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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.

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.

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.

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).

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

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

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