The plasmid BBa_K4907027 was transformed into <i>E. coli</i> BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and gene sequencing. The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> BL21(DE3).
<divclass="myPage-paragraph-fig-description"><b>Fig. 17 DNA gel electrophoresis of the colony PCR products of BBa_K4907027_pET-28a(+) in E. coli BL21(DE3).</b> Target bands (506 bp) can be observed at the position around 500 bp.
<divclass="myPage-paragraph-fig-description"><b>Fig. 14 DNA gel electrophoresis of the colony PCR products of BBa_K4907027_pET-28a(+) in E. coli BL21(DE3).</b> Target bands (506 bp) can be observed at the position around 500 bp.
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<pclass="myPage-paragraph-content">
After being cultivated and induced by 0.75 mM IPTG, the GE AKTA Prime Plus FPLC System was employed to collect purified protein from the lysate supernatant. CBM was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining (Fig. 28).
After being cultivated and induced by 0.75 mM IPTG, the GE AKTA Prime Plus FPLC System was employed to collect purified protein from the lysate supernatant. CBM was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining (Fig. 15).
<divclass="myPage-paragraph-fig-description"><b>Fig. 18 SDS-PAGE analysis of CBM-his protein.</b> Target bands (11.9 kDa) can be observed at the position around 10 kDa.
<divclass="myPage-paragraph-fig-description"><b>Fig. 15 SDS-PAGE analysis of CBM-his protein.</b> Target bands (11.9 kDa) can be observed at the position around 10 kDa.
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After purification, we got CBM-his successfully, although it was mixed with other proteins. Then the CBM-his was diluted to 10 M, and 10 M BSA was set as the negative control. 4 mL of 10 μM CBM and BSA was filtered three times by using cellulose filter paper and it was washed three times with Phosphate Buffered Saline (1×PBS). The eventual concentration of CBM and BSA was tested by the Bradford method after being diluted to the same volume. The result showed that CBM’s absorption at OD<sub>595</sub> is higher than BSA’s, illustrating that CBM can bind to cellulose effectively.
<divclass="myPage-paragraph-fig-description"><b>Fig. 19 The concentration of CBM and BSA filtered by cellulose filter paper.</b>
<divclass="myPage-paragraph-fig-description"><b>Fig. 16 The concentration of CBM and BSA filtered by cellulose filter paper.</b>
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<divclass="myPage-paragraph">
<h5class="myPage-paragraph-headline-h5"><b>mv<sup>140</sup>-linker-cbm-his and mv<sup>140</sup>-linker-his</b></h5>
<pclass="myPage-paragraph-content">
To verify whether MV<sup>140</sup> can display heterologous proteins on the surface of the engineered bacteria or not, a His-tag (6×His) was fused to the C-terminal of MV<sup>140</sup>. We used both <ahref=" http://parts.igem.org/Part:BBa_I0500"style="text-decoration:none;">BBa_I0500</a> (araC/pBAD) and <ahref=" http://parts.igem.org/Part:BBa_B0034"style="text-decoration:none;">BBa_B0034</a> to construct the expression system and obtained the composite parts <ahref=" http://parts.igem.org/Part:BBa_K4907136"style="text-decoration:none;">BBa_K4907136</a> and <ahref=" http://parts.igem.org/Part:BBa_K4907137"style="text-decoration:none;">BBa_K4907137</a> (Fig. 30) which are respectively assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmid was transformed into <i>E. coli</i> DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing (Fig. 31).
To verify whether MV<sup>140</sup> can display heterologous proteins on the surface of the engineered bacteria or not, a His-tag (6×His) was fused to the C-terminal of MV<sup>140</sup>. We used both <ahref=" http://parts.igem.org/Part:BBa_I0500"style="text-decoration:none;">BBa_I0500</a> (araC/pBAD) and <ahref=" http://parts.igem.org/Part:BBa_B0034"style="text-decoration:none;">BBa_B0034</a> to construct the expression system and obtained the composite parts <ahref=" http://parts.igem.org/Part:BBa_K4907136"style="text-decoration:none;">BBa_K4907136</a> and <ahref=" http://parts.igem.org/Part:BBa_K4907137"style="text-decoration:none;">BBa_K4907137</a> (Fig. 17) which are respectively assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmid was transformed into <i>E. coli</i> DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing (Fig. 18).
<divclass="myPage-paragraph-fig-description"><b>Fig. 20 Graphic description of the expression gene circuits for the protein display system. (a</b> BBa_K4907136 <b>b</b> BBa_K4907137)
<divclass="myPage-paragraph-fig-description"><b>Fig. 17 Graphic description of the expression gene circuits for the protein display system. (a</b> BBa_K4907136 <b>b</b> BBa_K4907137)
<divclass="myPage-paragraph-fig-description"><b>Fig. 21 DNA gel electrophoresis of the colony PCR products. a</b> BBa_K4907136 pSB1C3 in <i>E. coli</i> DH10β. Target bands (2188 bp) can be observed at the position between 3000 bp and 2000 bp. <b>b</b> BBa_K4907137_pSB1C3 in <i>E. coli</i> DH10β. Target bands (2515 bp) can be observed at the position between 3000 bp and 2000 bp.
<divclass="myPage-paragraph-fig-description"><b>Fig. 18 DNA gel electrophoresis of the colony PCR products. a</b> BBa_K4907136 pSB1C3 in <i>E. coli</i> DH10β. Target bands (2188 bp) can be observed at the position between 3000 bp and 2000 bp. <b>b</b> BBa_K4907137_pSB1C3 in <i>E. coli</i> DH10β. Target bands (2515 bp) can be observed at the position between 3000 bp and 2000 bp.
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2% <i>L</i>-arabinose solution was added to induce the expression of the surface-display system. Then, we use the FITC-labeled anti-His-Tag antibody to target the fused His-tag (6×His) displayed <i>via</i> MV<sup>140</sup>, followed by measuring the fluorescence intensity and OD<sub>600</sub> of the culture.
<divclass="myPage-paragraph-fig-description"><b>Fig. 22 The results of immunofluorescence to characterize the function of the two surface display systems.</b> Fluorescence intensity/OD<sub>600</sub> of <i>E. coli</i> DH10β whether express MV<sup>140</sup> or not. The left is for BBa_K4907136 (<i>p</i> = 0.004608) and the right is for BBa_K4907137 (<i>p</i> = 0.003578). <i>p</i>-value: no significance (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001(****).
<divclass="myPage-paragraph-fig-description"><b>Fig. 19 The results of immunofluorescence to characterize the function of the two surface display systems.</b> Fluorescence intensity/OD<sub>600</sub> of <i>E. coli</i> DH10β whether express MV<sup>140</sup> or not. The left is for BBa_K4907136 (<i>p</i> = 0.004608) and the right is for BBa_K4907137 (<i>p</i> = 0.003578). <i>p</i>-value: no significance (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001(****).
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<pclass="myPage-paragraph-content">
The results showed that the ratio of fluorescence intensity (fluorescence value/OD<sub>600</sub>) of positive control (bacteria harboring surface display system) is higher than that of negative control (bacteria without surface display system) (Fig. 32), which indicates that MV<sup>140</sup> can be successfully located on the surface of engineered bacteria.
The results showed that the ratio of fluorescence intensity (fluorescence value/OD<sub>600</sub>) of positive control (bacteria harboring surface display system) is higher than that of negative control (bacteria without surface display system) (Fig. 19), which indicates that MV<sup>140</sup> can be successfully located on the surface of engineered bacteria.
We attempted to regulate the ratio of these two engineered bacteria through antibiotics, which was preferentially carried out in the shake flask. So, we culture the <i>E. coli</i> BL21(DE3) harboring plasmid of J23100-B0034-<i>gfp</i>-B0015_pSB3C5 and EcNP harboring plasmid of J23100-B0034-<i>rfp</i>-T7t_pET-28a(+). Chloramphenicol and kanamycin were added to regulate the ratio of these two engineering bacteria, whose population quantity was characterized by the fluorescence intensity. After that, we could harvest the relationship between the concentration of antibiotics and the bacteria. As shown in Fig. 50 and Fig. 51 (data of EcNP and <i>E. coli</i> BL21(DE3) respectively), the various concentrations of antibiotics could exert different degrees of inhibiting effect on the bacteria quantity. The changes in fluorescence intensity were also consistent with that of bacteria quantity. As shown in Fig. 52 and Fig 53 (data of EcNP and <i>E. coli</i> BL21(DE3) respectively), various concentrations of antibiotics also have different effects on the growth rate of bacteria. These results will pave the way for the regulation of bacteria population quantity.
We attempted to regulate the ratio of these two engineered bacteria through antibiotics, which was preferentially carried out in the shake flask. So, we culture the <i>E. coli</i> BL21(DE3) harboring plasmid of J23100-B0034-<i>gfp</i>-B0015_pSB3C5 and EcNP harboring plasmid of J23100-B0034-<i>rfp</i>-T7t_pET-28a(+). Chloramphenicol and kanamycin were added to regulate the ratio of these two engineering bacteria, whose population quantity was characterized by the fluorescence intensity. After that, we could harvest the relationship between the concentration of antibiotics and the bacteria. As shown in Fig. 40 and Fig. 41 (data of EcNP and <i>E. coli</i> BL21(DE3) respectively), the various concentrations of antibiotics could exert different degrees of inhibiting effect on the bacteria quantity. The changes in fluorescence intensity were also consistent with that of bacteria quantity. As shown in Fig. 42 and Fig 43 (data of EcNP and <i>E. coli</i> BL21(DE3) respectively), various concentrations of antibiotics also have different effects on the growth rate of bacteria. These results will pave the way for the regulation of bacteria population quantity.
<divclass=" myPage-paragraph-fig-description"><b>Fig. 50 The effect of the antibiotic on the bacteria growth of EcNP. </b>The antibiotic concentration in <b>a</b>, </b>b</b>,</b> c</b>, <b>d</b>, and <b>e</b> was 0 mg/L, 34 μg/L. 68 μg/L. 136 μg/L ,and 340 μg/L, respectively.
<divclass=" myPage-paragraph-fig-description"><b>Fig. 40 The effect of the antibiotic on the bacteria growth of EcNP. </b>The antibiotic concentration in <b>a</b>, </b>b</b>,</b> c</b>, <b>d</b>, and <b>e</b> was 0 mg/L, 34 μg/L. 68 μg/L. 136 μg/L ,and 340 μg/L, respectively.
<divclass=" myPage-paragraph-fig-description"><b>Fig. 51 The effect of the antibiotic on the bacteria growth of <i>E. coli</i> BL21(DE3). </b>The antibiotic concentration in <b>a</b>, </b>b</b>,</b> c</b>, <b>d</b>, and <b>e</b> was 0 mg/L, 50 μg/L. 100 μg/L. 200 μg/L ,and 500 μg/L, respectively.
<divclass=" myPage-paragraph-fig-description"><b>Fig. 41 The effect of the antibiotic on the bacteria growth of <i>E. coli</i> BL21(DE3). </b>The antibiotic concentration in <b>a</b>, </b>b</b>,</b> c</b>, <b>d</b>, and <b>e</b> was 0 mg/L, 50 μg/L. 100 μg/L. 200 μg/L ,and 500 μg/L, respectively.
<divclass=" myPage-paragraph-fig-description"><b>Fig. 52 The effect of the antibiotic on the bacteria growth rate of EcNP. </b>The antibiotic concentration in <b>a</b>, </b>b</b>,</b> c</b>, <b>d</b>, and <b>e</b> was 0 mg/L, 34 μg/L. 68 μg/L. 136 μg/L ,and 340 μg/L, respectively.
<divclass=" myPage-paragraph-fig-description"><b>Fig. 42 The effect of the antibiotic on the bacteria growth rate of EcNP. </b>The antibiotic concentration in <b>a</b>, </b>b</b>,</b> c</b>, <b>d</b>, and <b>e</b> was 0 mg/L, 34 μg/L. 68 μg/L. 136 μg/L ,and 340 μg/L, respectively.
<divclass=" myPage-paragraph-fig-description"><b>Fig. 53 The effect of the antibiotic on the bacteria growth rate of <i>E. coli</i> BL21(DE3). </b>The antibiotic concentration in <b>a</b>, </b>b</b>,</b> c</b>, <b>d</b>, and <b>e</b> was 0 mg/L, 50 μg/L. 100 μg/L. 200 μg/L ,and 500 μg/L, respectively.
<divclass=" myPage-paragraph-fig-description"><b>Fig. 43 The effect of the antibiotic on the bacteria growth rate of <i>E. coli</i> BL21(DE3). </b>The antibiotic concentration in <b>a</b>, </b>b</b>,</b> c</b>, <b>d</b>, and <b>e</b> was 0 mg/L, 50 μg/L. 100 μg/L. 200 μg/L ,and 500 μg/L, respectively.
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<!--4 -Pave the way for co-culture fermentation --->
<h4class="myPage-paragraph-headline-h4"><b>Pave the way for co-culture fermentation</b></h4>
<pclass="myPage-paragraph-content">
The two engineered bacteria constructed were cultured in the shake flask to produce the hyaluronic acid and bacterial cellulose, which was mixed to produce water-retention material. As shown in Fig. 54, we can observe the flocculent water-retention material both in the mixed culture broth and sediment after centrifugation. Thus, we have successfully produced the water-retention material which has been applied in characterizing the water-retention efficiency in the soil (please see <ahref=" https://2023.igem.wiki/xmu-china/proof-of-concept "> Proof of concept </a> for details).
The two engineered bacteria constructed were cultured in the shake flask to produce the hyaluronic acid and bacterial cellulose, which was mixed to produce water-retention material. As shown in Fig. 44, we can observe the flocculent water-retention material both in the mixed culture broth and sediment after centrifugation. Thus, we have successfully produced the water-retention material which has been applied in characterizing the water-retention efficiency in the soil (please see <ahref=" https://2023.igem.wiki/xmu-china/proof-of-concept "> Proof of concept </a> for details).
<divclass=" myPage-paragraph-fig-description"><b>Fig. 54 Co-culture results of these two engineered bacteria. a</b> co-culture broth. <b>b</b> sediment after centrifugation.
<divclass=" myPage-paragraph-fig-description"><b>Fig. 44 Co-culture results of these two engineered bacteria. a</b> co-culture broth. <b>b</b> sediment after centrifugation.
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<h4class="myPage-paragraph-headline-h4">Anti-toxicity of <i>ccdA</i></h4>
We use pBAD (<ahref=" http://parts.igem.org/Part:BBa_K206001 "><b>BBa_K206001</b></a>), RBS (<ahref=" http://parts.igem.org/Part:BBa_B0034 "><b>BBa_B0034</b></a>), <i>ccdB</i> (<ahref=" http://parts.igem.org/Part:BBa_K3512001 "><b>BBa_K3512001</b></a>) to construct the composite part BBa_K4907139, which were assembled on pSB4K5 backbone by standard assembly. This constructed circuit was transformed into <i>E. coli</i> DH10β, followed by positive transformant selection using kanamycin and confirmation through colony PCR (Fig. 50) and sequencing.
We use pBAD (<ahref=" http://parts.igem.org/Part:BBa_K206001 "><b>BBa_K206001</b></a>), RBS (<ahref=" http://parts.igem.org/Part:BBa_B0034 "><b>BBa_B0034</b></a>), <i>ccdB</i> (<ahref=" http://parts.igem.org/Part:BBa_K3512001 "><b>BBa_K3512001</b></a>) to construct the composite part BBa_K4907139, which were assembled on pSB4K5 backbone by standard assembly. This constructed circuit was transformed into <i>E. coli</i> DH10β, followed by positive transformant selection using kanamycin and confirmation through colony PCR (Fig. 40) and sequencing.
<divclass="myPage-paragraph-fig-description"><b>Fig. 50 DNA gel electrophoresis of the colony PCR products of BBa_K4907139</b>
<divclass="myPage-paragraph-fig-description"><b>Fig. 40 DNA gel electrophoresis of the colony PCR products of BBa_K4907139</b>
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<pclass="myPage-paragraph-content">
We use pBAD (BBa_I13453), RBS (BBa_B0034), <i>ccdA</i> (BBa_K4907032) to construct the composite part BBa_K4907138, which were assembled on pSB1C3 backbone by standard assembly. This constructed circuit was transformed into <i>E. coli</i> DH10β, followed by positive transformant selection using chloramphenicol and confirmation through colony PCR (Fig. 51) and sequencing.
We use pBAD (BBa_I13453), RBS (BBa_B0034), <i>ccdA</i> (BBa_K4907032) to construct the composite part BBa_K4907138, which were assembled on pSB1C3 backbone by standard assembly. This constructed circuit was transformed into <i>E. coli</i> DH10β, followed by positive transformant selection using chloramphenicol and confirmation through colony PCR (Fig. 41) and sequencing.
Besides, the <i>E. coli</i> DB3.1 transformed with toxin controlled by pBAD promoter without antitoxin both grew better compared with <i>E. coli</i> DH10β. From these results, we can draw the conclusion that whether the <i>ccdA</i> is in plasmid or genome can play the role of neutralisation to <i>ccdB</i>.
</p>
<pclass="myPage-paragraph-content">
Therefore, in light of the serious leak of BBa_K206001,we use pRHa(BBa_K914003), RBS (BBa_B0034), <i>ccdB</i> (BBa_K3512001) to construct the composite part BBa_K4907131,which were assembled on pSB4k5 backbone by standard assembly. This constructed circuit was transformed into<i>E. coli</i> DH10β, followed by positive transformant selection using kanamycin through colony PCR (Fig. 52) and sequencing.
Therefore, in light of the serious leak of BBa_K206001,we use pRHa(BBa_K914003), RBS (BBa_B0034), <i>ccdB</i> (BBa_K3512001) to construct the composite part BBa_K4907131,which were assembled on pSB4k5 backbone by standard assembly. This constructed circuit was transformed into<i>E. coli</i> DH10β, followed by positive transformant selection using kanamycin through colony PCR (Fig. 42) and sequencing.
We use pBAD/araC (BBa_I0500), RBS (BBa_B0034), <i>ccdB</i> (BBa_K3512201) to construct the composite part BBa_K4907140, which were assembled on pSB4K5 backbone by standard assembly. This constructed circuit was transformed into <i>E. coli</i> DH10β, followed by positive transformant selection using kanamycin and confirmation through colony PCR (Fig. 53) and sequencing.
We use pBAD/araC (BBa_I0500), RBS (BBa_B0034), <i>ccdB</i> (BBa_K3512201) to construct the composite part BBa_K4907140, which were assembled on pSB4K5 backbone by standard assembly. This constructed circuit was transformed into <i>E. coli</i> DH10β, followed by positive transformant selection using kanamycin and confirmation through colony PCR (Fig. 43) and sequencing.
<pclass="myPage-paragraph-content">Given that BBa_K206001 and BBa_K4195005 both have serious leaks, we need to test if BBa_I0500 will leak. </p>
<pclass="myPage-paragraph-content">The <i>ccdB</i> circuit was induced by L-arabinose. Bacteria (<i>E. coli</i> DH10β harboring BBa_K4907140_pSB4K5 and BBa_I13453_pSB1C3) were induced with L-arabinose and glucose separately for 6 hours, with 3 replicates set for each. And the changes in OD<sub>600</sub> and CFU count before and after induction were measured.</p>
<divclass="myPage-paragraph-fig-description"><b>Fig. 54 The function of BBa_I0500 was characterized by survival.a</b> OD<sub>600</sub> values of bacteria upon addition of L-arabinose (left) and glucose (right). p value:<b>b</b> CFU values of bacteria upon addition of L-arabinose (left) and glucose (right)
<divclass="myPage-paragraph-fig-description"><b>Fig. 44 The function of BBa_I0500 was characterized by survival.a</b> OD<sub>600</sub> values of bacteria upon addition of L-arabinose (left) and glucose (right). p value:<b>b</b> CFU values of bacteria upon addition of L-arabinose (left) and glucose (right)
<pclass="myPage-paragraph-content">The experimental group (<i>E. coli</i> DH10β harboring BBa_K4907140_pSB4K5 and BBa_I13453_pSB1C3), and the control group (<i>E. coli</i> DH10β harboring BBa_K206001_pSB4K5 and BBa_I13453_pSB1C3 ) were separately induced with L-arabinose, with 3 replicates set for each. Then measure OD<sub>600</sub> and CFU count at 0, 2, 4, 6 and 8 hours.</p>
<divclass="myPage-paragraph-fig-description"><b>Fig. 55 Growth curve and survival assay for characterizing the function of <i>ccdB</i>. a</b> The value of OD<sub>600</sub> against time (h) for different groups. <b>b</b> CFUs/mL calculated of different groups are plotted against time (h).
<divclass="myPage-paragraph-fig-description"><b>Fig. 45 Growth curve and survival assay for characterizing the function of <i>ccdB</i>. a</b> The value of OD<sub>600</sub> against time (h) for different groups. <b>b</b> CFUs/mL calculated of different groups are plotted against time (h).
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<pclass="myPage-paragraph-content">We used promoter <partinfo>BBa_I0500</partinfo> to regulate the expression of <i>ccdB</i>. After induction by <i>L</i>-arabinose, OD<sub>600</sub> values were measured every two hours. The bacteria that had no <i>ccdB</i> expressed grew rapidly, while the one expressing <i>ccdB</i> showed a significant growth defect, as the optical density (at 600 nm) increased very slightly (Fig. 55a).</p>
<pclass="myPage-paragraph-content">We used promoter <partinfo>BBa_I0500</partinfo> to regulate the expression of <i>ccdB</i>. After induction by <i>L</i>-arabinose, OD<sub>600</sub> values were measured every two hours. The bacteria that had no <i>ccdB</i> expressed grew rapidly, while the one expressing <i>ccdB</i> showed a significant growth defect, as the optical density (at 600 nm) increased very slightly (Fig. 45a).</p>
<pclass="myPage-paragraph-content">At each time, the spot assay was also performed, then the cell viability was measured by CFU count (Fig. 55b). Consistent with the trend of OD<sub>600</sub> value against time, only the absence of <i>ccdB</i> allowed the host cells to survive. All these results indicated that <i>ccdB</i> was toxic enough to the engineered bacteria so that this toxin could be applied to the cases when the suicide of genetically engineered microorganisms (GEMs) were strongly needed.</p>
<pclass="myPage-paragraph-content">At each time, the spot assay was also performed, then the cell viability was measured by CFU count (Fig. 45b). Consistent with the trend of OD<sub>600</sub> value against time, only the absence of <i>ccdB</i> allowed the host cells to survive. All these results indicated that <i>ccdB</i> was toxic enough to the engineered bacteria so that this toxin could be applied to the cases when the suicide of genetically engineered microorganisms (GEMs) were strongly needed.</p>
<pclass="myPage-paragraph-content">We use pBAD/araC (BBa_I0500), inverter (BBa_Q04510), RBS (BBa_B0034 ), Terminator (BBa_B0015 ), mazF (BBa_K1096002) to construct the composite part BBa_K3332081.</p>
<divclass="myPage-paragraph-fig-description"><b>Fig. 56 CFU assay for characterizing the killing effect of kill switch in detection system</b>
<divclass="myPage-paragraph-fig-description"><b>Fig. 46 CFU assay for characterizing the killing effect of kill switch in detection system</b>
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<pclass="myPage-paragraph-content">We use pBAD/araC (BBa_I0500), RBS (BBa_B0034), terminator (BBa_B0015), mazF (BBa_K1096002) to construct the composite part BBa_K3332083, which were assembled on pSB1C3 backbone by standard assembly. </p>
