Part:BBa_K1919003

T7 promotor (under the control of lacI) and downstream CecropinXJ

Biobrick BBa_K1919003 is a device, consisting of T7 promotor, RBS, TrxA tag, 6xHis tag, thrombin site, S tag, enterkinase site and the coding sequence of antimicrobial peptide CecropinXJ. Besides, T7 promotor is under the control of lacI protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2008
  Illegal BamHI site found at 2051
  Illegal XhoI site found at 2252
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1418
  
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

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.

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 [9,10], 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.1A Schematic structure of CecropinXJ in pSB1C3

Fig.1B Schematic structure of CecropinXJ in pET32a(+)

After that, we selected positive clones which were resistant to ampicillin on LB plate to confirm the plasmid through PCR 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.

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.2), 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.3).

Fig.2 Expression of pET32a-cecropinXJ fusion protein analyzed by SDS PAGE.

Fig.3 Western Blotting result of cecropinXJ expression.

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.

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.

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.4).

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.4 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.5.

Fig.5 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 [11-13].

References

[1] Boman HG: Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 13: 61-92, 1995.

[2] Devine DA and Hancock RE: Cationic peptides: distribution and mechanisms of resistance. Curr Pharm Des 8: 703-714, 2002.

[3] Boman HG, Wade D, Boman IA, Wåhlin B and Merrifield RB: Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids. FEBS Lett 259: 103-106, 1989.

[4] Moore AJ, Devine DA and Bibby MC: Preliminary experimental anticancer activity of cecropins. Pept Res 7: 265-269, 1994.

[5] Hancock RE and Lehrer R: Cationic peptides: a new source of antibiotics. Trends Biotechnol 16: 82-88, 1998.

[6] Li JY, Zhang FC and Ma ZH: Prokaryotic expression of cecropin gene isolated from the silk worm Bombyx mori Xinjiang race and antibacterial activity of fusion cecropin. Acta Entomol Sin 47: 407-411, 2004 (In Chinese).

[7] Tang X, Wang H, Kelaimu R, Mao XF and Liu ZY: Molecular cloning, expression of cecropin-XJ gene from silkworm and antibacterial activity in Pichia pastoris. Biotechnology 21: 26-31, 2011 (In Chinese).

[8] Xia L, Zhang F, Liu Z, Ma J and Yang J: Expression and characterization of cecropinXJ, a bioactive antimicrobial peptide from Bombyx mori (Bombycidae, Lepidoptera) in Escherichia coli. Experimental and Therapeutic Medicine 5: 1745-1751, 2013.

[9] Ingham AB and Moore RJ: Recombinant production of antimicrobial peptides in heterologous microbial systems. Biotechnol Appl Biochem 47: 1-9, 2007.

[10] Cabral KM, Almeida MS, Valente AP, Almeida FC and Kurtenbach E: Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inef ciency of the STE13 protease. Protein Expres Purif 31: 115-122, 2003.

[11] Pokorny A, Kilelee EM, Wu D and Almeida PF: The activity of the amphipathic peptide delta-lysin correlates with phospholipid acyl chain structure and bilayer elastic properties. Biophys J 95: 4748-4755, 2008.

[12] Wimley WC: Describing the mechanism of antimicrobial peptide action with the interfacial activity model. ACS Chem Biol 5: 905-917, 2010.

[13] Rausch JM, Marks JR, Rathinakumar R and Wimley WC: Beta-sheet pore-forming peptides selected from a rational combinatorial library: mechanism of pore formation in lipid vesicles and activity in biological membranes. Biochemistry 46: 12124-12139, 2007.