July, 5th

LB agar plates prepared:

  • LB+Amp 100 (500ml)
  • LB+Cm 34 (500ml)
  • LB (250ml)
  • LB+Cm 12.5 (250ml)

July, 6th

We decided to perform a TECAN test to evaluate the strength of some promoters belonging to the Anderson Promoters Collection. Inoculum from glycerol stocks (8ul in 5ml LB) for:

  • <partinfo>BBa_J23101</partinfo>
  • <partinfo>BBa_J23110</partinfo>
  • <partinfo>BBa_J23116</partinfo>
  • <partinfo>BBa_J23118</partinfo>
  • <partinfo>BBa_J23114</partinfo>
  • <partinfo>BBa_B0033</partinfo>

TECAN experiment started at 10:20am.

In the morning one single colony was picked for BW53474 E. coli strain and glycerol stock was prepared after about 7 hours (37°C 220rpm)

Inoculum of:

  • BW53474 (falcon containing culture for glycerol stock was re-filled with 5ml LB)
  • MG1655 (from glycerol stock)
  • BW25141 (from glycerol stock)
  • BW25142 (from glycerol stock)

July, 7th

Competent cells preparation for:

  • MG1655
  • BW53474
  • BW25141
  • BW25142

PCR was performed to amplify Phasins:

All parts were correct!! :)

July, 8th

Transformation of 1ul (~4ng) of miniprepped <partinfo>BBa_J23118</partinfo> into 100ul of home-made competent cells

  • MG1655
  • BW25141
  • BW25142
  • BW53474
  • DH5-alpha (as control)

to verify the transformation efficiency.

Transformed bacteria were plated on LB+Amp and left overnight in oven, 37°C.

July, 9th

We checked the presence of colonies in plates. All plates showed red colonies!

MG1655 plate (with satellite colonies)
BW25141 plate
BW25142 plate
BW53474 plate
DH5-alpha plate

We calculated (thanks Nicolò) efficiency as #colonies/ug DNA

Strain #colonies Efficiency
MG1655 379 ~10^5
BW25141 1431 ~10^5
BW25142 2374 ~10^5
BW53474 9984 ~10^6
(very difficult to count correctly)
DH5alpha 7475
(only 100ul of cells plated)
~ 10^6
(very difficult to count correctly,
previous tests showed an efficiency of ~10^8)