Team:Cambridge/Notebook/Week2

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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
 +
 
 +
 
 +
 
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Revision as of 13:31, 23 July 2009


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


Retrieved from "http://2009.igem.org/Team:Cambridge/Notebook/Week2"