Team:ETH Zurich/Decoder/Results
From 2012.igem.org
Contents |
Hybrid promoters
Pre-decoder
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 pSEVA183-LacI-tetR plasmid to test our construct for 4 distinct expression conditions.
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
![](/wiki/images/3/37/Pre-Decoder_Results.png)
Single cell analysis revealed that the repression by LacI and TetR occurs and that the promoters work in absence of the repressors.
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.
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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