Difference between revisions of "Part:BBa K118011:Experience"
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====Characterization of PcstA with RFP Generator Part:BBa K081014====
 
====Characterization of PcstA with RFP Generator Part:BBa K081014====
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<img src="https://static.igem.org/mediawiki/2011/e/ee/RFP_Generator_Experience_01.jpg" width="750px" height="971px" />
 
<img src="https://static.igem.org/mediawiki/2011/e/ee/RFP_Generator_Experience_01.jpg" width="750px" height="971px" />
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===User Reviews===
 
===User Reviews===
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<partinfo>BBa_K118011 AddReview 4</partinfo>
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<I>SDU-Denmark</I>
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|width='60%' valign='top'|
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<br>
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[[File:K1135002_PcstAtranscriptionalactivity.png‎|330px|thumb|left|Transcriptional activity of PcstA during growth by measurering RNA. Negative control of MG1655Δ<i>cyaA</i> in LB, WT in LB+0.2% glucose, and WT in LB. Samples collected at different OD<sub>600</sub>measurements. Graph shows intensities of mRNA-<i>gfp</i> normalized according to intensity of 5S.]]
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<br>
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We set up to measure promotor activity of PcstA by measurering levels of <i>gfp</i>-mRNA with bacteria transformed with PcstA-<i>gfp</i> (<partinfo>BBa_K1135002</partinfo>). MG1655Δ<i>cyaA</i>, LB was used as a negative control. Samples were collected at different OD<sub>600</sub>-measurements. A single sample from the negative control was collected at OD<sub>600</sub> = 0.3. The RNA from the samples was purified and a Northen Blot with <i>gfp</i> and 5S probes was performed.
 +
<br><br>
 +
Generally during the exponential phase of the bacteria, they have a high level of transcriptional activity. However, levels of 5S rRNA are relatively constant at all times. The transcription of <i>gfp</i> increase as the cells enter exponential phase between the two OD<sub>600</sub> measurements 0.1 and 0.3. As expected, very low levels of <i>gfp</i> can be detected in the negative control. This strain lacks the ability to generate cAMP, and thus very little transcription is induced. The small amounts of <i>gfp</i> could be explained by leakiness of PcstA or that CAP alone initiates some transcription.
 +
<br><br>
 +
In the setup with WT, LB it is quite clear that the amount of <i>gfp</i> rises, compared to WT, LB+0.2% glucose. Transcription is clearly affected by the presence of glucose. One measurement WT, LB OD<sub>600</sub> = 0.8 stands out. The result is not readily explained, but is probably due to some error. But the tendency of the results correlates with the knowledge of the invert relationship between glucose and cAMP. Glucose signaling will repress adenylate cyclase-activity, thus intracellular levels of cAMP will be low in high-energy states, and little transcription of <i>gfp</i> will be initiated.
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<partinfo>BBa_K118011 2</partinfo>
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<I>WHU-China 2012</I>
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|width='60%' valign='top'|
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Firstly,by embedding the gene of RFP downstream the promoter PcstA,we have successfully certified that the promoter PcstA can be activated by CRP.In other words, it can be repressed by high concentration of glucose.
 +
What's more, we have constructed an indirect pathway by using an intermediate to change the function of the promoter. In the indirect pathway,PcstA is activated by high concentration of glucose.
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<html>
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<p align="center"><img src="https://static.igem.org/mediawiki/2012/d/d5/Indirect_regulation.png" width="500"
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height="250" hspace="2" vspace="1" align="middle" /></p>
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<p align="center"> The indirect regulatory pathway</p>
 +
After construction,the correct clones were cultured in 96-well plate at 37℃ for 24 hours,then the fluorescence and absorbance at 600 nm were recorded on a SpectraMax M2 plate reader.All fluorescence was normalized with absorbance at 600 nm.The cell density was characterized by the absorbance at 600nm,which increased with glucose concentration.The result is showed in the figure bellow. 
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<p align="center"><img name="" src="https://static.igem.org/mediawiki/2012/d/d5/Fluorescence_1n.png" width="520" height="409" alt=""></p>
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<p algin="justify">
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In this figure, the fluorescence of RFP generator promoted by PcstA decreased when concentration of glucose rose, meanwhile, the fluorescence of the indirect regulatory device increased with the glucose concentration. In another word, the indirect pathway can be activated by glucose</p>
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</html>
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For more information, please go to wiki of WHU-China 2012 on [http://2012.igem.org/Team:WHU-China/Project?catalog=2#Indirect_Pathway_Design Indirect pathway]
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<partinfo>BBa_K118011 3</partinfo>
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<I>UCL_PG 2013</I>
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<lol style="font-size:20px;">UCL_PG 2013 aka. SPECTRA</lol><br><br>
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pCSTA.RBS.mKeima.TT is compared with the preceding biobricks. We actually reconstructed pCSTA.RBS.eGFP.TT and pCSTA.RBS.mRFP.TT from 2013 distribution to test the modularity of the generator bricks. They are all successfully clone in few days.<br>
 +
During the comparison, we used a low volume of living <i>E.coli</i> for fluorescent detection. They are not lysed. The purpose is to demonstrate suitability of each fluorescent protein in tiny scale kinetic study. Please refer to our <a href="http://2013.igem.org/Team:UCL_PG/Project">project page</a> for more!
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Latest revision as of 16:11, 18 September 2015

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K118011

Preliminary tests of this part were conducted using the reporter gene xylE (BBa_K118021). Strong repression occurred in the presence of glucose, and partial repression in the presence of high concentrations of higher sugars. Results can be found [http://2008.igem.org/Team:Edinbrugh/Results/PcstA-xylE here].

Characterization of PcstA with RFP Generator Part:BBa K081014

User Reviews

UNIQ2b09f161953a3079-partinfo-00000001-QINU

••••

SDU-Denmark


Transcriptional activity of PcstA during growth by measurering RNA. Negative control of MG1655ΔcyaA in LB, WT in LB+0.2% glucose, and WT in LB. Samples collected at different OD600measurements. Graph shows intensities of mRNA-gfp normalized according to intensity of 5S.


We set up to measure promotor activity of PcstA by measurering levels of gfp-mRNA with bacteria transformed with PcstA-gfp (BBa_K1135002). MG1655ΔcyaA, LB was used as a negative control. Samples were collected at different OD600-measurements. A single sample from the negative control was collected at OD600 = 0.3. The RNA from the samples was purified and a Northen Blot with gfp and 5S probes was performed.

Generally during the exponential phase of the bacteria, they have a high level of transcriptional activity. However, levels of 5S rRNA are relatively constant at all times. The transcription of gfp increase as the cells enter exponential phase between the two OD600 measurements 0.1 and 0.3. As expected, very low levels of gfp can be detected in the negative control. This strain lacks the ability to generate cAMP, and thus very little transcription is induced. The small amounts of gfp could be explained by leakiness of PcstA or that CAP alone initiates some transcription.

In the setup with WT, LB it is quite clear that the amount of gfp rises, compared to WT, LB+0.2% glucose. Transcription is clearly affected by the presence of glucose. One measurement WT, LB OD600 = 0.8 stands out. The result is not readily explained, but is probably due to some error. But the tendency of the results correlates with the knowledge of the invert relationship between glucose and cAMP. Glucose signaling will repress adenylate cyclase-activity, thus intracellular levels of cAMP will be low in high-energy states, and little transcription of gfp will be initiated.

;

UNIQ2b09f161953a3079-partinfo-00000004-QINU

UNIQ2b09f161953a3079-partinfo-00000005-QINU

BBa_K118011 1 Not understood Kun


II09 CRP-GFP fluor different media.jpg

Cells with BBa_K200018 were grown overnight in various different media, and the GFP fluorescence was measured.

After overnight culture, the corrected fluorescence of glucose is almost negligible, showing that glucose represses the PcstA promoter strongly.

For all the other secondary carbon sources, 10% Casamino Acids in M9 shows the highest corrected fluorescence at 22000.

For more information, go to the Imperial iGEM 2009 E.ncapsulator project page on [http://2009.igem.org/Team:Imperial_College_London/Wetlab/Results/CRP_and_Media BBa_K200018 testing results]

;

UNIQ2b09f161953a3079-partinfo-00000007-QINU

UNIQ2b09f161953a3079-partinfo-00000008-QINU

BBa_K118011 2 Not understood WHU-China 2012

Firstly,by embedding the gene of RFP downstream the promoter PcstA,we have successfully certified that the promoter PcstA can be activated by CRP.In other words, it can be repressed by high concentration of glucose. What's more, we have constructed an indirect pathway by using an intermediate to change the function of the promoter. In the indirect pathway,PcstA is activated by high concentration of glucose.

The indirect regulatory pathway

After construction,the correct clones were cultured in 96-well plate at 37℃ for 24 hours,then the fluorescence and absorbance at 600 nm were recorded on a SpectraMax M2 plate reader.All fluorescence was normalized with absorbance at 600 nm.The cell density was characterized by the absorbance at 600nm,which increased with glucose concentration.The result is showed in the figure bellow.

In this figure, the fluorescence of RFP generator promoted by PcstA decreased when concentration of glucose rose, meanwhile, the fluorescence of the indirect regulatory device increased with the glucose concentration. In another word, the indirect pathway can be activated by glucose

For more information, please go to wiki of WHU-China 2012 on [http://2012.igem.org/Team:WHU-China/Project?catalog=2#Indirect_Pathway_Design Indirect pathway]

;

UNIQ2b09f161953a3079-partinfo-0000000B-QINU

UNIQ2b09f161953a3079-partinfo-0000000C-QINU

BBa_K118011 3 Not understood UCL_PG 2013

UCL_PG 2013 aka. SPECTRA

pCSTA.RBS.mKeima.TT is compared with the preceding biobricks. We actually reconstructed pCSTA.RBS.eGFP.TT and pCSTA.RBS.mRFP.TT from 2013 distribution to test the modularity of the generator bricks. They are all successfully clone in few days.
During the comparison, we used a low volume of living E.coli for fluorescent detection. They are not lysed. The purpose is to demonstrate suitability of each fluorescent protein in tiny scale kinetic study. Please refer to our project page for more!


UNIQ2b09f161953a3079-partinfo-0000000F-QINU