Team:ETH Zurich/Decoder/Results

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== Dual input promoters ==
 
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We succesfully cloned the dual input promoters. Results can be found on the partsregistry.
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== Hybrid promoters ==
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== PABA generator ==
 
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[[File: Pabamap.png|frameless|250px|right|thumb|''Fig.'': Plasmid map of <partinfo>BBa_K909014</partinfo> in pSB1C3. ]]
 
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<br style="clear: both" />
 
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[[File: Ohnepromoter.png|frameless|250px|right|thumb|''Fig.'':Plasmid map of <partinfo>BBa_K909015</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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=== Pre-decoder ===
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[[File: ethzurich_Decoderpart1_pseva.png|frameless|400px|right]]
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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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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& 2048 bp
 
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*Expected bands of <partinfo>BBa_K909015</partinfo>: 2678 bp & 2048 bp
 
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[[File: Geleelelelleleleleellllethzpaba2012.png|frameless|350px|left|thumb|''Fig.1.'': 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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[[File: Experimentsetuppart1ethz2012.png|frameless|300px|right]]
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<partinfo>BBa_K909014</partinfo>  was digested by all the restriction enzymes used while <partinfo>BBa_K909015</partinfo> was not digested by NheI as expected due to the lack of the promoter (only one band on lane 3).
 
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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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=== FACS===
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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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<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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== Full decoder ==
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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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* 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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* 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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* 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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* 4. IPTG & aTc: cI and YFP are expressed. cI represses mCherry and eCFP. Yellow output.
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TOP10 cells were transformed with the construct and the content of Pab exijijefwjioaaalame <partinfo>BBa_K909014</partinfo> <partinfo>BBa_K137055</partinfo> PabA <partinfo>BBa_S04039</partinfo> PabB/C
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<div class="eth_imagetable">
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l
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{|
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==== Plans ====
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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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Cloning of <partinfo>BBa_K909015</partinfo> downstream of the P<sub>L</sub> dual input promoter <partinfo>BBa_K909011</partinfo> containing the TetO<sub>1</sub> and LacO<sub>1</sub> operator sites and implementation.
 
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<br style="clear: both" />
 
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== Outlook ==
 
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After assembling all parts for the decoder, we want to test the circuit by simulating blue and red light with aTc and IPTG. Our output system is based on fluorescent proteins (eYFP, mCherry and GFP) giving us the possibility to analyse the result by single cell analysis (FACS).
 
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[[File:Proof-of-Principle.jpg|frameless|700px|center|thumb|Figure: Boolean logic for testing of Decoder.  ]]
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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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<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|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.


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