Contribution

In order to provide useful support and contribution to future iGEM, we have carried out the following three aspects of work:

• Characterization on the part BBa_K3576001 supplemented with new data.
• Provide new data of relevant literature for the part BBa_K3512001 .

This program designed a method for degradation of Polyethylene Terephthalate (PET) by an engineered bacteria which expresses PETase encoded in constructed plasmid transformed into the Escherichia coli. Whether engineered E. coli express PEATase as designed, is assessed by repeating the experimental process and comparisons of the catalytic ability.

We cThe purpose of our experiment is to produce engineered bacteria to express PET enzyme, so that the expressed PETase can catalyze the degradation of PET. The competent cells of DH5a and BL21 were first prepared in the main part of the experiment. The competent bacteria were further transformed with constructed plasmids containing resistance gene to ampicillin and coding gene for PETase. The transformed bacteria then expanded further. In order to produce adequate enzymes, we chosed the BL21 strain for PETase production, an engineering bacterium that were optimized for protein expression. In the whole experiment process, we validated all experimental procedures by a series of standard operations. To verify successful transformation of plasmid DNA, we used DH5a strain to do two related tests. The colony PCR and plasmid digestion experiments were carried out respectively. The products of PCR and digestion were then verified using DNA electrophoresis. For PETase production, we selected BL21 strain which were transformed with expression plasimid successfully. At last, SDS-PAGE verification was performed after lysis of the expressed bacteria and the untransformed negative control bacteria.onstruct the following gene circuit based on the principles of synthetic biology, as shown in Figure 1. The HS promoter was amplified from E. coli MG1655, the vector pRdegPLCP was linearized by PCR, and then replace the rDegP of pRdegPLCP by HS promoter to obtain pHSdegPLCP by homologous recombination, and the recombinant plasmid was verified by PCR to ensure the success of the recombinant.

The recombinant plasmid of PET enzyme uses pET21a (+) as shown in Figure 1. This series of vectors is the most commonly used prokaryotic expression system, which controls the expression of the target protein through lactose manipulation elements. The pET21 (+) vector is integrated with the protein sequence that can express Ampicillin resistance, which is convenient for us to screen positive clones. At the same time, we added a 6xHis tag to the target sequence, and added BamHI/EcoRI digestion sites at both ends to facilitate the subsequent digestion linking experiment and protein purification.

Figure 1 The recombinant plasmid of PETase-pET21a (+)

The transformed E. coli were subjected to plasmid extraction and agarose gel electrophoresis, and the bands marked in the figure appeared between 1000-1500 bp, indicating that E. coli from all six sample sources were probably successfully transformed. The PCR product size was considered as 1493bp as show in Figure2.

Figure2: Result of colony PCR Line1: DNA marker 1; Line2 and 9: DNA marker 2; Line3, 4, 5, 6, 7 and 8: plasmid extracted from transformed Escherichia coli

The extracted plasmid was electrophoresed after enzymatic digestion, and the size of the band labeled in the figure (1000-1500bp) is consistent with the nucleic acid sequence between the two restriction endonuclease specific binding sites of EcoR1 and BamH1(1249bp) and the absence of this band in the negative control, indicating that the nucleic acid sequence expressing PETase was probably successfully inserted in the plasmid. The Enzyme section size was considered as 1249 bp as show in Figure 3.

Figure3: Result of enzyme digestion Line 1 and 12: 1kb plus DNA marker; Line2: D2000 DNA marker; Line 3, 4, 5, 6, 7, 8, 9 and 10: plasmid samples after enzyme digestion; Line11: negative control group.

The protein extracted from the successfully transformed E. coli treated with Ripa lysate and PMSF showed bands between 40 and 55 kDa in size （which is the PETase band） after SDS-Page protein electrophoresis, and there was no such band in the negative control group, which was consistent with the expected results, indicating that E. coli successfully expressed PETase as show in Figure 4 .

Figure 4:Result of SDS-PAGE testing Line1&2: Page-Ruler Prestained Protein Ladder; Line3&4: negative control; Line5&6: Protein extracted by Ripa lysate and PMSF.

After three experiments to take the average value, the absorbance curve of the experimental group at 405nm wavelength showed an overall rising trend, with a fast growth rate in 0-15min, and leveled off to reach a plateau at about 45min; the curve of the control group was flat without fluctuation and at a lower value as show in Figure 5.

Blue line/red line: experimental groups containing PETase-expressing bacterial broth, acetonitrile and 4-nitrophenyl butyrate to explore the enzymatic activity of PETase at different concentrations.Gray line: control group containing bacterial solution and acetonitrile to exclude the effect of absorbance of 4-nitrophenyl butyrate on the experimental results. Yellow line: control group containing saline, acetonitrile and 4-nitrophenyl butyrate to exclude the effect of absorbance of the bacterial solution on the experimental results.

Figure 5 Result of pNp catalytical reaction.

We introduced the plasmid DNA expressing PETase into E. coli, and the presence of the target sequence was verified by PCR and following agarose gel electrophoresis. We further confirmed the success of the transformation after two experiments, enzyme digestion electrophoresis of the extracted plasmid and SDS-Page electrophoresis of the protein extracted from the successfully transformed bacterial broth. After that, we used p-NP assay to verify the enzyme activity by measuring the absorbance value of p-nitrophenol, the colored product of p-Nitrophenylbutyrate hydrolyzed by PETase, at 405 nm. After the experiments results were analyzed. The dynamic curve of the experimental group showed an overall increasing trend and was positively correlated with the concentration of the bacterial solution, reaching a plateau at about 45 min, while these performances did not appear in the control group. This means that the E. coli from the experimental group successfully expressed the PETase with hydrolytic activity.

The acetyltransferase ATF1 one of three known S. cerevisiae alcohol acetyl transferases responsible for the synthesis of volatile esters. In this paper, ATF1 is proved to also acetylate alcohols to make various acetates, which are the main component of moth pheromones. Therefore, we could use this enzyme ATF1 to produce moth pheromone compounds biologically, which could be used in pest control.

Our team inquired about a literature, sorted out the results, and supplemented BBa_ K3512001 with relevant information.

The origin and the evolution of toxin-antitoxin systems (TA) remain to be uncovered. TA are abundant in bacterial chromosomes and are thought to be part of the flexible genome that originates from horizontal gene transfer (HGT). To gain insight into TA evolution, They analysed the distribution of the chromosomally-encoded ccdO157 system in 395 natural isolates of E. coli. It was discovered in the E. coli O157:H7 strain in which it constitutes a genomic islet between 2 core genes (folA and apaH).

This literature revealed that Molecular evolution analysis showed that ccdBO157 is under neutral evolution, suggesting that this system is devoid of any biological role in the E. coli species.

In this paper, they found that the CcdB toxin proteins were much more diversified than the antitoxins. Among the 47 serogroups, 14 classes of alleles could be identified. Note that, as mentioned earlier, only one isolate for each serogroup was sequenced and tested (47 isolates). One class was composed of 4 isolates presenting sequence identical to the ccdBO157 gene of the O157:H7 EDL933 reference strain. The 2 most prevalent classes represented 13/47 and 8/47 isolates, and the corresponding alleles harboured either two variations (S10G and V28E) or 1 variation (S44I), respectively. The toxic activity of at least one representative CcdB protein of each class was tested using the toxicity plate assay (see materials and methods) and was comparable to that of the CcdBO157 protein. Four classes, together representing 7 isolates (7/47), contained from one to up to 5 variations (V28E; RH7-V28E; S10G-I26V-V28E; RH7-S10G-I26V-V28E- I93L). At least one representative of each class was assayed for toxicity using the toxicity plate assay.

Interestingly, expression of these variants led to cell killing, showing that the variations did not affect the toxic activity of the CcdB proteins. On the 7 classes remaining, 1 is composed of 2 isolates (representing serogroups O138 and O153) containing 4 variations in their ccdB gene (S10G-V28E-S44I-P54T). The CcdBO138 protein (from serogroup O138) was assayed for toxicity and surprisingly shown not to affect viability upon ectopic expression (Figure 1). The comparison of the variations among the different classes suggests that the P54T mutation is responsible for abolishing the toxic activity of the CcdBO138 variant. The 6 last classes, representing 13/47 isolates, were composed of truncated proteins. Deletions of the carboxy-terminal region were caused by amber mutations at various locations (E41, R42, E63, C84). Interestingly, all these truncated proteins contained several point variations that were also found in the full-length variants that were still toxic (S10G, I26V, V28E, S44I) or not (R7H, P54T). Two of these classes (7/47 isolates) contained 1 extra variation (K62S), while another class (1/47 isolates) contained 2 more variations (R11S and R32S). One representative of 4 out of these 6 classes was tested and was shown to be non toxic using the plate toxicity assay (data not shown). Figure 1 shows that ectopic overexpression of CcdBO51 in the liquid toxicity assay did not lead to the loss of viability. These truncated variants of CcdBO157 are thus inactive and it is likely to be the case for CcdBO7 and CcdBO102 since the active site of the CcdBO157 is located at the caboxy-terminus of the protein (BAHASSI et al. 1995; WILBAUX et al. 2007). The corresponding antitoxins were functional (Table 1).

Figure 6:The CcdBO51 and CcdBO138 variants are not toxic. SG22622/pBAD33 (diamond), SG22622/pBAD33-ccdBO51 (square), SG22622/pBAD33-ccdBO138 (circle) and SG22622/pBAD33-ccdBO157 (triangle) were grown in the presence of 1% arabinose. In A and B serial dilutions of the cultures were plated at regular time intervals on CCM plates without arabinose and incubated overnight at 37℃. Values correspond to the mean of results of three independent experiments and SDs are indicated.

Table 1: The variants of CcdAO157 and CcdBO157 proteins. NT: not tested, +: active for either toxicity or anti-toxicity, -: not active active for either toxicity or anti-toxicity. Amino acids variation as compared to amino acids sequence of the reference CcdAO157 and CcdBO157 proteins are indicated in red. The CcdA and CcdB variants that have been tested experimentally are represented in bold.