Caffeine disk results
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Caffeine Disk Results. Spring 2014. Procedure. Grow clone from glycerol stock Amp or amp + chlor 4 mM caffeine Measure A590 and standardize to 0.1 or 0.01 Spread 50 ul on Tet only plate with beads Prepare disks Commercial disks 35 ul solution per disk Up to 100 mM caffeine

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Caffeine Disk Results

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Caffeine disk results

Caffeine Disk Results

Spring 2014


Procedure

Procedure

  • Grow clone from glycerol stock

    • Amp or amp + chlor

    • 4 mM caffeine

  • Measure A590 and standardize to 0.1 or 0.01

  • Spread 50 ul on Tet only plate with beads

  • Prepare disks

    • Commercial disks

    • 35 ul solution per disk

    • Up to 100 mM caffeine

  • Place disks

    • Use sterile insulin needle and sterile tip

  • Incubate

    • 37 C overnight, then room temp for several days


Amp plasmid

Amp Plasmid

  • J119346

    • High promoter

    • High C-dog

    • RFP


Amp plasmid1

Amp Plasmid

  • J119347

    • Low promoter

    • Low C-dog

    • GFP


20 clones

20 Clones

  • Amp Plasmid

    • Promoter – C dog

      • High-High versus Low-Low

    • Origin

      • pSB1A2 High copy number

      • J119310 Low copy number

  • Chlor plasmid, pSB4C5

    • Chaperones

      • pG-Tf2

      • pTf16

      • pG-KJE8

      • pGro7

      • pKJE7


Too many bacteria a590 1 0

Too many bacteria, A590 = 1.0


Rfp on all 20

RFP on All 20


Origin pcr

Origin PCR

All are pSB1A2 All are J119310

with 500 bp with 750 bp


Chaperone pcr

Chaperone PCR

Marker

  • pG-Tf2

  • pTf16

  • pG-KJE8

  • pGro7

  • pKJE7


Procedure1

Procedure

  • Do Caffeine disk experiments with 0, 20, 40, 60, 100 mM

  • Take Pictures

  • Use loop to take colonies from near the disksand streak onto plates for colony isolation

  • Pick individual colonies and measure

    • RFP on Cytation

    • Chaperone PCR

    • Origin PCR


All 20 clones

All 20 Clones


High high vs low low

High-High vs. Low-Low


Origin pcr1

Origin PCR


Chaperone pcr1

Chaperone PCR


Genotypes

Genotypes


Sorted genotypes

Sorted Genotypes


Caffeine disk results 2615045

Grown only overnight at 37 C


1 20 with caffeine disks

1 + 20 with Caffeine Disks


Fitness clocks

Fitness Clocks


1 20 with caffeine disks1

1 + 20 with Caffeine Disks

0 mM

20 nM

40 mM

60 mM

80 mM

100 mM


Biosensor expts

Biosensor Expts


Too many bacteria a590 1 01

Too many bacteria, A590 = 1.0


Rfp and gfp on all 20

RFP and GFP on All 20


1 20 with theo disks

1 + 20 with Theo Disks


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