.png)

*Figure 20. OD 600 of S. aureus under different concentrations of endolysin GH15 with respect to time.*

*(some error bars are too small to be visible)*

The data emphasizes the bactericidal activity of endolysin GH15. As the experiment progressed, a significant decline in the OD600 measurements became evident, indicative of a diminishing *S. aureus* cell density. Notably, the rate of bacterial elimination was highest at the 1uM concentration, while efficiency reduced at 0.25uM and 0.05uM concentrations, especially during the initial 0-20 minutes. By the end of the one-hour period, both the 1uM and 0.25uM concentrations had markedly reduced bacterial counts, showing only small differences in their overall effectiveness. However, the 0.05uM concentration lagged considerably behind, displaying a lack of bactericidal capability.

To offer a more comprehensive perspective, cultures from the 1uM and elusion buffer group were diluted 300-fold and spread on *S. aureus* chromogenic agar post 120 minutes of reaction (the rest two groups were not joined because there weren't enough chromogenic plates). The colonies' density after an overnight incubation corroborated the insights derived from the OD600 assessments. Altogether, the findings confirm the successful execution of this part of the study.

![Overnight S.aureus chromogenic plate of S.aureus treated with different chemicals for 120 min.](https://static.igem.wiki/teams/4593/wiki/result/figure-18.png)

*Figure 21. Overnight S.aureus chromogenic plate of S.aureus treated with different chemicals for 120 min.*

*A) 1uM GH15 B) elution buffer*

#### *Characterization of Spn1s\_LysRZ*

---

To characterize the lytic efficiency of Spn1s\_LysRZ, we designed plasmid Spn1s\_LysRZ-pET28a (assembled by Genscript) (Fig. 19), which allows Spn1s\_LysRZ to be expressed under the presence of IPTG.

![The plasmid map of Spn1s_LysRZ-pET28a](https://static.igem.wiki/teams/4593/wiki/result/figure-19.png)

*Figure 22. The plasmid map of Spn1s\_LysRZ-pET28a*

To get a view of the self-lytic process at different expression levels over time, *E. coli* BL21 (ED3) with Spn1s\_LysRZ-pET28a was shaken overnight and then diluted 500-fold into fresh LB medium (K+). The culture is separated into 4 groups of different sterilized tubes, 5 ml each after its OD 600 reaches 1.1. Various amount of 1 M IPTG is added to each tube subsequently, resulting designated final concentration (0mM, 0.01mM, 0.1mM, 1mM). The OD 600 of each group is recorded (with 3 repeats each time) at 15, 30, 60, 120, and 240 minutes after IPTG is added (Fig. 20).

![OD 600 of E. coli with plasmid Spn1s_LysRZ-pET28a under different IPTG concentrations with respect to time.](https://static.igem.wiki/teams/4593/wiki/result/figure-20.png)

*Figure 23. OD 600 of E. coli with plasmid Spn1s\_LysRZ-pET28a under different IPTG concentrations with respect to time.*

*(Error bars are too small to be visible)*

As the increase in protein concentration is not instant, all 4 groups show an increase in OD 600 during the first 15-minute interval. However, the groups with inducer show a significant decrease in growth speed and even a decrease in OD600 after 30 minutes or longer of induction compared with the blank group. Although protein overexpression could result in slower growth of bacteria, it could not lead to such a significant decrease in bacteria density in a short amount of time. Therefore, the result reveals that Spn1s\_LysRZ is actively lysing *E. coli*.

To better visualize the result, after 240 min of induction, each group of culture is diluted 300-fold and 200 ul of each diluted bacteria culture is spread on K+ LB plates. The colony density on each plate after growing overnight is consistent with the relationship generated from OD 600 measurement. Overall, the result reveals a positive correlation between Spn1s\_LysRZ expression level and lysing speed of *E. coli*, proving that this part is working successfully.

![Overnight LB agar plate of E. coli with plasmid Spn1s_LysRZ-pET28a induced with different IPTG concentrations for 240 min.](https://static.igem.wiki/teams/4593/wiki/result/figure-21.png)

*Figure 24. Overnight LB agar plate of E. coli with plasmid Spn1s\_LysRZ-pET28a induced with different IPTG concentrations for 240 min.*

*A)0.01mM IPTG. B) 0.1 mM IPTG. C) 1 mM IPTG. D) 0 mM IPTG*

## The “Aptamer detect” Module

---

#### *EMSA*

---

EMSA is used to test the proper binding of the ssDNA and protein A. The samples are treated under the room temperature and incubated with EMSA reagents. An 6% polyacrylamide gel is used for electrophoresis step in EMSA. The protein a only and DNA only samples are set as control groups. The Gel running of EMSA is at 100V for 80 minutes with a pre-running of 10V for 10 minutes. Then, the gel is transferred to a pre-treated PVDF membrane under 160V for 60 minutes. The membrane is blocked to prevent non-specific binding, and then it is incubated with a strptavidin-HRP conjugate after the UV light for 15 minutes to achieve the highest conjugation effects. Finally, the membrane is developed after the chemiluminescent substrate treatment and displayed by X-ray film.

![Results of EMSA. Lane 1: only protein A, Lane 2: protein A and PA#2/8](https://static.igem.wiki/teams/4593/wiki/result/emsa-result-1.png)

*Figure 25. Results of EMSA. lane 1: only protein A
lane 2: protein A and PA#2/8
lane 3: PA#2/8*

The shift band of the binding of PA#2/8 and protein was displayed in between the shift band of the ssDNA only one and the protien A only one.The EMSA experiment results show that Protein A binds to the PA#2/8 DNA aptamer. A shift in the DNA band was observed on the gel when Protein A was added, indicating it was binding to and affecting the mobility of the PA#2/8. This confirms that Protein A can interact with and bind to the PA#2/8 through protein-DNA interactions.

#### *ELONA*

---

#### *ELONA test 1*

---

ELONA is used to verify the binding affinity of PA#2/8 to protien A. Generally, the protein A was loaded onto the wells at a concentration of 5 μg/mL and incubated for 2 hours at 37°C. Then BSA was then added at 1 mg/mL to block nonspecific binding. It was washed several times to remove unbound protein. EDC was used to crosslink the proteins for 10 minutes, and it was washed once. Ethanolamine was added next to quench any unreacted groups from the crosslinking, followed by three washes. The biotinylated aptamer library was then denatured and diluted according to the protocol before being added to the wells. Streptavidin-alkaline phosphatase was incubated to detect any bound biotins, washed off, and then luminescent substrate is applied. The luminescence was then tested for absorbance at the wavelength of 450 nm under the plate reader to detect binding between the proteins and the DNA library. The random biotinylated library and a sample that only contain portien A was set as controls.

![A qualitative test for the binding affinity of Protein A aptamer PA#2/8 traditional ELONA.](https://static.igem.wiki/teams/4593/wiki/result/figure-23.png)

*Figure 26. A qualitative test for the binding affinity of Protein A aptamer PA#2/8 traditional ELONA.*

The experiental group's absorbance has a greater absorbance than both other two controls, proving that PA#2/8 has more significant binding affinity with the protein A.

However, we were not satisfied with this result as we wanted to improve the binding affinity of PA#2/8. We conducted a truncation to considerably shorten aptamers while retaining conformational stability.

#### *ELONA test 2*

---

The second ELONA test is used to improve the possible higher affinity of PA#2/8, and the three test ssDNA (PA#2/8-a, PA#2/8-b, PA#2/8-c) was designed based on the 2D structure of PA#2/8. (See in Design page)

![Image during experiment](https://static.igem.wiki/teams/4593/wiki/result/figure-24.png)

*Figure 27. Image during experiment*

Table 1 lists the experimental and control groups. The aptamer-only groups were used to exclude background luminescence. The full-length aptamer was used for comparison with the three truncates to determine the optimal aptamer with the highest binding affinity.

*Table 1. The experimental and control groups' setting*

![The experimental and control groups' setting](https://static.igem.wiki/teams/4593/wiki/result/table-1.png)

All three truncates have been successfully designed to improve the aptamer-protein binding affinity, and the PA#2/8-C truncate has the slightly stronger binding affinity than PA#2/8-A and PA#2/8-B (Figure 25)

![Image during experiment](https://static.igem.wiki/teams/4593/wiki/result/figure-25.jpeg)

*Figure 28. The comparison of binding affinity between four aptamers*

{% endblock %}