Difference between revisions of "Part:BBa K1758324"
(→Results) |
|||
| Line 20: | Line 20: | ||
| − | <figure | + | <figure> |
| − | + | <img src="https://static.igem.org/mediawiki/2015/b/b2/Bielefeld-CebiTec_in_vivo_Copper.jpeg" width="90%"><figcaption>Figure 2: The concept of our <i>in vivo</i> copper sensor (<a href="https://parts.igem.org/Part:BBa_K1758324" target="_blank">BBa_K1758324</a>), which consists of the activator under the control of a constitutive promoter (<a href="https://parts.igem.org/Part:BBa_K1758320" target="_blank">BBa_K1758320</a>) and the operator and promoter sequence of the copper inducible promoter. An untranslated region in front of the sfGFP, which is used for detection, enhances its expression (<a href="https://parts.igem.org/Part:BBa_K1758323" target="_blank">BBa_K1758323</a>). </figcaption> | |
| − | <figcaption>Figure 2: The concept of our <i>in vivo</i> copper sensor (<a href="https://parts.igem.org/Part:BBa_K1758324" target="_blank">BBa_K1758324</a>), which consists of the activator under the control of a constitutive promoter (<a href="https://parts.igem.org/Part:BBa_K1758320" target="_blank">BBa_K1758320</a>) and the operator and promoter sequence of the copper inducible promoter. An untranslated region in front of the sfGFP, which is used for detection, enhances its expression (<a href="https://parts.igem.org/Part:BBa_K1758323" target="_blank">BBa_K1758323</a>). </figcaption> | + | |
</figure> | </figure> | ||
| − | + | <figure> | |
| − | + | <img src="https://static.igem.org/mediawiki/2015/9/90/Bielefeld-CeBiTec_Biolector_copper.jpg" width="90%"> <figcaption>Figure 3: Time course of the induction of a copper biosensor with sfGFP for different copper concentrations <i>in vivo</i>. The data are measured with BioLector and normalized on OD<sub>600</sub>. Error bars represent the standard deviation of two biological replicates.</figcaption> | |
| − | + | ||
| − | <figure | + | |
| − | + | ||
| − | <figcaption>Figure 3: Time course of the induction of a copper biosensor with sfGFP for different copper concentrations <i>in vivo</i>. The data are measured with BioLector and normalized on OD<sub>600</sub>. Error bars represent the standard deviation of two biological replicates.</figcaption> | + | |
</figure> | </figure> | ||
| − | + | <figure> | |
| − | + | <img src="https://static.igem.org/mediawiki/2015/4/4e/Bielefeld-CeBiTec_Biolector_copper_Balkendiagramm.jpeg" width="90%"></a> | |
| − | <figure | + | |
| − | + | ||
<figcaption>Figure 4: Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes. </figcaption> | <figcaption>Figure 4: Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes. </figcaption> | ||
</figure> | </figure> | ||
| − | |||
| − | |||
| − | |||
<p>We tested our <i>in vivo</i> ccopper sensor with sfGFP as reporter gene, to test the functionality of the system. Moreover we tested different copper concentrations. The kinetic of our sensors response to different copper concentrations is shown in figure 3. The first 10 hours show a strong increase in fluorescence. After that the increase in fluorescence is slower. For better visualization the kinetics of figure 3 are represented as bars in figure 4. A fluorescence level difference for 60 min, 150 min and 650 min is represented.</p> | <p>We tested our <i>in vivo</i> ccopper sensor with sfGFP as reporter gene, to test the functionality of the system. Moreover we tested different copper concentrations. The kinetic of our sensors response to different copper concentrations is shown in figure 3. The first 10 hours show a strong increase in fluorescence. After that the increase in fluorescence is slower. For better visualization the kinetics of figure 3 are represented as bars in figure 4. A fluorescence level difference for 60 min, 150 min and 650 min is represented.</p> | ||
| Line 46: | Line 36: | ||
<p><i>In vivo</i> we could show that the adding different concentrations of copper has effects on the transcription levels of sfGFP.</p> | <p><i>In vivo</i> we could show that the adding different concentrations of copper has effects on the transcription levels of sfGFP.</p> | ||
| − | |||
| − | |||
<p>The shown data suggest that sensing copper with our device is possible even if the detectable concentrations are higher than the desireble sensitivity limits. Therfore we tested the copper sensor in our <i>in vitro</i> transcription translation approach.</p> | <p>The shown data suggest that sensing copper with our device is possible even if the detectable concentrations are higher than the desireble sensitivity limits. Therfore we tested the copper sensor in our <i>in vitro</i> transcription translation approach.</p> | ||
| − | + | <p>Our sensors were cultivated in the BioLector. Due to the accuracy of this device we could measure our sample in duplicates.</br></br> | |
| − | + | <figure> | |
| − | <figure | + | <img src="https://static.igem.org/mediawiki/2015/9/90/Bielefeld-CeBiTec_Biolector_copper.jpg" width="90%"></a> |
| − | + | ||
<figcaption>Time course of the induction of a copper biosensor with sfGFP for different copper concentrations in vivo. The data are measured with BioLector and normalized on OD600. Error bars represent the standard deviation of two biological replicates.</figcaption> | <figcaption>Time course of the induction of a copper biosensor with sfGFP for different copper concentrations in vivo. The data are measured with BioLector and normalized on OD600. Error bars represent the standard deviation of two biological replicates.</figcaption> | ||
</figure> | </figure> | ||
| + | |||
<p><i>In vivo</i> we could show that the adding different concentrations of copper has effects on the transcription levels of sfGFP.</p> | <p><i>In vivo</i> we could show that the adding different concentrations of copper has effects on the transcription levels of sfGFP.</p> | ||
| − | <figure | + | <figure> |
| − | + | <img src="https://static.igem.org/mediawiki/2015/4/4e/Bielefeld-CeBiTec_Biolector_copper_Balkendiagramm.jpeg" width="90%"> | |
<figcaption>Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes.</figcaption> | <figcaption>Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes.</figcaption> | ||
</figure> | </figure> | ||
<p>The shown data suggest that sensing copper with our device is possible even if the detectable concentrations are higher than the desireble sensitivity limits. Therfore we tested the copper sensor in our <a href="http://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals" ><i>in vitro</i> transcription translation approach.</a> </p> | <p>The shown data suggest that sensing copper with our device is possible even if the detectable concentrations are higher than the desireble sensitivity limits. Therfore we tested the copper sensor in our <a href="http://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals" ><i>in vitro</i> transcription translation approach.</a> </p> | ||
Revision as of 05:41, 19 September 2015
Usage and Biology
For our copper sensor we used the native operator of cooper homeostasis from E.coli K12. This includes the promoter (CopAP) and its regulator CueR. CueR is a MerR like regulator, which stimulates the transcription of CopA, a P-type ATPase pump (Outten et al. 2000). CopA is the central component in obtaining copper homeostasis, it exports free copper from cytoplasm to the periplasm. This is enable by Copper induced activation of the operon transcription via CueR. The CueR-Cu+ is the DNA-binding transcriptional dual regulator which activates transcription(Yamamoto, Ishihama 2005) To sum it up CueR regulon plays an important role in aerobic copper tolerance in E.coli(Grass, Rensing 2001).In BBa_K1758324 we combined the codon optimized CueR (BBa_K1758320) under the control of a constitutive promoter with strong RBS(BBa_K608002)with CopAP and sfGFP (BBa_K1758321) for measuring output signals. Through the addition of a 5’UTR before the sfGFP we optimized the expression of sfGFP and increased.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 475
Illegal SapI.rc site found at 625
Results
in vivo
Our sensor for copper detection consists of CueR a MerR like activator and the copper specific promoter copAP. The promoter is regulated by CueR, which binds Cu 2+-ions. We also used a sfGFP downstream the promoter for detection through a fluorescence signal.
For our copper sensor we used the native operator of cooper homeostasis from E.coli K12. We constructed a part(BBa_K1758324) using the basic genetic structur showed in Our biosensors.The operator sequence, which includes the promoter (copAP), is regulated by the activator CueR. In BBa_K1758324 we combined a codon optimized version of cueR (BBa_K1758320) under the control of a constitutive promoter with sfGFP under the control of the corresponding promoter copAP (BBa_K1758321)(figure 2). Through the addition of a 5’UTR before the sfGFP we optimized the expression of sfGFP and increased fluorescence.
We tested our in vivo ccopper sensor with sfGFP as reporter gene, to test the functionality of the system. Moreover we tested different copper concentrations. The kinetic of our sensors response to different copper concentrations is shown in figure 3. The first 10 hours show a strong increase in fluorescence. After that the increase in fluorescence is slower. For better visualization the kinetics of figure 3 are represented as bars in figure 4. A fluorescence level difference for 60 min, 150 min and 650 min is represented.
In vivo we could show that the adding different concentrations of copper has effects on the transcription levels of sfGFP.
The shown data suggest that sensing copper with our device is possible even if the detectable concentrations are higher than the desireble sensitivity limits. Therfore we tested the copper sensor in our in vitro transcription translation approach.
Our sensors were cultivated in the BioLector. Due to the accuracy of this device we could measure our sample in duplicates.
In vivo we could show that the adding different concentrations of copper has effects on the transcription levels of sfGFP.
The shown data suggest that sensing copper with our device is possible even if the detectable concentrations are higher than the desireble sensitivity limits. Therfore we tested the copper sensor in our in vitro transcription translation approach.