Contents 1 May 1.1 8th 1.2 23rd 2 June 2.1 25th 2.2 26th 3 July 3.1 16th 3.2 22nd 3.3 23rd 3.4 24th 3.5 25th 3.6 28th 3.7 29th 4 August 4.1 1st 4.2 2nd 4.3 3rd 4.4 4th 4.5 5th 4.6 6th 4.7 25th 4.8 26th 4.9 27th 5 September 5.1 3rd 5.2 4th 5.3 5th 5.4 6th 5.5 7th 5.6 15th 5.7 16th 5.8 17th 5.9 18th 6 October	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.

More information will be available soon!

May

8th

• SOE-PCR step 2

23rd

• we transformed some BioBricks and did a colony PCR

Colony PCR picture todo

June

25th

• 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

26th

• 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

July

16th

• 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

22nd

• 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

23rd

• 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)

24th

• 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 [http://openwetware.org/wiki/M9_medium/supplemented 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

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:

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.

25th

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:

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:

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:

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.)

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

28th

29th

• ...

August

1st

• Stefan and Vera made electrocompetent E.coli.
• Arne prepared cultures for Saturday, 14-8-2 of Renés iLOV and K131026

2nd

• Testing the OD measurement device and comparing it to the Spektrophotometer and the Platereader.
• Also testing K131026 and K731520 iLOV for fluorescence in the Platereader.
• Heat shock Transformation into NEB TOP 10 cells of I746909.
• Electroshock transformation of pET17-Gal3 into E.coli rosetta.

3rd

• OD Measurements of the iGEM device in comparison to the Spektrometer.
• Preparing culutures for cryos of K131026 and K731520 iLOV

4th

• Arne and Michael made cryo-stocks of K731520 iLOV and K131026 in NEB/BL21/DH5alpha, I746909 in BL21 and pET17-His-SNAP-YFP-Gal3 in E. coli Rosetta (DE3)
• Michael plasmid-prepped most of them using 1.5 ml culture and eluted with 1x 50 µl of ddH₂O.

To confirm the quality of pET17-Gal3-transformations, the purified plasmids were test-digested:

Test digest clones were test-digested

All pET17-Gal3 clones were positive and clone #2 was selected for further experiments.

5th

• Stefan, Arne and Michael assembled a V_R=2.5 L bioreactor for cultivation of a 1 L expression culture
• Two precultures of 20 ml LB+A were inoculated at 19:00
• Anna transformed K746909 into BL21 cells and K1319000 into NEB10beta

6th

• Eshani and Arne transformed J04450 in pSB1K3 and pSB1A3 in NEB Beta cells
• They also did a plasmid prep of J04450 in pSB1C3 and Flo's vectors
• Arne made precultures of NEB Beta and DH5 alpha cells
• Arne and Michael inoculated the fermenter at 11:40, induced the fermentation of pET17-Gal3. The fermentation is expected to run 24 h

25th

• Plasmid restriction of I20260 (EcoRI,PstI), J23115 (EcoRI, SpeI), K516032 (XbaI,PstI), and J23101 (EcoRI, SpeI)

26th

• Ligation of J23115 and K516032 to J23115.K516032 and J23101 and K516032 to J23101.K516032
• Plasmid prep of I20260, K516032 and B0034
• Restriction of Plasmid I20260, K516032, B0034 with EcoRI and PstI
• Gel with restricted I20260, K516032 and B0034
• purification of vector backbone, pSB1A2, pSB3K3 and pSB1C3
• Restriciton of snythesized TEV-Protease with EcoRI and PstI

27th

• Ligation of J23101.K516032 into pSB3K3 and J23115.K516032 into pSB3K3 and K1319004 into pSB1C3
• Transformation of K1319004 in pUC and pSB1C3 as well as J04450 in pSB1K3 and pSB1A3

September

3rd

Michael, Vera and Eshani prepared 50 ml LB+antibiotic overnight-cultures of pSBX-vectors from Heidelberg.

4th

• In the morning, at 10:15, Anna inoculated the precultures for the interlab study experiment.
• Michael prepared cryo-stocks of the pSBX-carryng E. coli from the overnight cultures. He also purified each pSBX-vector, eluting with 15+30 µl water:

• Anna did PCRs for Gibson-Assembly of K1319003 into pET17. Duplicates of 25 µl reaction volume (12.5 µl Q5 2x Master Mix, 2x 1.25 µl primers, 2 µl template)

The PCR conditions:

Finally, Florian did the Gibson assembly and heat shock transformation into NEB10beta cells.

At 10:15 Arne inoculated the primary cultures of the Interlab Study experiment and began with regular fluorescence measurements.

5th

• Anna made masterplates of yesterdays transformed cells.

6th

• Anna made precultures of 3 clones from each prepared master palte and inoculated precultures for OD/F measurements as well as chip production on the 7th.

7th

• Anna made cryos of from the precultures and Michael and Arne purified the plasmids:

• Elution was performed with 2 * 15 µl of nuclease free water.

15th

Arne and Michael were able to analyze the sequencing data from the clones of GFP_Reach 1, GFP_Reach 2 and K1319008.

GFP_Reach 2 clone number 3 and 5 were fine. With the included Leu to Ile mutation. GFP_Reach 1 clone number 4 and 5 were fine and did not contain the Leu to Ile mutation. Clone 6 was fine but contained the Leu to Ile mutation from the Reach 1 quick change mutations.

Further we will use the GFP_Reach 1 clone 4 and the GFP_Reach 2 clone 4.

Transformation of GFP_Reach 1 clone 3 and GFP_Reach 2 clones number 3 and 5 were performed together with the TEV-Protease to create two plasmid construct.

The GFP_Reach 1 and GFP_Reach 2 constructs were also restricted and ligated into the pSB1C3 vector from the pSB3K3 vector.

16th

Vera made master plates for the transformation from the day before.

17th

18th

October

...