Team:Cambridge-JIC/Progress

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Cambridge iGEM 2014

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Labwork

Status, protocols and safety considerations for our everyday wetwork

Chromoprotein Constructs

Design

Aim of the construct

    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.

    We decided to test the following chromoprotein constructs:

    • 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

    By expressing the chromoproteins in the pGreen vector with the 35s promoter and the nosT terminator, 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:

    Date: 18.07.2014

    UIDs of primers/plasmids used:

    • 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

    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.

PCR Excell Protocol

Gel Picture


Attempt 2:

    Date: 21.07.2014

    UIDs of primers/plasmids used:

    • 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

    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.

PCR Excell Protocol

Gel Picture

Attempt 3:

    Date: 22.07.2014

    UIDs of primers/plasmids used:

    • 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

      • 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.

    PCR Excell Protocol

    Gel Picture

Gibson

Attempt 1:

  • Date: 21.07.2014
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E-Coli transformation

Attempt 1:

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Agrobacteria transformation

Attempt 1:

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Spore preparation

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Spore transformation

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Evaluation

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Gal4 - Hap1GR - H2BmRFP

Design

Aim of the construct

  • We'll try out 35s->GAL4->HAP1GR->H2BmRFP.
  • Since H2BmRFP has not been tried in Mp, we'll also try 35s->H2BmRFP. A positive control for lab technique will be 35s->Venus N7
  • We'll also use FF's original plasmid to try 35s-Hap1GR-mRFP, as a control for the Hap1GR. I expect low transformation rate as in the original plasmid, we're using 35s as promoter for HygR selection marker.
  • Not sure whether 35s->Gal4 has been tried (check with BP), so 35s-Gal4-venus n7 is doable with BP's construct as a control
  • As another control, we'll try 35s-Gal4-H2mRFP

End goal plasmid

Experimentation (up to date)

PCR

Attempt 1:

Gibson

Attempt 1:
  • Start date/time: 25/07
  • Comments: seemed to be fine.

E-Coli transformation

Attempt 1:
  • Start date/time: 25/07
  • Comments:
Negative control negative, positive control positive.

Selection

Attempt 1:
  • Start date/time: 28/07
  • Comments:
Selected initially just 3 colonies. Grew them in LB and miniprepped on 27/07. Performed digest with SalI to check for template carryover and see that assembly was done. DEXX3, pb>RVe each have a unique site. Following digest with Sal1, we should have a 12kb fragment and a 2kb fragment. After digest, 2kb fragment was visible, but 12kb fragment was not. Very mysterious.
Some error in the digestion protocol is likely to have occurred.
To insert a promoter, a digest with Kpn1 was performed. Gel was illegible, although a faint band at 13kb may have been visible. No extraction was made due to poor quality of the gel. Attempt 2:
  • Start date/time: 30/07
  • Comments:
Grew up in LB 8 more colonies. Attempted colony PCR using inoculation loop in dry PCR tube - will wait to see results from GEL. Expect an 8kb fragment: unlikely that this will show results. Will perform a SalI digest with correct protocol on 31/07 following miniprep from colonies.

Agrobacteria transformation

Attempt 1:
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Spore preparation

Attempt 1:
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Spore transformation

Attempt 1:
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Evaluation

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