Difference between revisions of "Part:BBa K3350862"

Latest revision as of 12:22, 27 October 2020

yqjF3rd (promoter)

We used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the yqjF (BBa_K1316002) promoter and obtained its mutant versions yqjF1st and yqjF2nd (BBa_K3350859 and BBa_K3350860) with reduced 2,4-dinitrotoluene (DNT) detection thresholds. After combining the favorable mutation sites of the yqjF1st and yqjF2nd (BBa_K3350859 and BBa_K3350860), we generated the yqjF3rd promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type yqjF (BBa_K1316002) promoter.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

  Landmines pose a great threat to human lives and health. In our project, we designed a Bio-optical Landmine Detection device to achieve landmine detection with high sensitivity.
  Our yqjF promoter (BBa_K1316002) was originally involved in the metabolism of aromatic compounds in bacteria and was later found to respond to chemicals, DNT constantly released from landmines. To improve the sensitivity of the yqjF (BBa_K1316002) promoter to DNT for its practical application, We used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the yqjF (BBa_K1316002) promoter and obtained its mutant versions yqjF1st and yqjF2nd (BBa_K3350859 and BBa_K3350860) with reduced DNT detection thresholds. After combining the favorable mutation sites of the yqjF1st and yqjF2nd (BBa_K3350859 and BBa_K3350860), we generated the yqjF3rd promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type yqjF (BBa_K1316002) promoter. And we overexpressed yhaJ1st (BBa_K3350858) in the engineered bacteria, we reduced the DNT detection threshold from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity.
  Finally, aromatic compounds are the main components of water pollutants, and we hope that other iGEM teams can use this basic part to achieve highly sensitive detection of different water pollutants.

Model

Our modeling analyses of the yqjF (BBa_K1316002) promoter showed that DNT could induce the yqjF (BBa_K1316002) promoter, and there was a three-way relationship between the fluorescent protein expression and DNT concentration, with an R² of 0.990, which was higher than 0.7. Thus, our proposed model could well fit the experimental data, suggesting that there was a strong correlation between fluorescent protein expression and DNT concentration , and the 95% confidence interval for the slope of the data was 1263.861-1716.902, with a slope of the p-value 0.000. Therefore, the difference between the slope value and 0 was statistically significant, and there was a strong linear relationship between fluorescent protein expression and DNT concentration(Fig. 1)

Y=11472.18-3552.68x+383.15x²-8.72x³

Fig. 1. DNT could induce the yqjF promoter.

Characterization and Measurement

We amplified the wild-type yqjF promoter from the genomic DNA of bacteria, inserted the EGFP, as a reporter, at its downstream, and thus generated the reporter plasmid pYB1a-yqjF-EGFP. This reporter was transformed into Escherichia Coli DH5α for the following experiments. We then tested the induction of the reporter system by different concentrations of DNT (20, 25, 30, 40 and 50 mg/L). EGFP intensity in response to DNT changes was shown in Fig. 2. The results indicated that the pYB1a-yqjF-EGFP system could be induced by DNT; However, when the DNT concentration dropped to 20 mg/L, the system showed no response, suggesting that the DNT detection limit of the wild-type yqjF promoter was around 25 mg/L, consistent with the reported value in previous literaturer ^[1].

Fig. 2. Assay to evaluate the DNT detection threshold of the wild-type yqjF promoter.

Based on the data above, we need to optimize our system and improve the sensitivity of the reporter expression induced by DNT. For this purpose, we will focus on the following aspects, including (1) modifying the essential elements of the yqjF promoter to enhance transcription strength, (2) promoting the conversion from DNT to 2,4,5-trihydroxytoluene (THT), which is the key molecule to trigger the promoter ^[2], and (3) Optimizing the transcription activator YhaJ to elevate its responsiveness to the inducer THT ^[3].

(1) Mutagenesis study of the yqjF promoter: Using site-direct mutagenesis, we combined the mutations of theyqjF1st and yqjF2nd (BBa_K3350859 and BBa_K3350860) to generate a newer version of the promoter, designated as the yqjF3rd promoter. We tested the response of the reporter containing the yqjF3rd promoter to the induction of different concentrations of DNT (2.5, 5, 10, 15 and 20 mg/L), and found that the yqjF3rd promoter showed a DNT detection threshold of 5 mg/L (Fig. 3). Thus, with the combination of the yqjF1st and yqjF2nd, we generated the yqjF3rd promoter that exhibited 5-fold increase of DNT detection sensitivity compared to the wild-type yqjF promoter.

Fig. 3. DNT detection by the reporter containing the yqjF3rd promoter.

(2) Promoting the DNT-to-THT conversion: Based on previous literature, the yqjF promoter is likely activated by THT, which is a metabolite of DNT, and when binding to THT, the transcription factor yhaJ can activate the yqjF promoter. Based on these evidence, we generated three types of bacteria to further understand the connection between the metabolic processes of DNT and its regulatory mechanism. The nemA-nfsA-nfsB genes are responsible to convert DNT to THT. However, when we overexpressed the nemA-nfsA-nfsB (BBa_K1316006) genes and simultaneously introduced the yhaJ gene in the engineered bacteria, the fluorescence intensity was markedly reduced compared to the bacteria carrying the yhaJ gene alone (Fig. 4). We predicted that the co-expression of all these proteins could cause undetermined conflicts or overload the bacteria, and thus the combination of the two strategies would not be able to further increase the sensitivity.

Fig. 4. The effects of increasing THT production and overexpressing YhaJ individually or combinatorially on DNT detection sensitivity.

(3) Overexpression of the transcription activator: We further investigated whether we could optimize the transcription activator YhaJ to further improve the detection sensitivity. Employing random mutagenesis approach, we generate yhaJ1st (BBa_K3350858),and we overexpressed yhaJ1st in the engineered bacteria. We reduced the DNT detection threshold from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity (Fig.5).

Fig. 5. YhaJ1st overexpression could remarkably reduce the DNT detection threshold to 0.1 mg/L.

References

[1]Yagur-Kroll, S., Lalush, C., Rosen, R., Bachar, N., Moskovitz, Y., & Belkin, S. (2014). Escherichia coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene. Applied microbiology and biotechnology, 98(2), 885–895.

[2]González-Pérez, M. M., van Dillewijn, P., Wittich, R. M., & Ramos, J. L. (2007). Escherichia coli has multiple enzymes that attack TNT and release nitrogen for growth. Environmental microbiology, 9(6), 1535–1540. https://doi.org/10.1111/j.1462-2920.2007.01272.x

[3]Palevsky, N., Shemer, B., Connolly, J. P., & Belkin, S. (2016). The Highly Conserved Escherichia coli Transcription Factor YhaJ Regulates Aromatic Compound Degradation. Frontiers in microbiology, 7, 1490.

@@ Line 4: / Line 4: @@
 <html>
 <br>
-<p>We used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the <i>yqjF</i>(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter and obtained its mutant versions <i>yqjF1st </i>and <i>yqjF2nd</i> (<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859</a>and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>) with reduced DNT detection thresholds. After combining the favorable mutation sites of the <i>yqjF1st</i> and <i>yqjF2nd</i>(<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a>and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>), we generated the <i>yqjF3rd</i> promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type  <i>yqjF</i>(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter.
+<p>
+&nbsp;&nbsp;We used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the <i>yqjF</i>&nbsp;(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>)&nbsp;promoter and obtained its mutant versions <i>yqjF1st </i> and <i>yqjF2nd</i> (<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859</a> and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>) with reduced 2,4-dinitrotoluene (DNT) detection thresholds. After combining the favorable mutation sites of the <i>yqjF1st</i> and <i>yqjF2nd</i>&nbsp;(<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a> and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>), we generated the <i>yqjF3rd</i> promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type  <i>yqjF</i>&nbsp;(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter.
 </p>
 </html>
@@ Line 20: / Line 21: @@
 ===Usage and Biology===
 <html>
-Landmines pose a great threat to human lives and health. In our project, we designed a Bio-optical Landmine Detection device to achieve landmine detection with high sensitivity.
+&nbsp;&nbsp;Landmines pose a great threat to human lives and health. In our project, we designed a Bio-optical Landmine Detection device to achieve landmine detection with high sensitivity.
 <br>
-Our <i>yqjF</i> promoter (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) was originally involved in the metabolism of aromatic compounds in bacteria and was later found to respond to chemicals, such as 2,4-dinitrotoluene (DNT) constantly released from landmines. To improve the sensitivity of the<i> yqjF</i> (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter to DNT for its practical application,
+&nbsp;&nbsp;Our <i>yqjF</i> promoter (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) was originally involved in the metabolism of aromatic compounds in bacteria and was later found to respond to chemicals, DNT constantly released from landmines. To improve the sensitivity of the<i> yqjF</i> (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter to DNT for its practical application,
-We used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the <i>yqjF</i>(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter and obtained its mutant versions <i>yqjF1st </i>and <i>yqjF2nd</i> (<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a>and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>) with reduced DNT detection thresholds. After combining the favorable mutation sites of the <i>yqjF1st</i> and <i>yqjF2nd</i>(<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a>and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>), we generated the <i>yqjF3rd</i> promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type  <i>yqjF</i> (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>)promoter. With overexpressed <i>yhaJ1st</i> (<a href="https://parts.igem.org/Part:BBa_K3350858">BBa_K3350858</a>)in the engineered bacteria, we reduced the DNT detection threshold from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity.
+We used semi-rational mutagenesis and error-prone PCR mutagenesis to randomly mutate the <i>yqjF</i> (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter and obtained its mutant versions <i>yqjF1st </i>and <i>yqjF2nd</i> (<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a> and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>) with reduced DNT detection thresholds. After combining the favorable mutation sites of the <i>yqjF1st</i> and <i>yqjF2nd</i> (<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a> and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>), we generated the <i>yqjF3rd</i> promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type  <i>yqjF</i> (<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter. And we overexpressed <i>yhaJ1st</i> (<a href="https://parts.igem.org/Part:BBa_K3350858">BBa_K3350858</a>) in the engineered bacteria, we reduced the DNT detection threshold from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity.
 <br>
-Finally, aromatic compounds are the main components of water pollutants, and we hope that other iGEM teams can use this basic part to achieve highly sensitive detection of different water pollutants.
+&nbsp;&nbsp;Finally, aromatic compounds are the main components of water pollutants, and we hope that other iGEM teams can use this basic part to achieve highly sensitive detection of different water pollutants.
 </html>
 ===Model===
 <html>
-<br>
+&nbsp;&nbsp;Our modeling analyses of the <i>yqjF</i>&nbsp;(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter showed that DNT could induce the <i>yqjF</i>&nbsp;(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter, and there was a three-way relationship between the fluorescent protein expression and DNT concentration, with an R<sup>2</sup> of 0.990, which was higher than 0.7. Thus, our proposed model could well fit the experimental data, suggesting that there was a strong correlation between fluorescent protein expression and DNT concentration , and the 95% confidence interval for the slope of the data was 1263.861-1716.902, with a slope of the p-value 0.000. Therefore, the difference between the slope value and 0 was statistically significant, and there was a strong linear relationship between fluorescent protein expression and DNT concentration(Fig. 1)
-Our modeling analyses of the <i>yqjF</i>(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter showed that DNT could induce the <i>yqjF</i>(<a href="https://parts.igem.org/Part:BBa_K1316002">BBa_K1316002</a>) promoter, and there was a three-way relationship between the fluorescent protein expression and DNT concentration, with an R<sup>2</sup> of 0.990, which was higher than 0.7. Thus, our proposed model could well fit the experimental data, suggesting that there was a strong correlation between fluorescent protein expression and DNT concentration , and the 95% confidence interval for the slope of the data was 1263.861-1716.902, with a slope of the p-value 0.000. Therefore, the difference between the slope value and 0 was statistically significant, and there was a strong linear relationship between fluorescent protein expression and DNT concentration(Fig. 1)
 <img src="https://static.igem.org/mediawiki/parts/e/e1/T--NEFU_China--%E5%BB%BA%E6%A8%A11.png
@@ Line 45: / Line 45: @@
 ===Characterization and Measurement===
 <html>
-<br>
+&nbsp;&nbsp;We amplified the wild-type <i>yqjF </i>promoter from the genomic DNA of bacteria, inserted the <i>EGFP</i>, as a reporter, at its downstream, and thus generated the reporter plasmid pYB1a-<i>yqjF</i>-<i>EGFP</i>. This reporter was transformed into  <i>Escherichia Coli DH5α</i> for the following experiments. We then tested the induction of the reporter system by different concentrations of DNT (20,&nbsp;25,&nbsp;30,&nbsp;40&nbsp;and&nbsp;50&nbsp;mg/L). EGFP intensity in response to DNT changes was shown in Fig. 2. The results indicated that the pYB1a-<i>yqjF</i>-<i>EGFP</i> system could be induced by DNT; However, when the DNT concentration dropped to 20 mg/L, the system showed no response, suggesting that the DNT detection limit of the wild-type yqjF promoter was around 25 mg/L, consistent with the reported value in previous literaturer&nbsp;&nbsp;<sup>[1]</sup>.
-Previously reported detection limit of the<i> yqjF</i> promoter is as high as 25 mg/L of DNT<sup>[1]</sup>(Fig.2), thus, we must improve the sensitivity of the <i>yqjF</i> promoter for its practical application.
 <div>
@@ Line 56: / Line 56: @@
 <br>
 <p>
-Based on the data above, we need to optimize our system and improve the sensitivity of the reporter expression induced by DNT. For this purpose, we will focus on the following aspects, including (1) modifying the essential elements of the<i> yqjF</i> promoter to enhance transcription strength, (2) promoting the conversion from DNT to THT, which is the key molecule to trigger the promoter<sup>[2]</sup>, and (3) optimizing the transcription activator YhaJ to elevate its responsiveness to the inducer THT.(3) optimizing the transcription activator YhaJ to elevate its responsiveness to the inducer THT<sup>[3]</sup>.
+&nbsp;&nbsp;Based on the data above, we need to optimize our system and improve the sensitivity of the reporter expression induced by DNT. For this purpose, we will focus on the following aspects, including (1) modifying the essential elements of the<i> yqjF</i> promoter to enhance transcription strength, (2) promoting the conversion from DNT to 2,4,5-trihydroxytoluene (THT), which is the key molecule to trigger the promoter&nbsp;&nbsp;<sup>[2]</sup>, and (3) Optimizing the transcription activator YhaJ to elevate its responsiveness to the inducer THT&nbsp;&nbsp;<sup>[3]</sup>.
 </p>
 <br>
-<br>
+<p> &nbsp;&nbsp;(1) Mutagenesis study of the <i>yqjF</i> promoter:&nbsp;Using site-direct mutagenesis, we combined the mutations of the<i>yqjF1st </i>and <i>yqjF2nd</i> (<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a>and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>)  to generate a newer version of the promoter, designated as the <i>yqjF3rd</i> promoter. We tested the response of the reporter containing the <i>yqjF3rd </i>promoter to the induction of different concentrations of DNT (2.5,&nbsp;5,&nbsp;10,&nbsp;15&nbsp;and&nbsp;20 mg/L), and found that the <i>yqjF3rd</i> promoter showed a DNT detection threshold of 5 mg/L (Fig. 3). Thus, with the combination of the <i>yqjF1st </i>and <i>yqjF2nd</i>, we generated the <i>yqjF3rd </i>promoter that exhibited 5-fold increase of DNT detection sensitivity compared to the wild-type<i> yqjF</i> promoter.
-<p> (1)	Mutagenesis study of the <i>yqjF</i> promoter:&nbsp;Using site-direct mutagenesis, we combined the mutations of the<i>yqjF1st </i>and <i>yqjF2nd</i> (<a href="https://parts.igem.org/Part:BBa_K3350859">BBa_K3350859 </a>and <a href="https://parts.igem.org/Part:BBa_K3350860">BBa_K3350860</a>)  to generate a newer version of the promoter, designated as the <i>yqjF3rd</i> promoter. We tested the response of the reporter containing the <i>yqjF3rd </i>promoter to the induction of different concentrations of DNT (2.5, 5, 10, 15 and 20 mg/L), and found that the <i>yqjF3rd</i> promoter showed a DNT detection threshold of 5 mg/L (Fig. 3). Thus, with the combination of the <i>yqjF1st </i>and <i>yqjF2nd</i>, we generated the <i>yqjF3rd </i>promoter that exhibited 5-fold increase of DNT detection sensitivity compared to the wild-type<i> yqjF</i> promoter.
 </p>
 <div>
@@ Line 70: / Line 68: @@
 <br>
 <p>
-(2)Promoting the DNT-to-THT conversion:&nbsp;Based on previous literature, the <i>yqjF</i> promoter is likely activated by THT, which is a metabolite of DNT, and when binding to THT, the transcription factor yhaJ can activate the <i>yqjF</i> promoter. Based on these evidence, we generated three types of bacteria to further understand the connection between the metabolic processes of DNT and its regulatory mechanism. The <i>nemA-nfsA-nfsB</i> genes are responsible to convert DNT to THT. However, when we overexpressed the<i> nemA-nfsA-nfsB </i> (<a href="https://parts.igem.org/Part:BBa_K1316006">BBa_K1316006</a>)genes and simultaneously introduced the<i> yhaJ </i>gene in the engineered bacteria, the fluorescence intensity was markedly reduced compared to the bacteria carrying the <i>yhaJ</i> gene alone(Fig.4). We predicted that the co-expression of all these proteins could cause undetermined conflicts or overload the bacteria, and thus the combination of the two strategies would not be able to further increase the sensitivity.
+&nbsp;&nbsp;(2) Promoting the DNT-to-THT conversion:&nbsp;Based on previous literature, the <i>yqjF</i> promoter is likely activated by THT, which is a metabolite of DNT, and when binding to THT, the transcription factor yhaJ can activate the <i>yqjF</i> promoter. Based on these evidence, we generated three types of bacteria to further understand the connection between the metabolic processes of DNT and its regulatory mechanism. The <i>nemA-nfsA-nfsB</i> genes are responsible to convert DNT to THT. However, when we overexpressed the<i> nemA-nfsA-nfsB </i> (<a href="https://parts.igem.org/Part:BBa_K1316006">BBa_K1316006</a>) genes and simultaneously introduced the<i> yhaJ </i>gene in the engineered bacteria, the fluorescence intensity was markedly reduced compared to the bacteria carrying the <i>yhaJ</i> gene alone (Fig. 4). We predicted that the co-expression of all these proteins could cause undetermined conflicts or overload the bacteria, and thus the combination of the two strategies would not be able to further increase the sensitivity.
 </p>
 <div>
@@ Line 79: / Line 77: @@
 <br>
 <p>
-(3)Overexpression of the transcription activator YhaJ:&nbsp;We further investigated whether we could optimize the transcription activator YhaJ to further improve the detection sensitivity. Employing random mutagenesis approach, we generate the YhaJ mutant library and screen over 500 clones to evaluate their effects on <i>yqjF</i> promoter-mediated EGFP expression in response to 10 mg/L DNT. we discovered that one YhaJ mutant showed markedly increased EGFP production compared to the wild-type YhaJ. Based on DNA sequencing analysis, this mutant, designed as  <i>yhaJ1st</i> (<a href="https://parts.igem.org/Part:BBa_K3350858">BBa_K3350858 </a>). We overexpressed <i>yhaJ1st</i> in the engineered bacteria, we reduced the DNT detection threshold from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity(Fig.5).
+&nbsp;&nbsp;(3) Overexpression of the transcription activator:&nbsp;We further investigated whether we could optimize the transcription activator YhaJ to further improve the detection sensitivity. Employing random mutagenesis approach, we generate  <i>yhaJ1st</i> (<a href="https://parts.igem.org/Part:BBa_K3350858">BBa_K3350858</a>),and we overexpressed <i>yhaJ1st</i> in the engineered bacteria. We reduced the DNT detection threshold from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity (Fig.5).
 </p>
 <div>
 <img src="https://static.igem.org/mediawiki/parts/8/86/T--NEFU_China--%E9%98%88%E5%80%BC%E9%99%8D%E4%BD%8E%E5%88%B00.1%281%29.png
 " style="width:35%;height=35%; "></img></div>
-Fig. 5. Evaluation of overexpression of <i>yqjF3rd</i> and <i>YhaJ1st</i>.
+Fig. 5. <i>YhaJ1st</i> overexpression could remarkably reduce the DNT detection threshold to 0.1 mg/L.
 </html>
 ===References===
-[1]S. Yagur-Kroll, S. Belkin et al., “<i>Escherichia Coli </i>bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene”, Appl. Microbiol. Biotechnol. 98, 885-895, 2014.
+[1]Yagur-Kroll, S., Lalush, C., Rosen, R., Bachar, N., Moskovitz, Y., & Belkin, S. (2014). Escherichia coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene. Applied microbiology and biotechnology, 98(2), 885–895.
 <br>
 <br>
-[2]González-Pérez MM, van Dillewijn P, Wittich RM, Ramos JL. <i>Escherichia Coli </i> has multiple enzymes that attack TNT and release nitrogen for growth. Environ Microbiol. 2007 Jun;9(6):1535-40.
+[2]González-Pérez, M. M., van Dillewijn, P., Wittich, R. M., & Ramos, J. L. (2007). Escherichia coli has multiple enzymes that attack TNT and release nitrogen for growth. Environmental microbiology, 9(6), 1535–1540. <html>https://doi.org/10.1111/j.1462-2920.2007.01272.x</html>
 <br>
 <br>
-[3]Palevsky N, Shemer B, Connolly JP, Belkin S. The Highly Conserved<i>Escherichia Coli </i> Transcription Factor YhaJ Regulates Aromatic Compound Degradation. Front Microbiol. 2016 Sep 22;7:1490.
+[3]Palevsky, N., Shemer, B., Connolly, J. P., & Belkin, S. (2016). The Highly Conserved Escherichia coli Transcription Factor YhaJ Regulates Aromatic Compound Degradation. Frontiers in microbiology, 7, 1490.