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Team:INSA Toulouse/contenu/lab practice/notebook/calendar/red sensor
From 2013.igem.org
Calendar
Red Light Sensor Characterization
The first necessary construction for red light sensor is the following:
Ho1 and PcyA are two genes required for phycocyanobilin (PCB) biosynthesis. PCB associates with EnvZ fusion protein, Cph8, to transduce a light signal into a genetic response.
The light sensor Cph8 should be inhibited by tetR and expressed in presence of aTc (anhydrotetracycline). Atc binds to TetR lift the inhibition, allowing Cph8 production.
The second necessary construction for red light sensor characterization is the following:
This is a test Biobrick for the Red light responsive system with the RFP protein as output. The RFP is positively regulated by OmpR-controlled promoter. Phosphorylated OmpR binds to the operator sites and activates transcription of RFP.
EnvZ phosphorylates OmpR to OmpR-P that activate the promoter.
June 2013
- Week 3 (24-30 June)
Amplification of new biobricks for cloning :
BBa_I15008: ho1,
BBa_K081017: rbs PcyA term,
BBa_K592018: rbs Cph8,
BBa_R0082: pOmpc
We tested a new method for midipreps using kits: alkaline lysis with spin columns. Results were significantly better in terms of purity. Cloning can start!!!
July 2013
- Week 4 (1-7 July) 07/01
- Week 5 (8-14 July) 07/08
- Week 6 (15-21 July) 07/15
- Week 7 (22-28 July) 07/23
Planning of cloning and study of the available methods for biobricks assembly.
07/02 First cloning
We decided to start the construction for the characterisation and the light responsive domain. Assembly of first biobricks using iGEM 3A Assembly method:
- ptet + rbs Cph8 (2318 bp) in PSB1K3
- pOmpc + rbs RFP term (890 bp) in PSB1K3
Restriction of parts, ligation and then transformation in DH5 α.
On the electrophoresis after restriction pOmpc and pTetr digested plasmids seems to be at lower concentration than the other.
07/03
Bacterial layers were obtained: transformation with the remaining 5 µL ligation of 02/07.
07/04
2 mL liquid cultures of 4 clones for each cloning.
Miniprep after 8 hours of incubation.
The electrophoresis of the digested plasmids with EcoRI and PstI did not confirm the presence of wanted constructions for all clones.
Cloning for red light sensor characterisation keeps going. New round of 3A cloning:
- ptet + rbs Cph8 (2318 bp) in PSB1K3
- pOmpc + rbs RFP term (890 bp) in PSB1K3
07/09
2 mL liquid cultures of 6 clones for each cloning and then minipreps.
0,8% gel was run to confirm cloning success.
07/10A 1,5% gel was also run as reference to confirm the assembly. Small parts like promoters had been indeed added only with the RFP.
Cloning failed for ptet + rbs-Cph8, it seems to be only Cph8 without ptet.
07/11
Standby because of a lack of plasmids backbones...
Because a lack of plasmids backbones, assembling of rbs Cph8 (PSB1C3) into ptetR (PSB1A3).
Transformation and spreading on petri dish with ampicillin.
07/16
2 mL liquid cultures of 6 clones for each cloning and then minipreps.
0,8% gel was run to confirm cloning success.
07/18
Plasmids backbones are available again. Changing of vector for ho1 from the PSB1C3 to PSB1A3 after EcoRI/PstI restriction.
07/19
2 mL liquid cultures of 6 clones and then minipreps.
0,8% gel was run to confirm cloning success. Ho1 in pSB1A3 is now available.
Sample confirmed by restriction was send to sequencing:
- ptet + rbs Cph8 in PSB1A3
- pOmpc + rbs RFP term in PSB1K3
07/24
Assembling of PcyA (PSB1C3) into ho1 (PSB1A3).
Transformation and spreading on petri dish with ampicillin.
07/252 mL liquid cultures of 6 clones and then minipreps.
0,8% gel was run to confirm constructions by restriction.
07/26
Assembling of TetR (PSB1C3) into ho1 PcyA (PSB1A3).
Transformation and spreading on petri dish with ampicillin.
August 2013
- Week 8 (29-4 August) 07/29
- Week 9 (5-11 August) 08/06
- Week 10 (12-18 August)
- Week 11 (19-25 August) 08/20
- Week 12 (26-31 August) 08/26
2 mL liquid cultures of 6 clones and then minipreps.
0,8% gel was run to confirm constructions by restriction.
There was not enough DNA in the miniprep.
Culture overnight for DNA amplification.
07/30
Assembling of ho1 PcyA TetR (PSB1A3) into strong promoter and strong rbs (PSB1C3).
Transformation and spreading on petri dish with chloramphenicol.
08/01
Result of sequencing confirmed cloning success for pOmpc + rbs RFP term but not for ptet + rbs Cph8.
2 mL liquid cultures of 6 clones and then minipreps.
0,8% gel was run to confirm constructions by restriction but it failed
08/02
New try to cloning of ho1 PcyA TetR into strong promoter strong rbs (spsr) and strong promoter and medium rbs (spmr).
Minipreps then 0,8% gel : ho1 PcyA TetR in spmr success, others failed.
08/07
Others verifications by restriction :
- Linearization with EcoRI
- Linearization with AvrII (cutting into strong promoter and medium rbs)
- Restriction with AvrII and PstI.
Restriction confirmed the construction
08/08
Cloning for red light sensor characterisation keeps going. New round of cloning:
- spmr ho1 PcyA TetR in pOmpC rbs RFP term
- rbs Cph8 in ptetR
08/09
Minipreps and verification of constructions by restriction.
0,8% gel confirmed cloning success
08/12Further verifications by restriction :
- Xho I confirmed ptet + Cph8 cloning success
- BstXI et SnabI invalid spmr ho1 PcyA TetR in pOmpC rbs RFP term cloning
08/13
Assembling of pOmpc rbs RFP Term (PSB1K3) into ptet rbs Cph8 term (PSB1A3).
Restriction of parts, ligation and then transformation in DH5-1.
08/14
Minipreps and 0.8% gel: cloning success.
Assembling of pOmpc rbs RFP term ptet rbs Cph8 term (PSB1A3) in spsr ho1 PcyA TetR to obtain the final characterisation construction.
08/21
4 mL liquid cultures overnight of 6 clones.
08/22
Minipreps and 0.8% gel: cloning failed.
New tentative of cloning.
08/27
4 mL liquid cultures overnight of 6 clones.
08/28
Minipreps and 0.8% gel: cloning success after EcoRI and PstI restriction.
08/29
Further verifications by restriction :
- Xho I confirmed the presence of ptet Cph8
- BstXI confirmed the presence of ho1 and mrfp
- SnabI confirmed the presence of PcyA and TetR
September 2013
- Week 13 (1-8 September)
09/01
For the characterisation a transformation in ΔEnvZ cells is necessary. But due to time limitation, the experiences stopped. The parts were not further characterized.
