Team:SCU-China/Rihanna
Rihanna
You can stand on my umbrella to ANTI microorganisms.
Antimicrobial peptide CecropinXJ is synthesizing in “Rihanna” anytime. When your foot is on the insole, the temperature of your foot induces the temperature sensitive promoter to drive the expression of self-kill gene hokD to lyse the “Rihanna” to release CecropinXJ.
Visit Project “Rihanna” for more.
Overview
The best way to control foot odor and tinea pedis is to control the microorganisms on our feet. It’s well known that fungus can cause tinea pedis: most commonly caused by Trichophyton rubrum, a fungi species originated from Southeast Asia. But in different areas, the species of fungi causing infection varies sometimes. [1]. For foot odor, researches have shown that gram positive microorganisms contribute to the foot odor (Figure 1) [2].
Figure 1
So we decided to use engineered bacteria to secret chemical components to inhibit them. Antibiotics are not a wise choice because the abuse of them are very serious now and it’s useless for fungus inhibition. As a result, we decide to utilize antimicrobial peptide (AMP) to deal with the problem. It’s important to find a antimicrobial peptide which has a wide antimicrobial spectrum towards gram positive bacteria and fungus and can be expressed in E. coli successfully. At last, we find CecropinXJ. It can be expressed in E. coli in fusion protein form and has a wide antimicrobial spectrum (Figure 2) [3], which is suitable for our goal.
Figure 2
But another problem is that, how to release the antimicrobial peptide? The secretion system in E. coli is not easy for releasing of protein synthesized in E.coli even with signal peptide. As a result, we decide to lyse the cell directly to release the content by temperature inducement.
The Rihanna system works as follows.
Figure 3
[1] Michael J Wells et al. "Tinea Pedis" http://emedicine.medscape.com/article/1091684-overview. 2016/10/4.
[2] James, A. Gordon, D. Cox, and Kathryn Worrall ‡. "Microbiological and biochemical origins of human foot malodour †." Flavour & Fragrance Journal 28.4(2013):231–237.
[3] Xia, Lijie, et al. "Expression and characterization of cecropinXJ, a bioactive antimicrobial peptide from Bombyx mori (Bombycidae, Lepidoptera) in Escherichia coli." Experimental & Therapeutic Medicine 5.6(2013):1745-1751.
CecropinXJ
Overview
Goal: To make AMP CecropinXJ being expressed by E.coli strain Rihanna.
Achievements: Successful prokaryotic expression of CecropinXJ was indicated by the results of SDS-PAGE and Western blot. Unfortunately, the results of antimicrobial test and inhibition zone assay exhibits effective limited antimicrobial activity of this peptide towards Staphylococcus aureus.
Parts: BBa_K1919000, BBa_K1919001, BBa_K1919002, BBa_K1919003
Introduction
Antimicrobial peptides (AMPs) are a group of peptides that play roles in the innate immune system to protect the host from invading pathogens [1]. AMPs have minimal toxicity and low sensitivity effects to the host [2], which means antimicrobial peptides have the potential to be used to replace antibiotics in the future. Thus, the detrimental effects of antibiotics overuse will be released.
Cecropins, a group of small AMPs mainly found in the hemolymph of insects, consist of 31‑39 amino acid residues and have a broad spectrum, high heat stability and potent bacteriostatic activity [3-5]. CecropinXJ (Part BBa_K1919000) is a member of the Cecropin family, which was first cloned from the larvae of the Xinjiang silkworm (Bombyx mori). Previous researches have determined the complete amino acid sequence of this molecule [6]. It has been demonstrated that CecropinXJ could be expressed in eukaryotic expression system such as Pichia pastoris [7] or prokaryotic expression system such as E.coli [8]. What’s more, CecropinXJ exhibited to have various activities such as antibacterial activity against both Gram‑positive and Gram-negative bacteria, as well as antifungal activity [8]. These characteristics indicate that CecropinXJ is an ideal antimicrobial substance to be used to treat foot diseases caused by microbes.
In recent years, studies concerning the expression of antimicrobial peptides have mainly focused on the use of fusion partners [9]. For example, Thioredoxin, a heat-stable and low molecular weight soluble protein in the prokaryotic cytoplasm, has been shown to display chaperone‑like activity [10]. Fusion proteins of antimicrobial peptides generated in E. coli reduce the toxic effect of antimicrobial peptides to the host cells and protect the small antimicrobial peptides from proteolytic degradation.
Design
Based on the various antimicrobial activities performed by CecropinXJ, we aimed to use hire this peptide as the main antimicrobial substrate in our insole. In order to realize it, CecropinXJ was expressed in our E.coli strain Rihanna, which is the main attacker grow in our insole.
We employed 3 different promotors to construct 3 individual expression plasmids respectively. These promotors are: T7 promotors (inductive), promotor J23105 (constitutive), promotor J23106 (constitutive). Downstream of promotors, the coding sequences are consistent: TrxA tag/6 x His tag/Thrombin site/S-tag/enterokinase site/CecropinXJ CDS. The 5-tag system exists in commercial plasmid pET32a(+), which could be easily constructed through PCR. The final plasmid products are BBa_K1919001, BBa_K1919002 and BBa_K1919003
After plasmids construction, our target proteins are expressed using E.coli BL21(DE3) under appropriate conditions. Protein expression level was determined through SDS-PAGE and Western Blot (Anti-6 x His tag). Finally, the antimicrobial activities are determined through inhibition zone assay. We aimed to test the inhibition level on the growth of Staphylococcus aureus (Foot odor pathogen), Bacillus subtilis (Foot odor pathogen), Klebsiella pnuemoniae (Quality control strain), Escherichia coli (Quality control strain), Enterococcus faecalis (Quality control strain), Microsporum canis (Tinea Pedis pathogen) and Trichophyton rubrum (Tinea Pedis pathogen). Due to lab condition limitation, we performed S. aureus and E. coli inhibition tests in our lab, while the tests on other microbes were performed by our collaborator TMMU_China.
Results
Considering the fact that an antimicrobial peptide expressed in bacteria may be cytotoxic to the host or subjected to degradation by host-derived peptidases [11,12], we used the recombinant expression system to overcome the potential problems ——fusing the DNA coding sequence of CecropinXJ with the sequence of a bacterial thioredoxin gene exists in the pET32a(+) expression system [3,4]. To construct recombinant pET32a-cecropinXJ expression vector (Fig.1B), we used artificially synthetic CecropinXJ synthesized by Tsingke Ltd. (Fig.1A). Subsequently, CecropinXJ and pET32a plasmid were subjected to enzymatic digestion with EcoRI and XhoI, and ligated using T4 DNA ligase.
Fig.1 Schematic structure of CecropinXJ in pSB1C3(A) and CecropinXJ in pET32a (B).
After that, we selected positive clones which were resistant to ampicillin on LB plate to confirm the plasmid through PCR (Fig.2) and DNA sequencing. The verified recombinant plasmid was transformed into the E. coli strain BL21(DE3) pLYsS competent cells [8], which encodes a chromosomal T7 RNA polymerase under the control of a tac promoter.
Fig.2 The result of colony PCR confirmed the successful construction of CecropinXJ expression plasmid.
When the optical density at OD600nm of the culture reached 0.6-0.8, we added 0.8mM IPTG to induce the cells, by which time the tac promoter was activated and drove expression of pET32a-CecropinXJ. After 5 hours induction at 37 ̊C, the expression level of recombinant CecropinXJ was detected through SDS-PAGE and western blot analysis. In the result of SDS-PAGE (Fig.3), an obvious band at the size of 25 kDa compared with control was observed, which was as expected. The result of western blot analysis provided subsequent confirmation of expression (Fig.4).
Fig. 3 Expression of pET32a-cecropinXJ fusion protein analyzed by SDS‑PAGE.
However, since the western blot analysis was used on the whole cell proteins, some non-specific bindings were also obtained. Nonetheless we can still make a clear determination through the comparison between the induced cell and the control based on the high quantity of the expression product. Besides, in the lane of the pET32a factor without CecropinXJ gene, we also obtained a clear expression band at the size of about 20kDa, which is the protein product of the 5-tag system encoded by pET32a expression system itself. In addition, the purification of recombinant protein by Ni-NTA was performed under the assistance of TMMU_China.
Fig.4 Western Blotting result of cecropinXJ expression
According to the results, we can see that the E.coli recombinant expression system is a good way to produce AMPs because of its easy culture, fast growth and larger quantities than those purified from their natural sources. What’s more, this system costs less money compared with chemical synthesis.
Besides, another two vectors which express cecropinXJ continuously downstream of two different constitutive promoters, J23105(Fig. 5A) and J23106(Fig. 5B) were also constructed.
Fig.5 Schematic structure of CecropinXJ with J23105 (A) and CecropinXJ with J23106(B)
The combination of cecropinXJ and constitutive promoters were determined by PCR.( Fig. 6)
Fig.6 The result of colony PCR confirmed the successful combination of CecropinXJ and constitutive promoters J23105(A) /J23106(B)
To detect the antimicrobial activity of the recombinant CecropinXJ, we did the inhibition zone assay towards Staphylococcus aureus (as well as Bacillus subtilis, Klebsiella pnuemoniae, Escherichia coli, Enterococcus faecalis, Microsporum canis and Trichophyton rubrum with the assistance of TMMU_China) and detected the growth situation of them after recombinant CecropinXJ was added into the bacterial culture solution. To obtain recombinant CecropinXJ solution, 10ml culture was centrifuged at 8,000 x g for 5 min after induction. The pellet was resuspended in 10 ml PBS and placed in an ice bath for ultrasonic lysis (200 W, 5 sec, 5 sec). The lysate was centrifuged at 10,000 x g for 5 min and supernatant was collected for further work.
To simulate the real environment of our insole, we used the raw ultrasonic cell lysate for bacterial inhibition test. Staphylococcus aureus were grown in LB at 37℃. A dilution of Staphylococcus aureus (20µl; OD600=0.5) was taken and added to 1.5ml, 1.0ml and 0.5ml ultrasonic lysate containing recombinant CecropinXJ respectively and then 0.5ml, 1.0ml and 1.5ml of fresh LB was added accordingly. (Table 1) After incubation at 37℃ for 0.5h, 1.0h and 1.5h, the absorbance of culture at 600nm (OD600) was detected respectively using spectrophotometer (Fig.7).
| Number | Staphylococcus aureus culture/μl | ultrasonic lysate of pET32-XJ BL21 /ml | ultrasonic lysate of wild BL21（pLyss)/ml | LB/ml | PBS/ml | Chloromycetrin/μl |
|---|---|---|---|---|---|---|
| 1.1(1.5XJa) | 50.0 | 1.50 | 0 | 0.50 | 0 | 0 |
| 1.2(1.5XJa) | 50.0 | 1.50 | 0 | 0.50 | 0 | 0 |
| 2.1(0.5XJa) | 50.0 | 0.50 | 0 | 1.50 | 0 | 0 |
| 2.2(0.5XJa) | 50.0 | 0.50 | 0 | 1.50 | 0 | 0 |
| 3.1(1.5XJ1b) | 50.0 | 1.50 | 0 | 0.50 | 0 | 0 |
| 3.2(1.5XJ1b) | 50.0 | 1.50 | 0 | 0.50 | 0 | 0 |
| 4.1(0.5XJ1b) | 50.0 | 0.50 | 0 | 1.50 | 0 | 0 |
| 4.2(0.5XJ1b) | 50.0 | 0.50 | 0 | 1.50 | 0 | 0 |
| 5.1(1.5w) | 50.0 | 0 | 1.50 | 0.50 | 0 | 0 |
| 5.2(1.5w) | 50.0 | 0 | 1.50 | 0.50 | 0 | 0 |
| 6.1(0.5w) | 50.0 | 0 | 0.50 | 1.50 | 0 | 0 |
| 6.2(0.5w) | 50.0 | 0 | 0.50 | 1.50 | 0 | 0 |
| C+(1.5) | 50.0 | 0 | 0 | 0.50 | 1.50 | 2.0 |
| C+(0.5) | 50.0 | 0 | 0 | 1.50 | 0.50 | 2.0 |
Table 1 Different conditions of bacterial inhibition test
“XJa” and “XJb” were the ultrasonic lysate of two repeated independent induced experiments. “Wild” was the ultrasonic lysate of wild BL21(pLysS) strain. Detailed component of each tube was listed. Chloromycetin (C+) was used for positive control (PC) . The ultrasonic lysate of wild BL21(pLyss) was used for negative control (NC). Each tube was cultured at 37℃ with rotation in a speed of 300rpm.
Fig.7. The Antimicrobial Test Results. “XJa” and “XJb” were the ultrasonic lysate of two repeated independent induced experiments. “Wild” was the ultrasonic lysate of wild BL21（pLyss). “1.5” and “0.5” means the adding amount of corresponding ultrasonic lysate. Detailed components in each tube were listed in table 1. Chloromycetin (C+) was used for positive control (PC). The ultrasonic lysate of wild BL21(pLysS) was used for negative control (NC). Each tube was cultured at 37℃ with rotation in a speed of 300rpm. The measuring error is within ±0.02.
For the inhibition zone assay, we used the filter paper method. The filter paper was soaked in the raw ultrasonic lysate for 10min and put on the medium after plate coating of Staphylococcus aureus. The antibacterial efficacy and inhibition zone was shown in Fig.8.
Fig.8. Antimicrobial activity of recombinant CecropinXJ using inhibition zone assays. Staphylococcus aureus were plated on LB medium. (A)①25μg/ml chloromycetin was used as positive control. ②250μg/ml chloromycetin was used as positive control. ③10μg/ml kanamycin was used as positive control. ④Ultrasonic lysate of wild BL21(pLyss) was used as negative control. ⑤,⑥Ultrasonic lysates from two repeated independent induced experiments. (B)①Ultrasonic lysates of pET32-CecropinXJ BL21(pLyss). ②Ultrasonic lysate of wild BL21(pLyss) was used as negative control. ③250μg/ml Chloromycetin was used as positive control. ④25μg/ml chloromycetin was used as positive control.
Compared with the high bacterial inhibition effects of prokaryotic expressed CecropinXJ, shown in previous researches, [8] our results show that the recombinant CecropinXJ has limited antimicrobial activity. Even we used three specialized E.coli BL21 strain (RIPL, Rosetta and pGr07) provided by SCUT-China_A, the results are still not ideal. We hypothesized that the induction condition used in our experiments were slightly different with the original research, which lead to serious consequence. However, due to time limits, our experiments on the prokaryotic expression of CecropinXJ had to be paused here. Further experiments using different induction condition are required. Earlier studies had indicated that CecropinXJ shares a similar structure with ABP-CM4, which has the ability to form specific amphipathic α-helices which allows targeting of nonpolar lipid cell membranes. Upon membrane targeting, the helices form ion-permeable channels, subsequently resulting in cell depolarization, irreversible cytolysis and cell death [15-17].
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Lysis
Overview
Goal:To lyse E.coli stains above 42℃ and test mutant temperature-sensitive device.
Achievement:We built a new temperature-sensitive device and designed the mutant type of CIλrepressor.For measurement , we finished qualitative measurement and quantitative measurement of these parts and devices.Results indicate that temperature-sensitive killing device can lyse E.coil above 42℃ .
Part:BBa_K1919100
BBa_K1919101
BBa_K1919102
Introduction:
Background:To achieve the goal that once people wear shoes, the CecropinXJ(BBa_K1919000) can be released from cystolic, we design a temperature sensitive killing device (BBa_K1919100) .This part consists of a composite promoter (BBa_K608351)and hokD (BBa_K1497008).Composite promoter (BBa_K608351) is a part of CIλrepressor system. When the temperature is raised to 35℃, CIλrepressor start to denature and can’t bind with promoter ,then hokD gene will be expressed to kill the bacteria. At 42℃, the repressor is totally denatured.
Otherwise, human body temperature is about 37℃(In modeling part, we did more experiment for the acute temperature), obviously, wide-type temperature-sensitive promoter can not express efficiently at this temperature. For searching essay ,we found that some sites were important for CIλ repressor, then, we design a new mutant type having five important changed sites. In the later measurement, it expressed better than wild type temperature-sensitive promoter.
Design
1) Part: BBa_K1919101
2)
Function: This is based on part(BBa_K608351).Composite promoter (BBa_K608351) include the mutant type CIλrepressor so that it can express above 37℃.Compared with the part(BBa_K608351),it is more sensitive at 37℃.According to the essay, we choose five important sites A44G, G92A, C188T, G378A, A629T, which may enhance more than tenth times expression at 37℃，compared with wild type(BBa_K608351).
As for the measurement of this part, we choice eGFP and FCM to test the expression at 37℃.
But it’s not finished in the end
Device
a. BBa_K1919100
Introduction: Composite part consists of a composite promoter (BBa_K608351)and hokD (BBa_K1497008).Composite promoter (BBa_K608351) is a part of CIλrepressor system.When the temperature is raised to 42°C, CIλrepressor can not bind with promoter ,then hokD gene will be expressed to kill the bacteria.
Qualitative measurement
After 16h incubation of E.coli with device BBa_K1919100 at 30℃, we take 1ml culture in 2ml EP tube and incubate one night .We use Methylene blue solution to identify the difference between 30℃ team and 42℃ team.The result shows that 42℃ team bacteria were almost died and dyed with Methylene blue when 30℃team bacteria survived.
Quantitative measurement
1) OD600:We build two kinds of plasmids to test the expression of temperature-sensitive device(BBa_K1919100-team and BBa_K1919100 with reporter E0450-RFP team).Comparing the same E.coli strain, . Despite of the temperature factor, the OD 600 of 30℃team and 42℃team all rose before 10 hours. Then 30℃team remained the same trend until 14 hours and have a platform for 8 hours then rose again to OD600 1.6. But the 42℃team’s OD 600 showed a significant decline after that rise phase during 10 to 14 hours and then grown steadily.
The OD 600 of 30℃team-RFP and42℃team-RFP also rose before 10 hours together. Then the The OD 600 of 30℃team-RFP held this trend and rose until 14 hours then it still rose in a more gentle way. But the OD 600 of 42℃team-RFP showed a steady trend after 10 hours and grew slightly.
The result of control team shows normal BL21 strains have a similar K-value in different temperature in 23 hours. Compared with BL21 which has lysis system, we can assure that temperature sensitive lysis system has worked.
2)Fluorescence microplate system:
Result
The results shows this device can work in 42℃, but it doesn’t work high-efficiently. Combined with OD600 measurement, we found two results for its low efficuent. Firstly, the expression of RFP protein is lower than normal strain, which only has reporter part in the plasmid. Secondly, the RFP protein has a similar emission wave when test OD600, and that may be the most biology nosie in this measurement. What’s more, we changed eGFP reporter to test this part, but it didn’t finish.
