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Team:Mingdao/projet.html
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Food wastes can be recycled as fertilizers but leaving strong odors (e.g., NH3, H2S, etc.) from metabolizing by food spoilage bacteria.
Antimicrobial peptides (AMPs) with effect against bacteria, viruses and fungi are small, cationic peptides that bind anionic membrane surfaces of microbes, resulting in forming channels or pores and leaking cell contents.
Stenotrophomonas maltophilia is an environmental bacterium with beneficial effects for plant growth. Several extracellular proteins such as proteases, lipases, nucleases, chitinases and elastases have been identified as decomposing enzymes.
In the design of our genetically engineered bacteria, we’ve created biobricks of (1) AMPs (cecropin and magainin) to attack spoilage bacteria, and (2) several secreted decomposing enzymes directed by the secretion signal of E. coli OmpA to enhance the digestion of food wastes, as well as set (3) a self-destructive device inside to sacrifice when completing the mission, in which ccdB lethal gene expression is regulated by light.
1. Food wastes & Fertilizers
Food wastes are serious problems for humans and the environment alike. Accordingly, one-third of all food produced on earth is wasted before it goes into a human stomach. The average person produces around 475 pounds of food waste every year and the world wastes 11 billion metric tons annually. In addition to educating people to take food carefully and reduce food wastes, another way is to recycle the food wastes. One of them is converting the wastes to fertilizers through composting. However, the composting process not only grows a lot of kinds of bacteria but also leave strong odors produced from bacterial metabolism.
2. Odor & Spoilage Bacteria
Based on food nutrient composition and on the chemical and physical parameters, various microorganisms emerge in the food wastes. Typical spoilage bacteria are given in Table 1, classified by spoilage substrates and metabolites found in spoiled foods. (L. Gram et al., 2002)
Table 1.
To reduce the odor from food wastes, the spoilage bacteria have to be controlled or eliminated. Strains of bacteria are benefit and applied for composting food wastes.
Some grow well in the special environment (e.g., aerobic and acid environment) where the spoilage bacteria cannot survive such as Trichoderma sp. Some are able to produce antibiotics against the spoilage bacteria such as Actinobacteria. And some producing extracellular strong digestive enzymes are capable of decomposing food efficiently before used by the spoilage bacteria such as Bacillus spp.
Reference:
- L. Gram et al., 2002. Food spoilage—interactions between food spoilage bacteria. International Journal of Food Microbiology 78, 79– 97
3. Antimicrobial peptides
- Antimicrobial peptides (AMPs) are an important component of the natural defenses of living organisms against surrounding microbes. AMPs contain a broad spectrum of antimicrobial activities against Gram-positive and Gram-negative bacteria, mycobacteria, fungi, and viruses. AMPs are small peptides and generally less than 10 kDa. They have an overall net positive charge and target on the anionic membrane surfaces of bacteria to form channels or pores, which is leading to leakage of cell contents and the death of the cell.
- Cecropin, an antimicrobial peptide originally isolated from the moth, Hyalophora cecropia. Cecropins are 3~4 kDa linear amphipathic peptides composed of about 31~37 amino acid residues. They have broad activity against both Gram-positive and Gram-negative bacteria through lysing bacterial cell membranes, inhibiting proline uptake and causing leaky membranes. Cecropins constitute a main part of the cell-free immunity of insects.
- Magainin, another antimicrobial peptide found from the skin of the African clawed frog Xenopus laevis. Magainins composed of 23 residues are positively charged and amphiphatic. They preferentially bind to anionic phospholipids abundant in bacterial membranes with the formation of dynamic peptide-lipid supramolecular pore and cell permeabilization.
References:
- Antimicrobial peptides - Wikipedia, the free encyclopedia
- K.V.R. Reddy et al. 2004. Antimicrobial peptides: premises and promises. International Journal of Antimicrobial Agents 24, 536–547
4. Stenotrophomonas maltophilia
Stenotrophomonas maltophilia is a soil, Gram-negative bacterium. They are aerobic, nonfermentative. They are found throughout the environment, particularly in close association with plants with a dominant beneficial effect for plant growth and health. They have strong decomposing enzymes and been applied to the breakdown of natural and man-made pollutants for bioremediation. Extracellular enzymes such as proteases, lipases, nucleases, chitinases and elastases have been identified and recognized as important factors for plant growth and colonization by other microorganisms.
References:
- Robert P. Ryan, et al. 2009. The versatility and adaptation of bacteria from the genus Stenotrophomonas. Nat Rev Microbiol. 7(7):514-25.
5. BioBrick design & Genetically Engineered Bacteria
Fertilizers converted from food waste make odors from the metabolism of the spoilage bacteria. In the iGEM project this year (2014), we’ve designed a genetically engineered bacterium which can produce antimicrobial peptides, cecropin and magainin, to kill the bacteria grown in the food wastes. And the sterile food wastes would be decomposed by a genetically engineered bacterium which produces extracellular decomposing enzymes from Stenotrophomonas maltophilia. And finally the genetically engineered bacteria will be self-destructive with a regulated device carrying ccdB lethal gene induced by the light.
1. Food waste experiment- sniff test
To test the hypothesis of food waste odor produced from the spoilage bacteria, we treated the food wastes with an antibiotic, chloramphenicol, to eliminate bacteria and keep sterile. The fresh food wastes were took from the recycling system in Mingdao High School. The 2-ml aliquots were supplemented with 10 or 50 ug/ml of chloramphenicol (Table 1), followed by culturing in a 37°C incubator for 3 hours.
14 participants in grade 11 were subject to the sniff test and gave a score from 1 to 10. As shown in Figure 1, food wastes treated with chloramphenicol had fewer scores than those without treatment, suggesting that keeping food sterile can reduce the odor. The results implied that preventing food wastes growing bacteria by antibiotics would eliminate the food spoilage odor.
Table 1. Food wastes supplemented with various concentrations of chloramphenicol.
Figure 1. The result of sniff test (score from 1 to 10) from food wastes supplemented with various concentrations of chloramphenicol.
2. Zone of inhibition assay
To test the efficiency of inhibiting bacterial growth by antibiotics, we performed a zone of inhibition assay. 200 µl of E. coli from overnight culture were spread on a agar plate, followed by covering 4 papers with 5, 10, 30, 50 µg of chloramphenicol, respectively.
The bacteria were grown at 37°C for 24 hours. The zones of inhibition appeared and were measured. As shown in Figure 1 and 2, the sizes of zones were correlated with the concentrations of chloramphenicol, demonstrating the efficiency of antibiotics against bacteria.
Figure 1. Zone of inhibition assay Figure 2. Zones of inhibition diameter were measured.
3. Bacillus transformation
Bacillus spp. are soil bacteria and have been applied to food waste composting. In order to genetically engineer Bacillus strains for further application, we’d like to develop a system for transforming Bacillus.
We collaborated with LMU-Munich to develop the system. We obtained several vectors for Bacillus transformation from LMU-Munich and iGEM HQ. Following the transformation protocol developed by LMU-Munich, we’ve tried 6 vectors and successfully generate a Cm-resistant strain of Bacillus subtilis 168 (DB2) transformed with the vector of pBS1C (Figure 1). Bacillus/pBS1C survives in the media supplemented with 10 g of chloramphenicol but not wild-type strain (Figure 2.)
Figure 1. Bacillus transformation with 6 vectors Figure 2. Wild-type and Cm-resistant Bacillus strains
4. Stenotrophomonas maltophilia & strain identification
Stenotrophomonas maltophilia is a soil bacterium with a close association with plant growth and health. Many extracellular enzymes have been identified such as proteases, lipases, nucleases, chitinases and elastases.
In order to clone these genes, we purchased the strains of Stenotrophomonas maltophilia form Bioresource Collection and Research Center (BCRC) in Taiwan. Unfortunately, the strains with full genome sequences in the database (e.g., KEGG, BioCyc, NCBI, etc.) are currently unavailable in the stock. However, we got two strains from the soil isolates (BCRC #11901, BCRC #15550) and culture them in four different media, followed by genomic DNA extraction and PCR for a conserved protein ChiA1.
As seen in Figure 1, the PCR data showed that ChiA1 gene can be amplified from the strain of BCRC #15550 but not BCRC #11901.
Figure 1. PCR for ChiA gene with the genomic DNAs from BCRC #11901 and BCRC #15550 cultured in 4 different media.
PCR products were subjected to sequencing after gel extraction of the band around the size of 1191 bp (the predicted size of ChiA1).The sequence data was subjected to phylogenic tree analysis on an online web server of Phylogeny.fr.
The result of phylogenic tree analysis showed that the strain of BCRC #15550 is the outgroup to other strains, whose full genome sequences are available in the database (Figure 2).
Figure 2. Phylogenic tree analysis between strains of Stenotrophomonas maltophilia.
In addition, we performed NCBI Nucleotide BLAST with the ChiA1 sequence of BCRC #15550. As shown in Figure 3, the sequence can align 4 strains of Stenotrophomonas maltophilia with 98% query cover and 91% identity.
Figure 3. The result of NCBI Nucleotide BLAST
Therefore, we designed primers for extracellular enzymes based on the strains of D457 and K279a and try to clone these gene by PCR.
5. gene cloning of decomposing enzymes
The genomic DNAs of Stenotrophomonas maltophilia BCRC #15550 and Bacillus subtilis 168 (DB2) were extracted. The primers for the genes of DNase, Protease, Lipase, Chitinase were designed based on the Stenotrophomonas maltophilia strains of D457 and K279a, and the primers for the genes of Glucanases were designed based on Bacillus subtilis 168 (DB2).
Figure 1 showed the PCR result for DNase, Protease, Lipase, Chitinase of Stenotrophomonas maltophilia, and Figure 2 for Glucanases of Bacillus subtilis 168 (DB2).
Figure 1. PCR for genes of DNase, Protease, Lipase, ChitinaseFigure 2. PCR for genes of Glucanases
The PCR products were subjected to cleanup and ligated to the pSB1C3-based vector (pSB1C3-Plac-SS-NB,Part: BBa_K1256003) with a secretion signal of OmpA of E. coli designed by us.
6. BioBrick construction
Figure 1 shows the standard backbone of pSB1C3, which is a basic BioBrick part for cloning. pSB1C3 contains RFP coding device composed of subparts of R0010 (Plac), B0034 (RBS), E1010 (RFP) and B0015 (Terminator).
Figure 1. pSB1C3
If you want to clone a gene under a given promoter, you have to do two rounds of cloning, that is, you have to clone your interest gene on pSB1C3, followed by assembling a promoter on the part you’ve generated.
To facilitate the gene cloning process, we’ve introduced NheI and BamHI sites just behind RBS (B0034) and in front of the terminator (B0015) on the vector of pSB1C3 (pSB1C3-Plac-NB (Part: BBa_K1256001), Figure 2). Therefore, the gene of interest can now can be cloned using NheI and BamHI restriction enzyme sites and driven under the front promoter (e.g., Plac (R0010) in this case).
Figure 2. pSB1C3-Plac-NB (Part: BBa_K1256001)
In addition, we further introduced MfeI and NsiI sites just following the antibiotic resistance cassette (i.e., chloramphenicol resistance cassette) (Figure 3). And now you can easily add another BioBrick part cut by EcoRI and PstI and ligate to this region with MfeI and NsiI (EcoRI and MfeI are compatible, PstI and NsiI are compatible), driving the gene part either by the promoter of Cm cassette or independently by the respective promoter.
Figure 3. pSB1C3-Plac-NB-Cm-MN (Part: BBa_K1256002)
7. AMP construction
To produce antimicrobial peptides, cecropin and magainin were chosen as candidates because they are applied in medicine and food preservative and are sold as chemical forms by Sigma-Aldrich.
We obtained the amino acid sequences from Sigma-Aldrich webpage and optimized the nucleotide sequences for gene expression and engineering in the host of Escherichia coli. We performed codon optimization on the web server of Integrated DNA Technologies (IDT). The figure 1 showed the amino acid sequences and optimized nucleotide sequences for cecropin and magainin, respectively.
Figure 1. The amino acid sequences and optimized nucleotide sequences of cecropin and magainin
In order to synthesize the DNA sequences on the vector without scars or any restriction enzyme sites, Gibson assembly method was performed with T5 exonuclease. The optimized DNA sequences of cecropin and magainin were synthesized on the primers, followed by PCR with the template of pSB1C3-Plac-SS-MprF. Colony PCR (Figure 2) and sequencing were performed to check the correction of the plasmid.
Figure 2. The results of colony PCR for checking the plasmids carrying the DNA sequences of cecropin and magainin.
8. gene regulation
To express a lethal gene (e.g., ccdB) in an engineered bacterium, the gene has to be tightly regulated and expressed at an appropriate time. Lac operon is a well-studied gene regulation system. Lac promoter is inhibited at the presence of LacI repressor while induced at the presence of IPTG inducer.
We’ve amplified the LacI repressor gene from the BioBrick part of Part:BBa_C0012 (Figure 1) and cloned it into the vector of pSB1C3-Plac-NB-Cm-MN we designed and constructed. The LacI gene is following Cm cassette and driven by the promoter of Cm, checked by colony PCR (Figure 2). As shown in Figure 3, RFP gene driven by Plac was inhibited in the absence of IPTG and expressed in the presence of IPTG.
Figure 1. PCR amplification of LacI and ccdB geneFigure 2. Colony PCR for check the plasmid carrying LacI
Figure 3. RFP gene expression regulated in the Lac operon system. RFP gene expression was induced by IPTG
In addition, we’ve amplified the ccdB gene (Figure 1) from the the BioBrick part of Part: BBa_K145151. The cloning of ccdB gene onto the vector of pSB1C3-Plac-NB-Cm-LacI carrying LacI repressor is ongoing.
