Team:ETH Zurich/Decoder/Results

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{{:Team:ETH_Zurich/Templates/SubmenuDecoder}}
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== PABA generator ==
 
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=== Cloning ===
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== Hybrid promoters ==
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[[File: Pabamap.png|frameless|250px|right|thumb|''Fig.1'': Plasmid map of <partinfo>BBa_K909014</partinfo> in pSB1C3. ]]
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We were succesful in constructing both parts <partinfo>BBa_K909014</partinfo> (2699bp) and <partinfo>BBa_K909015</partinfo> (2656bp) based on the already existing parts <partinfo>BBa_K137055</partinfo> and <partinfo>BBa_S04039</partinfo>. The vector backbone is <partinfo>pSB1C3</partinfo> (2079bp).
 
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They differ from each other due to the fact that <partinfo>BBa_K909014</partinfo> contains the constitutive promoter <partinfo>BBa_J23100</partinfo> with two NheI restriction sites.
 
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To verify the sizes of the constructs, the plasmids containing <partinfo>BBa_K909014</partinfo> and <partinfo>BBa_K909015</partinfo> were digested with
 
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====1. NheI & PstI ====
 
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*Expected bands of <partinfo>BBa_K909014</partinfo>: 2685 bp & 2061 bp
 
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*Expected bands of <partinfo>BBa_K909015</partinfo>: 4724 bp
 
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====2. XbaI & PstI====
 
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*Expected bands of <partinfo>BBa_K909014</partinfo>: 2721 bp & 2048 bp
 
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*Expected bands of <partinfo>BBa_K909015</partinfo>: 2678 bp & 2048 bp
 
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[[File: Ohnepromoter.png|frameless|250px|right|thumb|''Fig.2'':Plasmid map of <partinfo>BBa_K909015</partinfo> in pSB1C3.  ]]
 
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=== Pre-decoder ===
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[[File: ethzurich_Decoderpart1_pseva.png|frameless|400px|right]]
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[[File: Geleelelelleleleleellllethzpaba2012.png|frameless|350px|left|thumb|''Fig.3.'': Digests left to right: Ladder, <partinfo>BBa_K909014</partinfo> (NheI/PstI), <partinfo>BBa_K909014</partinfo> (XbaI,PstI),<partinfo>BBa_K909015</partinfo> (NheI/PstI), <partinfo>BBa_K909015</partinfo> (XbaI,PstI). ]]
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In a first step two promoters were cloned together.
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The promoter controling mCherry expression is repressible by TetR and cI while the promoter controling eCFP expression is repressible by LacI and cI. To verify the expectations we used the pSEVA-LacI-tetR to test our construct for 4 distinct expression conditions.
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[[File: Experimentsetuppart1ethz2012.png|frameless|300px|right]]
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TOP10 cells cotransformed with the pSEVA-LacI-tetR and the pre-decoder plasmid were grown in 250 mL shaking flask in LB containing  Ampicillin and Kanamycin. After a certain time, each pre-culture was aliquoted into 4 shaking flasks containing:
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* 1. No inducer: LacI as well as TetR (due to the leakiness of the tac promoter) are present and bind to the Lac & Tet operator regions. No output.
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* 2. IPTG: LacI cannot repress the tac promoter anymore, TetR is expressed. TetR represses the expression of mCherry. On the other hand the promoter controling eCFP is active. Blue output.
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* 3. aTc: LacI is expressed, TetR is not there, mCherry is expressed. eCFP cannot be produced as LacI binds to the LacO operator region. Red output.
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* 4. IPTG & aTc: mCherry as well as eCFP are expressed. Red and blue output.
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As it is visible in ''Fig.3.'' the band sizes of the <partinfo>BBa_K909014</partinfo> & <partinfo>BBa_K909015</partinfo> digestion match the expectation.
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=== FACS===
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In a further step <partinfo>BBa_K909015</partinfo> is joined to the P<sub>L</sub> hybrid promoter <partinfo>BBa_K909011</partinfo> for implementation.
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<br style="clear: both" />
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Single cell analysis revealed that the repression by LacI and TetR occurs and that the promoters work in absence of the repressors.
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== Hybrid promoters ==
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<div class="eth_imagetable">
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{|
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|[[File: 2012ethz_part1decGrowth_curve.png|frameless|300px|right|thumb|Growth curve of the cotransformed TOP10 cells. All of them grew well and similarly fast.  The time after induction was measured after aliquotation of the precultures into the 4 differently treated flasks.]]
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|[[File:New-Pre-decoder-ohne.png|frameless|300px|right|thumb|The peaks of the four conditions show the expected properties, which is also visible in the scatterplot (over 90% of each population shown). Induction with IPTG leads to eCFP expression, while mCherry is not expressed. Addition of aTc leads to mCherry expression, while no eCFP is produced. IPTG together and aTc lead to expression of both XFPs. No induction does not lead to any XFP expression. The graphs show the data set obtained 4.5 hours after induction.]]
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|-
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|}
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</div>
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This is a proof that the both promoters are working and that the operator sites can indeed be repressed by LacI and TetR. With that knowledge we could start assembling the whole decoder and check if the promoters can also be repressed by cI.
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=== Assembly part 1===
 
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In a first step two promoters were cloned together.
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== Full decoder ==
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The promoter controling mCherry expression is repressible by TetR and cI while the promoter controling eFCP expression is repressible by LacI and cI. To verify the expectations we used the following plasmid to introduce 4 distinct expression conditions:
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{|class='tablenoborder'
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| [[File: ETHZ2012decoderpart1.png|frameless|200px|right]]|[[File: Pseva 183.jpg|frameless|200px|right]]
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|}
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Condition1:  No induction: LacI as well as TetR (due to the leakiness of the tac promoter) are present and bind to the LacO & TetO operator regions.
 
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Condition2: IPTG: LacI cannot repress the tac promoter anymore, TetR is expressed. TetR represses the expression of mCherry. On the other hand the promoter controling eCFP is active.
 
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Condition3: aTc: LacI is expressed, TetR is not there, no repression of mCherry occurs. eCFP cannot be produced as LacI binds to the LacO operator region.
 
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Condition4: IPTG & aTc: mCherry as well as eCFP are expressed.
 
 +
The following construct was cloned to the first part of the decoder. By that, the third repressor cI is introduced to the system. If the promoter is turned on, cI is expressed and represses the other two hybrid promoters. To test the decoder we used the same procedure as mentioned above. The four conditions are then the following ones:
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[[File: 2012ethzDecoderodffacs.png|frameless|400px|center]]
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* 1. No inducer: LacI as well as TetR (due to the leakiness of the tac promoter) are present and bind to the Lac & Tet operator regions. All the promoters are turned off. No output.
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Single cell analysis revealed that the repression by LacI and TetR occurs and that the promoters work in absence of the repressors. The second step (full decoder assembly) was carried out after the FACS experiments.
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* 2. IPTG: LacI cannot repress the tac promoter anymore, TetR is expressed. TetR represses the expression of mCherry, cI and YFP. On the other hand the promoter controling eCFP is active. Blue output.
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[[File: 2012ethDecoderall.png|frameless|200px|right]]
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* 3. aTc: LacI is expressed, TetR is not there, mCherry is expressed. eCFP, cI and YFP cannot be produced as LacI binds to the LacO operator region. Red output.
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A first glimpse at the plates revealed already that the hybrid promoters are repressible by cI. While colonies containing only the hybrid promoters controling mCherry and eCFP were shining mainly red, the ones containing the whole decoder were not red. This means the promoter controling cI and YFP is working and repression by cI works as well.
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PICTURE FOTO!
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* 4. IPTG & aTc: cI and YFP are expressed. cI represses mCherry and eCFP. Yellow output.
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<div class="eth_imagetable">
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{|
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|[[File: ethz2012Tetlacciyfp1.png|thumb|400px|center]]
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|[[File: Decoder_wiki_ethz2012.png|thumb|240px|right|Hybrid promoters combined to the decoder.  ]]
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|}
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</div>
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[[File: Decoder_gel_2012ethz.png|frameless|350px|left|thumb|''Fig.3.'': Digests left to right: Ladder, 3 screened colonies containing the decoder plasmid.  ]]
 
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{|class='tablenoborder'
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A first glimpse at the plates revealed already that the hybrid promoters are repressible by cI. While colonies containing only the hybrid promoters controling mCherry and eCFP were shining mainly red, the ones containing the whole decoder were hardly red.  
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| [[File: Decoder_wiki_ethz2012.png|frameless|350px|left|thumb|''Fig.3.'': Digests left to right: Ladder, 3 screened colonies containing the decoder plasmid.  ]]
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| [[File: Pseva 183.jpg|frameless|400px|center]]
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|}
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<div class="eth_imagetable">
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{|
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|[[File: 2012ethDecoderall.png|frameless|250px|left|thumb| Top: decoder part1; Left: P(TetO,LacO) controling cI and YFP; Right:Final decoder.]]
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|[[File: Decoder_gel_2012ethz.png|frameless|300px|right|thumb|Left to right: Ladder, 3 screened colonies containing the decoder plasmid.  ]]
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|}
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</div>
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[[File:Proof-of-principle.jpg|frameless|700px|center|thumb|''Fig.4:'' Boolean logic for testing of Decoder.  ]]
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[[File:Proof-of-principle.jpg|frameless|700px|center|thumb|Boolean logic for testing of Decoder.  ]]
{{:Team:ETH_Zurich/Templates/Footer}}
{{:Team:ETH_Zurich/Templates/Footer}}

Latest revision as of 02:27, 27 October 2012

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Navigate through Overview, Design and Results


Contents

Hybrid promoters

Pre-decoder

Ethzurich Decoderpart1 pseva.png

In a first step two promoters were cloned together. The promoter controling mCherry expression is repressible by TetR and cI while the promoter controling eCFP expression is repressible by LacI and cI. To verify the expectations we used the pSEVA-LacI-tetR to test our construct for 4 distinct expression conditions.


Experimentsetuppart1ethz2012.png


TOP10 cells cotransformed with the pSEVA-LacI-tetR and the pre-decoder plasmid were grown in 250 mL shaking flask in LB containing Ampicillin and Kanamycin. After a certain time, each pre-culture was aliquoted into 4 shaking flasks containing:


  • 1. No inducer: LacI as well as TetR (due to the leakiness of the tac promoter) are present and bind to the Lac & Tet operator regions. No output.
  • 2. IPTG: LacI cannot repress the tac promoter anymore, TetR is expressed. TetR represses the expression of mCherry. On the other hand the promoter controling eCFP is active. Blue output.
  • 3. aTc: LacI is expressed, TetR is not there, mCherry is expressed. eCFP cannot be produced as LacI binds to the LacO operator region. Red output.
  • 4. IPTG & aTc: mCherry as well as eCFP are expressed. Red and blue output.



FACS

Single cell analysis revealed that the repression by LacI and TetR occurs and that the promoters work in absence of the repressors.

Growth curve of the cotransformed TOP10 cells. All of them grew well and similarly fast. The time after induction was measured after aliquotation of the precultures into the 4 differently treated flasks.
The peaks of the four conditions show the expected properties, which is also visible in the scatterplot (over 90% of each population shown). Induction with IPTG leads to eCFP expression, while mCherry is not expressed. Addition of aTc leads to mCherry expression, while no eCFP is produced. IPTG together and aTc lead to expression of both XFPs. No induction does not lead to any XFP expression. The graphs show the data set obtained 4.5 hours after induction.

This is a proof that the both promoters are working and that the operator sites can indeed be repressed by LacI and TetR. With that knowledge we could start assembling the whole decoder and check if the promoters can also be repressed by cI.


Full decoder

The following construct was cloned to the first part of the decoder. By that, the third repressor cI is introduced to the system. If the promoter is turned on, cI is expressed and represses the other two hybrid promoters. To test the decoder we used the same procedure as mentioned above. The four conditions are then the following ones:

  • 1. No inducer: LacI as well as TetR (due to the leakiness of the tac promoter) are present and bind to the Lac & Tet operator regions. All the promoters are turned off. No output.
  • 2. IPTG: LacI cannot repress the tac promoter anymore, TetR is expressed. TetR represses the expression of mCherry, cI and YFP. On the other hand the promoter controling eCFP is active. Blue output.
  • 3. aTc: LacI is expressed, TetR is not there, mCherry is expressed. eCFP, cI and YFP cannot be produced as LacI binds to the LacO operator region. Red output.
  • 4. IPTG & aTc: cI and YFP are expressed. cI represses mCherry and eCFP. Yellow output.
Ethz2012Tetlacciyfp1.png
Hybrid promoters combined to the decoder.


A first glimpse at the plates revealed already that the hybrid promoters are repressible by cI. While colonies containing only the hybrid promoters controling mCherry and eCFP were shining mainly red, the ones containing the whole decoder were hardly red.

Top: decoder part1; Left: P(TetO,LacO) controling cI and YFP; Right:Final decoder.
Left to right: Ladder, 3 screened colonies containing the decoder plasmid.
Boolean logic for testing of Decoder.


References

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  • Tschowri, N., Lindenberg, S., & Hengge, R. (2012). Molecular function and potential evolution of the biofilm-modulating blue light-signalling pathway of Escherichia coli. Molecular microbiology.
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