Revision as of 13:23, 26 July 2014 by Mjoppich (Talk | contribs)


In order to detect the pathogens fast, specifically and inexpensively we are building sensor cells to detect these pathogens. These sensor cells can identify pathogens in very low concentration by responsing to specific extracellular molecules either secreted by or displayed on the pathogens. These molecules trigger a fast fluorescence response by our immobilized sensor cells which will be measured by our device. Aachen Cellock standingup.png

More information will be available soon!



  • SOE-PCR step 2


  • we transformed some BioBricks and did a colony PCR
Aachen 14-05-23 COLONY-PCR.jpg
Colony PCR picture



  • preculture for competent NEB10β cells
  • submit samples for sequencing
  • make masterplates of transformed BioBricks
  • centrifuge and freeze overnight expression culture of J23101.E0240 and K516132


  • made competent NEB10β cells - several things went wrong.. we'll see (remember: pre-cool centrifuge, always check if it's indeed spinning, frequently check OD of the culture)
  • colony PCR on the transformed clones looked awful. There were too many cells in the 10 µl reaction volume. Some seals weren't fully closed. Basically only primers and smear except for the positive control which contained a plasmid template instead of cells
  • 2x 500 ml of LB and three sterile flasks have been made



  • plasmid preps
  • transformation of different reporter strains
    • NEB
      • pSEVE641_BsFbFP
      • pSEVA234_LasR
      • pSEVE641_BsFbFP pSEVA234_LasR
      • pSEVE641_BsFbFP pSB1C3_C0179
    • BL21
      • pSEVE641_BsFbFP pSEVA234_LasR
      • pSEVE641_BsFbFP pSB1C3_C0179


  • Philipp and Michael made 100x stocks for Hartmans media
  • Michael and Vera made about 25 HM+C-plates without Glucose
  • K731520 iLOV was plated on a HM+C plate


  • Michael made 25 new HM+C plates with 1 % agar and 4 g/L glucose
  • He also added 170 µl of the Glucose stock (500 g/L) to the Glucose-free HM+C-plates
  • 1 L of sterile HM+Glucose was prepared
  • K731520 iLOV was plated on HM+C+Glucose and HM+C+Glucose drops, 3x 5 ml HM+C+Glucose precultures were inoculated (17:00)


  • K731520 iLOV did not grow, neither on the plates, nor on in the liquid media. The most probable cause is that the E. coli is missing some vitamins
  • based upon the M9 recipes by the Knight and Endy labs on OpenWetWare, we made a 20 ml supplement stock solution that can be added to 1x HM media
Component supplement for 1x HM [g/L] final 1x concentration
Casamino acids 2 2 g/L
Thiamine hydrochloride 0.3 300 mg/L
MgSO4 0.208 2 mmol/L
CaCl2 0.011 0.1 mmol/L

The powders for 1 L of 1x HM were dissolved in 20 ml of a 10 % w/v Casamino acid stock solution and sterile-filtered (.22 µm PES). To make agar chips, we can add 1 ml to the hot agar mixture, together with the three 500 µl 100x stocks for the HM.

We made 250 ml of HM+C+Glucose+Supplements plates with 1.5 % agar.

Then we plated the following combinations:

Construct HM+C+Glucose+Supplements LB+C
K731520 iLOV - +
J23101.E0240 + +

We also prepared a 150 ml HM+C+Glucose+Supplements culture with K731520 iLOV to hopefully make agar chips on Friday.

6x of last weeks J23115.E0240 clones were plated on LB+C and 5 ml LB+C precultures were inoculated for plasmid preparation.


The K731520 iLOV strain did not grow on the HM plate, while another strain with the J23101.E0240 construct did. Both have the pSB1C3 backbone. To confirm the plasmids and inserts, Michael set up a colony PCR:

ID Template Product Length Result
1 J23101.E0240 #5 1233
2 J23101.E0240 #6 1233
3 J23101.E0240 #5 plasmid 1233
4 K731520 iLOV LB colony 2053
5 K731520 iLOV plasmid 2053
6 K731520 GFP plasmid 2437
7 water none

Reaction volume per tube is 15 µl, GoTaq Green Mastermix and the VF2 and VR primers. You can find the durations and temperatures are in this table:

parameter duration temp [°C]

Florian mini-prepped the 6 overnight cultures of J23115.E0240 clones. He used 3 ml instead of 1.5 and eluted twice with 25 µl nuclease-free water. Everything else was according to the protocol of the illustra plasmidPrep Mini-Spin Kit.

The resulting DNA concentrations are listed in this table:

clone # concentration [ng/µl]
1 73.5
2 94.5
3 100.5
4 53
5 82
6 138

In the evening, we plated some strains on the different media to investigate why the K731520 iLOV did not grow on the HM+suppl. plate. (Note: in the morning, Michael re-inoculated the HM+C+suppl. shake flask with cells from the LB-plate and by afternoon the culture did grow to a high cell density.)

Construct Host strain LB+C HM+C+Glucose HM+C+Glucose+supplements
J23101.E0240 NEB10beta ++ - ++
K731520 iLOV DH5alpha ++ - (+)
K131026 NEB10beta ++ - -
K131026 DH5alpha ++ - +

Philipp inoculated J23101.E0240 in LB and HM+C+Glucose+supplements


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