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Team:Cambridge-JIC/Constructs/progress template
From 2014.igem.org
Chromoprotein Constructs
Design
Aim of the construct
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Chromoproteins represent ideal reporter genes. Unlike fluorescent protein, from which chromoproteins were originally derived, they require no specialised optical equipment for detection. Our aim is to use a selection of such proteins, donated by the iGEM 2012 Uppsala team, as our most basic and main output in regards to the Marchantia framework.
- 35s - eforRed - nosT
- 35s - eforRed - N7 - nosT
- 35s - amilCP - N7 - nosT
- 35s - tsPurple - nosT
- 35s - tsPurple - N7 - nosT
- 35s - asPink - N7 - nosT
- 35s - aeBlue - N7 - nosT
We decided to test the following chromoprotein constructs:
By expressing the chromoproteins in the pGreen vector with the 35s promoter and the nosT terminaor, we control for the possiblity of the promoter/terminator not functioning in our chassis. The N7 fragment allows for the selected constructs to localise chromoprotein to the nucleus. It is believed by Bernardo Pollak that this may end up increasing the likelihood of detecting the chromoproteins with the naked eye.
End goal plasmid
- insert image here
Experimentation (up to date)
PCR
Attempt 1:- Backbone Fragment 1 (35s - Hyg promoter): B14, P16, P14. Tubes: CpI 1
- Backbone Fragment 2(Hyg promoter - nosT): B14, P15, P17. Tubes: CpI 2
- N7 fragment: B15, P13, P22. Tubes: CpI 3
- eforRed gene(cytoplasmic): B13, P1, P2. Tubes: CpI 4
- eforRed gene(nuclear): B13, P1, P3. Tubes: CpI 5
- amilCP gene(nuclear): B10, P4, P5. Tubes: CpI 6
- tsPurple gene(cytoplasmic): B12, P6, P7. Tubes: CpI 7
- tsPurple gene(nuclear): B12, P6, P8. Tubes: CpI 8
- asPink gene(nuclear): B9, P9, P10. CpI 9
- aeBlue gene(nuclear): B11, P11, P12. Tubes: CpI 10
Date: 18.07.2014
UIDs of primers/plasmids used:
After running the gel, the concentrations of CpI 1, CpI 2 and CpI 3 were much lower compared to the chromoprotein genes (see gel picture). Amplification of CpI 3 failed. The problem was an alteration to the primers necessary for amplification. Re-ran PCR for CpI 3. Modification: Appropriate primers (P13 & P22) and the extension phase of PCR has been reduced from 2 min to 15 sec since N7 is only ~300bp long. The DNA was then purified using the QIAGEN QiaQuick Gel Purification Kit. The DNA was eluted in 50 ul of EB buffer. Due to the large elution volume and the lower yield of the backbone DNA, the concentrations (measured using Nanodrop) were lower than the suggested 10ng/ul threshold for Gibson.
Attempt 2:
- Backbone Fragment 1 (35s - Hyg promoter): B14, P16, P14. Tubes: CpII 1
- Backbone Fragment 2(Hyg promoter - nosT): B14, P15, P17. Tubes: CpII 2
- N7 fragment: B15, P13, P22. Tubes: CpII 3
- eforRed gene(cytoplasmic): B13, P1, P2. Tubes: CpII 4
- eforRed gene(nuclear): B13, P1, P3. Tubes: CpII 5
- amilCP gene(nuclear): B10, P4, P5. Tubes: CpII 6
- tsPurple gene(cytoplasmic): B12, P6, P7. Tubes: CpII 7
- tsPurple gene(nuclear): B12, P6, P8. Tubes: CpII 8
- asPink gene(nuclear): B9, P9, P10. CpII 9
- aeBlue gene(nuclear): B11, P11, P12. Tubes: CpII 10
Date: 21.07.2014
UIDs of primers/plasmids used:
Due to the low yield of the DNA purification fro Attempt 1, the entire PCR was repeated. Upon gel separation we realised the PCRs were nearly identical (see Gel Picture), which is what we were expecting. However, the DNA was euted into 10ul of EB buffer instead of 50ul during the gel purification step, which increased the concentrations of CpII 4-10 significantly. However, CpII 1-3 concentrations were still below the 10 ng DNA/ul range.
- Backbone Fragment 1 (35s - Hyg promoter): B14, P16, P14. Tubes: CpIII 1
- Backbone Fragment 2 - Full Size(Hyg promoter - nosT): B14, P15, P17. Tubes: CpIII 2 RBLB
- Backbone Fragment 2 - LB(Hyg promoter - KanR): B14, P18, P17. Tubes: CpIII 2 LB
- Backbone Fragment 2 - RB(KanR - nosT): B14, P15, P19. Tubes: CpIII 2 RB
- N7 fragment: B15, P13, P22. Tubes: CpIII 3
Date: 22.07.2014
UIDs of primers/plasmids used:
The backbone fragments were re-aplified to allow for future assemblies to be made. The PCR was altered to run for 35 amplification cycles instead of 30, which increase the yield of DNA.
