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