Team:Cambridge/Notebook/Week2

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Project :

Overview Sensitivity Tuner --- Characterisation --- Modelling Colour Generators --- Carotenoids (Orange/Red) --- Melanin (Brown) --- Violacein (Purple/Green) The Future Safety

Notebook :

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10

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The Project

Monday 20 July

Finalized our project into a few objectives

• Pigment production
  • Transform E. coli to produce pigment
  • Hook up the pigmentation genes to inducible promoters
• Shutter mechanism
  • build, test a shutter mechanism
  • Hook up pigmentation genes under inducible promoters to the shutter mechanism
• Improve on previous iGEM projects
  • arsenic sensor?
  • bacterial photography?
• Modelling
  • shutter mechanism
  • Growth rate regulation

Goals for Wet Work

• Inducible pigment production integrated with a shutter mechanism
• Possibly attach the arsenic bio-sensor to our green/red pigment output

Tuesday 21 July

Proceedure to make competant cells started

• One colony of TOP10 E. coli added to 50ml of LB broth. Incubated overnight in the shaking incubator

Planned and prepared for transformation

• Media made up
  • Two litres of 0.1 mM HEPES
  • One litre of LB broth (for making competant cells)
  • 100ml of 10% glycerol
• Ampicillin made up with water to make 8 x 1ml aliquots of 100mg/ml
• 15 LB agar and 100ug/ml Ampicillin plates made up

Dry work

Plan for tomorrow

• prepare trimethoprim stock solution
• make LB agar and trimethoprim plates as well as LB agar and copper + tyrosine plates
• Complete proceedure for making competant cells (transfer bacteria into big flask, wash with HEPES, and aliquot into glycerol)
• Attempt transformations by electroporation
  • pPSX plasmid (violet)
  • melA plasmid (brown)
  • biobricks

Wednesday 22 July

competent cell procedure

• completed (see protocol page for details of the procedure)

planned and prepared for electroporation tomorrow

• made up 4 LB agar and 100ug/ml Ampicillin, 0.5ug/mL tyrosine, 15 ug/mL CuSO4 plates
• plan to transform TOP10 competent bacteria with
  • melanin plasmid, pTRCmelA plasmid --plate onto tyrosine/copper/ampicillin plate
  • violacein plasmid,Plasmid pPSX-Vio+ -- plate onto trimethoprim plate
  • orange biobrick, BBa_K152005 -- plate onto ampicillin plate
  • promoter, R0011 -- plate onto ampicillin plate
  • more?
• need to make trimethoprim stock solution 10mg/mL acetone, and plate at 50ug/mL
  • tried to make the stock solution today, couldn't get the solid trimethoprim to dissolve in acetone
• need to work out exact volumes of plasmid to be added to the cell (concentrations written on the side of the bottles)

Thursday 23 July

Electroporation

• transformed Top10 competent cells, incubated the following plates:
  • Top10 + pTRCmelA plasmid on tyrosine/copper/ampicillin plate
  • Top10 + Plasmid pPSX-Vio+ on trimethoprim plate
  • Top10 + BBa_K152005 on ampicillin plate
  • Top 10 + R0011 on ampicillin plate
  • 1:10 dilutions of each as well