Difference between revisions of "Part:BBa K3350862"

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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;(3) Optimizing the transcription activator YhaJ to elevate its responsiveness to the inducer THT&nbsp;&nbsp;<sup>[3]</sup>.
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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>.
 
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(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.
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(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.
 
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(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).
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(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  <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).
  
  

Revision as of 11:47, 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 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 landmines 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, such as 2,4-dinitrotoluene (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. With 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


Previously reported detection limit of the yqjF promoter is as high as 25 mg/L of DNT [1]  (Fig. 2), thus, we must improve the sensitivity of the yqjF promoter for its practical application.

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 YhaJ: 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). 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. Evaluation of overexpression of yqjF3rd and YhaJ1st.

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.