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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) 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.
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).
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.
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.
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), 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.
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).
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.
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.
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.
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.
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.
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) 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.
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.
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.
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.
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). The result is shown below.
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.
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.) 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.
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)
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.
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/