April

1st

Organization

Where to get:

• key cards and keys for the lab
• cooled centifuge
• space in -80 °C
• bottles for 70 % ethanol
• ... (problems of a first year team ^^)

12th

• Chemically competent NEB Top10, DH5α and BL21 were made

15th

• 800 ml LB for plates with 1.5% agar and kanamycin (50 µg/L) were prepared(Stefan)
• The efficiency of our competent cells was tested(Anna)

Colonies on the agar plates were counted after transformation with 1 µl DNA 147 ng/µl and afterwards incubated in SOC or LB medium.

• BL21 cells were centrifuged and plated. Only 128 colonies grew in total indicating a very bad efficiency (~482.11).

Following formula was used to calculate the efficiency: Efficiency = (CFU /µg DNA) / dilution

• for DH5α: medial efficiency in SOC: 1864.36 and in LB: 440.68
• for NEB: medial efficiency in SOC: 6779.66 and in LB: 4698.30

  Higher efficiency when using SOC!

22th

Pcq lab PCR advanced primus 25, 96

Program name IGEM.cyc

1. RFC25_F RFC25_R
2. RFC25_F REACh1_R
3. REACh1_F RFC25_R
4. RFC25_F REACh2_R
5. REACh2_F RFC25_R

23th

• 5 µl of the PCR products were run on a 1,5% agarose gel
• DNA bands were purified from gel with the High Pure Product Purification Kit. Full length contaminated with REACh1

• SOE
• ca. 30 ng/µL

Anneling: 52 °C

Elongation: 20 sec

• PCR with Phusion polymerase

• Digestion for restriction with NEB enzymes

• Ligation with NEB Kit

28th

• Anna measured the DNA concentration of SOE2 after purification

  1. 57 ng/µL
  2. 69.5 ng/µL

29th

• PCR SOE1 and SOE2

30th

• A PAGE of the SOE2 products were run on agarose gel for checking

  → no positive results; only full length products were amplified

• The SOE PCRs was run again

  → more template

  → hot start

  → faster

  → elongation: 15 min

  → 20 cycles

• SOE1

template A+B → 20 µL → 13.8 + 6.2 5.3 + 14.7

• SOE2

May

1st

• gel with M - full -full - REACh1 SOE3.2 - REACH2 SOE3.2 - M

  → 120 V, 30 min

  → cut out the bands

5th

• Anna made chemical competent DH5α and BL21 cells

8th

• The efficiency of our competent cells was tested

  → BL21: 6.6 x 10⁴

  → DH5α: 2.59 x 10⁷

• The SOE-PCR step 2 like on 30.04. with the template of SOE1 from 30.04. was re-done
• The SOE2 product was run on a gel for checking (5 µL)

  → restriction, (dephosphorylation of vector)

  → purification on a gel with high pure kit

14th

• Florian made some LB agar plates with chloramphenicol and some with ampicillin
• REACh2 purification on 1.2 % agarose gel
• After that: purification of the 778 bp fragment with High Pure PCR Product Purification Kit

19th

• Transformation of K131026 in DH5α and NEB
• Transformation of K731520 in DH5α

20th

• Stefan made master plates on chloramphenicol (cam) → at least 6 clones on each plate
• Stefan prepared 2x 5 mL LB + cam
• Anna made sterile 50 % glycerol

21th

• Cryo stocks of clones 1 & 2 of each BioBrick/ host were prepared
• 15 mL cultures in 250 mL flasks, inoculated with 1.5 mL preculture (3 cultures A B C from clones 1; 1 + 2; 2)
• 250 µl Chloramphilicol from a 35 mg/mL stock was added
• The cultures were grown until OD₆₀₀= 0.6, then one was induced with IPTG

→ a negative control without plasmid was left out

23rd

• transformation of some BioBricks
• colony PCR (s. picture below)

  → K131026: 1807 bp

  → K731520: 2123 bp

  → full length EYFP: 778 bp

Colony PCR todo

June

3rd

• transformation of 34 BioBricks

4th

Preperation of consumables:

• Fresh 50 % glycerol
• New LB plates with cam and with kanamycin (kan)
• 60 glas tubes
• 2 L LB^-
• 100 mL steril glas beads for plating

Master plates (6 clones per BioBrick) were made

5th

• A colony PCRs on all transformed BioBricks were conducted

  → 2 clones from each master plate were picked

• Overnight cultures in freshly prepared 5 mL LB + cam were inoculated

  → 1 culture per BioBrick

6th

• made 2 glycerol stocks of each BioBrick

14th

PCR of E0030 to K1319000

• Q5 and Phusion polymerase are used

→ 30 cycles

17th

• Colony PCR on following constructs:

→ anneling: 50°C

→ elongation: 02:57

20th

• Vera did colony PCR of K325108, K325218, C0062

→ 30 cycles

• PAGE: 30 min on 1.2 % agarose gel at 110 V

→ weak bands for K325108 a & b at 4.5 kb and 9 kb respectively

23rd

• Repetition of yesterdays colony PCR
• Preculture for plasmid prep were inoculated
  • K1319000 → sequencing
  • K592100 → BFP
  • S01022 → CFP
  • J23101.E0240 → sequencing
  • J23115.E0240 → sequencing
• K516132 and J23101.E0240 were plated on LB + cam plates

24th

• 8 plasmid preps were conducted
• Overnight cultures of K516132 and J23101.E0240 were inoculated

25th

• A preculture for competent NEB10β cells was set up
• Samples for sequencing were submitted
• Master plates of transformed BioBricks were made
• Overnight expression cultures of J23101.E0240 and K516132 were centrifuged and frozen
• Fresh 50% glycerol was prepared

26th

• Competent NEB10β cells were made, however, several things went wrong. (For future reference: pre-cool centrifuge, always check if it is 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 tubes were not fully sealed during the PCR. Basically, only primers and smear, except for the positive control, which contained a plasmid template instead of cells.
• 2x 500 mL of fresh LB and three sterile flasks were prepared and autoclaved.

27th

• Plasmid preps

• Two cryos stocks of each were prepared

28th

• Sequencing
• Trafo of 17 BioBricks

30th

• Master plates

July

1st

• Transformation efficiency kit is broken

→ run an agarose gel with the kit plasmids to check for nuclease activity/ bands

• An inventory of the -80°C freezer was done

2nd

• Overnight cultures (LB) of K731520 and K1319042 for chips were inoculated

3rd

• OD of the over night culture of
  • K731520: 2.3
  • K1319042: 2.5
• Anna made chips with K1319042 and K731520 in NA and M9 medium images were taken every 30 min with the Geldoc
• Print K1319042 Chip on LB plate
• Over night culture of E0030 for plasmid prep

4th

• Plasmid prep of E0030: 159.5 ng/µl (concentration)
• Print of K1319042 Chip on LB plate made 1 and 7 colonies

7th

• transformation of 13 Biobrick
• over night culture of K103006 for plasmid prep
• made colony PCR master mix

8th

• over night culture of K1319042 for chips

9th

• Anna made chips with K1319042 in M9, M9 + casamino acids, Hartman medium (HM). Images were taken every 30 min with the Geldoc
• Vera made OmpA-linker_iLOV 3A-Assambley
• colony PCR
• transformation for backbones

10th

• master plates of the strains for backbone isolation

14th

• Anna built again J23115.E0240 for interlab study

15th

• Philipp made 500 ml LB and LB plates
• Florian and Stefan made the transformation of J23115.E0240
• over night culture of K1319042 for chips

16th

• did 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
• Anna made chips with K1319042 in Hartman, Hartman + 20 % glycerol, M9 medium images were taken every 30 min with the Geldoc

22nd

• Philipp and Michael made 100x stocks for Hartmans minimal medium.
• Michael and Vera made about 25 HM+C plates without glucose.
• K1319042 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
• K1319042 was plated on HM+C+Glucose and HM+C+Glucose drops, 3x 5 mL HM+C+Glucose precultures were inoculated (17:00)

24th

• K1319042 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 on 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 filter-sterilized (.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 K1319042 to hopefully make agar chips on Friday, July 25th.

Six of last weeks J23115.E0240 clones were plated on LB+C, and 5 mL LB+C precultures were inoculated for plasmid preparation.

25th

The K1319042 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 was 15 µL. GoTaq Green Mastermix and the VF2 and VR primers were used. You can find the durations and temperatures the table below:

Florian mini-prepped the 6 overnight cultures of J23115.E0240 clones. He used 3 mL instead of 1.5 mL culture medium and eluted twice with 25 µL nuclease-free water. Everything else was according to the protocol of the [http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/de/GELifeSciences/28904269 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 K3139042 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.

29th

• inoculate 2x 150 ml cultures HM+Glucose+C
• prepare 200 ml 1.5 % agar
• pre-cool centrifuge
• centrifuge 1x 50, 1x 100 and 1x 150 ml
• make 3 kinds of chips - OOx1, ODx2, ODx3
• pre cultures of K1319042 and K131026 in LB

30th

• Anna made chips with K1319042 and K131026 in HM medium images were taken every 30 min with the Geldoc
• print chips on LB and LB + Cam , count 10-30 colonies

31th

• Anna transformed K1319042 and K131026 in DH5α, BL21 and NEB

August

1st

• Stefan and Vera made electrocompetent E.coli rosetta cells.
• Arne prepared cultures of K1319042 and K131026 for Saturday, August 2nd.

2nd

• tested the OD measurement device and compared it to the spectrophotometer and the plate reader.
• tested K131026 and K1319042 for fluorescence in the plate reader
• did a heat shock transformation of I746909 into NEB TOP 10 cells
• did an electroshock transformation of pET17-Gal3 into E.coli rosetta

3rd

• OD measurements of the iGEM device in comparison to the spectrophotometer were taken.
• cryo cultures of K131026 and K1319042 were prepared
• master plates of Gal3 #1-#10 and I746909 #1-#4 and overnight cultures

4th

• Arne and Michael made cryo stocks of K1319042 and K131026 in NEB/BL21/DH5alpha, I746909 in BL21 and pET17-His-SNAP-YFP-Gal3 in E. coli rosetta (DE3), respectively.
• Michael did a plasmid prep, most of them using 1.5 mL culture medium, and eluted with 1x 50 µL of ddH₂O. The resulting DNA concentrations are shown below.

To confirm the quality of pET17-Gal3 transformations, the purified plasmids were tested by carrying out a digest. Results are shown in the below picture and table.

Test digest clones were test-digested

All pET17-Gal3 clones were positive and clone #1 was selected for further experiments.

• Arne prepared an over night cultur of K1319042 for chips

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 NEB10β cells.
• Anna made Chips with K1319042 in HM. Images were taken every 30 min with the Geldoc
• Anna made alliquots of HM, 1 L HM + glucose + supplements and 500 ml LB

6th

• Eshani and Arne transformed J04450 in pSB1K3 and pSB1A3 in NEB10β cells.
• They also did a plasmid prep of J04450 in pSB1C3 and Flo's vectors.
• Arne made precultures of NEB10βand DH5 alpha cells
• Arne and Michael inoculated the fermenter at 11:40, and induced the fermentation of pET17-Gal3. The fermentation is expected to run 24 h.

7th

• Philipp made media for Pseudomonas flourescens
  • Nutrient Broth
  • Pseudomonas-F
  • Pseudomonas-P
• Anna made 2 L LB

8th

• Anna prepared 2x 60 ml (LB + cam + IPTG) with K1319042
• Florian prepared 5 ml K131026 and C0179
• Vera and Michael made SDS page

9th

• Anna and Florian made a plate reader experiment with K131026 in LB and LB + HM

11th

• Anna plated on LB + antibiotics
  • K131026
  • I746909
  • K13190042
  • I04450 in pSB1C3
  • I04450 in pSB1A3
  • I04450 in pSB1K3
  • Florian's construct (pSEVA 641_FP pSEVA 234-LasR)

13th

• Anna made chips with
  • K131026 and I746909 in LB and HM
  • K1319042 and Florians two plasmid construct in HM
• Images were taken every 30 min with the Geldoc

18th

• Anna made over night cultures of I746909 and K131026 in LB, TB, 2x HM+
• plated 3x J23101.E0240

19th

• Anna made Chips with K131026 and I746909 in HM. Images were taken every 30 min with the Geldoc
• made PCR of J23101.E0240, K1319000, K1319001, K1319002 and run a 1,2 % agarose gel

20th

• repeat PCR for REACh1 and J23101.E0240 and run a gel
• plasmid prep of pSEVA BfsB, pSEVA lasR and I746909

24th

• Anna made 2.5 L LB
• Anna made chips of K131026 in NEB , DH5α and BL21 in LB and additionally K131026 in DH5α in HM+. Images were taken every 30 min with our own device

25th

• did a plasmid restriction of I20260 (EcoRI,PstI), J23115 (EcoRI, SpeI), K516032 (XbaI,PstI), and J23101 (EcoRI, SpeI)
• Nina tested the growth of Pseudomonas fluorescens in different liquid media for high OD and strong fluorescence. She tested Standard I medium, Cetrimide medium and Pseudomonas-F medium, and Pseudomonas-F medium supplemented with 300 µL Fe3+ in 500 mL flasks with a filling volume of 30 mL. The flasks were inoculated with P. fluorescens cells on Standard I agar, and incubated at 30 °C at 250 rpm.
• Anna prepared over night cultures of K131026 in DH5α and NEB for chips
• Anna prepared 2x 5 ml of pSB1C3, psB3K3 and pSB1A2 for plasmid prep

26th

• ligation of J23115 and K516032 to J23115.K516032, and J23101 and K516032 to J23101.K516032, respectively.
• plasmid prep of I20260, K516032 and B0034
• restriction of plasmids I20260, K516032, B0034 with EcoRI and PstI
• A gel with restricted the I20260, K516032 and B0034 was run.
• purification of vector backbones pSB1A2, pSB3K3 and pSB1C3
• restriciton of synthesized TEV protease with EcoRI and PstI
• Nina qualitatively tested the Pseudomonas fluorescens that had grown over night for OD and fluorescence. She determined that Pseudomonas-F medium is the most adequate for the cultivation of the strain we use, since both OD and fluorescence were best in the flask containing the respective medium. Growth in the Pseudomonas-F medium supplemented with 300 µg/L Fe3+ was weaker, however, fluorescence was also successfully suppressed.
• Anna made chips with K131026 in DH5α and NEB, in LB and LB + 10 % glycerol. Images were taken with our own device every 30 min.

26th

• plasmid prep of the back bones, restriction and gel purification

27th

• transformation of some BioBricks
• Ligation of J23101.K516032 into pSB3K3 and J23115.K516032 into pSB3K3 and K1319004 into pSB1C3
• Transformation of K1319004 into pUC and pSB1C3, and J04450 into pSB1K3 and pSB1A3, respectively

September

3rd

Michael, Vera and Eshani prepared 50 mL LB+antibiotic overnight-cultures of pSBX-vectors that were sent in by team Heidelberg.

4th

• In the morning, at 10:15, Anna inoculated the precultures for the interlab study experiment.
• Michael prepared cryo stocks of the pSBX-carrying E. coli from the overnight cultures. He also purified each pSBX-vector, eluting with 15+30 µL water, and resulting in the following DNA concentrations:

• Anna did PCRs for Gibson assembly of K1319003 into pET17. Duplicates of 25 µL reaction volume (12.5 µL Q5 2x Master Mix, 1.25 µL per primer, 2 µL template)

The PCR conditions:

• Finally, Florian did the Gibson assembly and a heat shock transformation into NEB10β cells.

• At 10:15, Arne inoculated the primary cultures of the interlab study experiment and began with regular fluorescence measurements.

5th

• Anna made master plates of yesterday's 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 stocks of the precultures.
• Michael and Arne purified the following plasmids:

Elution was performed twice with 15 µL of nuclease free water each time.

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 #3 and #5 were fine, including the Leu to Ile mutation. GFP_Reach 1 clone #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.

For future experiments, 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 #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 of the transformation from the day before.

• Also PCRs were made from pSBXA3, I20260 and K131900 for a Gibson assembly. The PCRs were checked with a gel electrophoresis.

17th

• Nina prepped and autoclaved 33 500 mL shake flasks.

18th

• Nina tested Pseudomonas fluoresence if they are suitable for a growth experiment that is planned for our collaboration with the NEAnderLab next week. Therefore, she filled 2 500 mL flasks with 30 mL LB Pseudomonas-F medium, and inoculated each one with 1 mL culture medium of the overnight preculture. Flasks were inoculated at 30 °C at 250 rpm. However, after 5 hours no exponential growth could be shown (s. plot below). Thus, it was decided to use a E. coli K12 derivate strain in TB medium instead, and 30 mL of TB medium in a 500 mL flask were inoculated with E. coli DH5alpha cells and incubated at 37 °C at 300 rpm over night. According to the DSMZ E . coli K12 strain derivates, such as DH5alpha, are adequate for the kind of school experiment we are planning with the NEAnderLab.

Growth Curves Unfortunately, P. fluorescens did not show a nice exponential growth curve over the observed 5 hours.

19th

• René, Arne and Philipp made flask cultures of K1319013, K1319013 + K1319008, K1319013 + K1319008 + iPTG, K1319014, K1319014 + K1319008, K1319014 + K1319008 + iPTG, B0015 (negative control) and I20260 (positive control). iPTG was added at an OD of ~0.5. Inoculation was done via precultures in 500 ml shake flasks (50 ml filling volume). Media was always LB. Cultivation was done at 37 °C and 300 rpm. The starting OD was aimed to be 0.1. Inoculation occured directly from the precultures. Samples were taken every hour and checked for OD and fluorescence using a spectrophotometer and plate reader, respectively.

• Vera did a plasmid preparation from the cultures of the day before (K1319013, K1319013 + K1319008, K1319013 + K1319008 + iPTG, K1319014, K1319014 + K1319008, K1319014 + K1319008 + iPTG, B0015 and I20260). The plasmid were then be cut with EcoRI and PstI, and the results were be put on an agarose gel in order to perform a restriction test. Also plasmids of K1319013 and K1319014 will be cut with EcoRi and SpeI. K1319008 will be cut with XbaI and PstI. These will then be ligated together and then ligated into a pSB1A3 vector via the 3A assembly (vector cut with EcoRI and PstI). These constructs will be transformed into BL21 (and NEB as a backup). The created construts will be known as K1319018 (K1319013.K1319008) and K1319019 (K1319014.K1319008).

• Florian made precultures of the master plates from the day before (K1319008, K1319013, K1319015 and pSBX1A3 with Gal3).

• Arne also inoculated 4 cultures for the further testing of the OD/F device (the F part). The cultures are 2 shake flasks of I20260 and 2 shake flasks of B0015.

• Furthermore, Nina did a growth experiment with DH5alpha for the NEAnderLab school experiment. 3 500 mL shake flasks were filled with 50 mL TB medium, and inoculated to an OD of 1.5 with the overnight preculture. Samples were taken every 30 minutes and tested for OD using our own device as well as the spectrophotometer. The resulting growth curve is shown below. Nina concluded that the growth was fast enough for these growth conditions to be used for the school experiment on the 24th.

Growth Curves Growth under these conditions was sufficient for the school experiment to be carried in 5 hours. And our device did a good job measuring, too!

• Anna also made chips with K1319013 + K1319008, K1319014 + K1319008, K1319013, K1319014, B0015 and K131026. These will be inocubated for an hour at 37 °C. Then they will be induced with iPTG/HSL and fotos will be made every 30 minutes to check for fluorescence (GFP).

• Florian tested our OD/F device with a dilution test. Samples were checked with the spectrophotometer (OD), our OD/F device (fluorescence) and platereader (fluorescence).

• Anna made SDS gels.

• Stefan inoculated a culture of K1319008, B0015 as well as I20260 to check whether the results from our construct are from a wrongly done Gibson assembly with a still functioning superfolded GFP (the TEV protease was inserted in a backbone that formely contained superfolded GFP.)

20th

22nd

• Nina poured several Pseudomonas-F agar plates with 0, 150 and 300 µg/L for the NEAnderLab school experiment. She also autoclaved 12 500 mL shake flasks, partly to be used for the school collaboration on Wednesday.

26th

• Michael did a check PCR on several cryo cultures. All samples with G00100_Alternative+K1319004_check_R combinations resulted in a strong band at ~2300 bp that we cannot explain. All G00100_Alternative+K1319004_check_R combinations resulted in a strong band at 900 bp that we cannot explain either. We concluded that the annealing temperatures were wrong and favored unspecific products. Therefore, we decided to do a gradient PCR to find out the optimal annealing temperatures for our new primers.

Gradient PCR to test new primers

Florian and Michael did gradient PCR with these new primers:

Three PCRs with different primer combinations were run. In all of them the templates were K1319004 in pSB1C3, K1319008 in pSB1C3 and I746909 in pSB1C3.

The first gradient PCR tested the G00100_Alternative + G00101_Alternative combination:

Gradient PCR 1 the primers were G00100_Alternative and G00101_Alternative and they worked well at all temperatures from 55-65 °C.

The second gradient PCR tested the G00100_Alternative + I746916_check_R combination:

Gradient PCR 2 the primers were G00100_Alternative and I746916_check_R and they worked well at all temperatures from 55-65 °C. Apparently the K1319008 template contained I746916.

The third gradient PCR tested the G00100_Alternative + K1319004_check_R combination:

Gradient PCR 3 The primers were G00100_Alternative and K1319004_check_R and they worked well at all temperatures from 60-68.1 °C. To our disappointment, the K1319008 template did not contain K1319004. It is unclear why the 5 bands of K1319008 and I746916 look different.

The results of these three PCRs are:

1. The KAPA2G Fast ReadyMix worked well
2. All three primers work well at >65 °C annealing temperature
3. The K1319008 template was contained I746916 instead of the intended K1319004 ORF

It was concluded that a similar check PCR with 65 °C annealing temperature will be done on all plasmids and cryos of K1319008.

27th

• First Michael transformed K1319001, K1319002, K1319003 and K1319004 (all in pSB1C3) into NEB10β cells. He tested the PCR machine for semi-automated heat-shocking by splitting the 50 µL cells with the plasmid into 2x 25 µL. All 100 µL were plated for all construct/machine combinations.

• Vera transformed several constructs into chemically competent BL21(DE3) cells.

• Philipp and Michael did colony-PCR on all plasmids, cryos and colonies that should contain the K1319004 sequence.

• Vera also made check a PCR on galectin-constructs:

28th

• Michael made a restriction of BioBrick K1319020 and vector pSB1C3 with restriction enzymes EcoRI and PstI. Then Vera ligated the restricted parts and made a transformation using E. coli NEB 10ß cells.

29th

• Eshani made cryo cultures and plasmid preparation of K1319010, K1319011, K1319012, K1319021 and K1319042. Vera determined the contentration of plasmids and made did a restriction digest of K1319010, K1319011, K1319012, pSB1C3, K1319021, K1319013 and K1319014, followed by a ligation in K1319010.pSB1C3, K1319011.pSB1C3, K1319012.pSB1C3, K1319021.K1319013.pSB1A3 and K1319021.K1319013.pSB1A3. All constructs were transformed into E. coli NEB 10ß.

• Nina prepared 3 500 mL flasks with 30 mL LB medium which were inoculated with a Pseudomonas putida strain. The cells were cultured over night at 28 °C and ~300 rpm. The cultures are supposed to be used to test our OD device.

30th

• Sequencing samples were sent in for K1319020 clone #2, 3 & 5 (in pSB1C3), K1319017 clone #1 (in pSB1C3), K1319010 clone #2 (in pSB3K3), K1319011 clone #1 (in pSB3K3), K1319012 clone #2 (in pSB3K3), K1319013 clone #1 (in pSB1C3), K1319014 clone #1 (in pSB1C3), K1319001 (in pSB1C3) and K1319002 (in pSB1C3).

• A plasmid prep of K1319013 and K1319014 was run.

• A Gibson assembly with the K1319015 from the I20260 backbone and the K1319000 insert, forming K3139015, was conducted. The product was subsequently transformed into NEB10β cells.

• The pSB1C3 plasmid backbones were amplified via PCR and purified.

• Colony-PCRs of K1319008 and K1319012 master plates were made to confirm the colony's identity. Subsequently, pre-cultures were inoculated.

• A transformation of K1319010 and K1319010 in pSB1C3 was conducted.

• Another plasmid prep of K1319010 clone #2, K1319011 clone #1, K1319012 clone #2 (all in pSB3K3), K1319013 clone #4, K1319014 #3, K139020 #2, 3, 5 (all in pSB1C3) was run.

• The OD device was tested with a dilution series of a Pseudomonas putida culture.

October

1st

• Prepartations for sensor-chip production the following day (2014-10-02) was done accoringly to the sensor chip manufacturing protocol:
  • At 18:30 Patrick prepared over-night cultures from K1319042, B0015 and K131026 by inoculating 250 ml Erlenmeyer flasks each containing 50 ml LB medium . The flasks were incubated for ~12 hours at 37 °C on a shaker.

2nd

• At 8:30, Nina and Arne did a plasmid prep of dublicate samples of K1319011 clone #1 and #6. Florian measured the DNA yield, and the higher concentrated sample of clone #1 and #6, respectively, were sent in for sequencing.
• Nina made precultures and a master plate of 6 colonies of K3139008 in psB1C3 in NEB10β cells that had been plated at 5:30 this morning.

• Production of sensor-chips was done accordingly to sensor chip manufacturing protocol. Briefly:
  • At 10:00 Patrick prepared 150 ml 1.5 % (w/v) LB+agarose solution. The LB+agarose solution was autoclaved and subsequently tempered to 45 °C. Precultures (50 ml each) of K1319042, B0015 and K131026 were spun down at 3000 g for 10 min at 21 °C and re-suspended in 1 ml pretempered (21 °C) LB medium. The re-suspended cultures were mixed with 50 ml LB+agarose and poured onto three sensor-chip-templates (one template per culture). Sensor chips were cut out from the template and incubated at 37 °C for 1 h.
  • K1319042 and B0015 were induced with 0.2 µl IPTG (100 mM) subsequently to incubation and K131026 was induced with 0.2 µl homoserinlacton stock solution (50 µM) 30 minutes after induction of the K1319042 and B0015. The induced sensor-chips were read out every 30 minutes for 180 minutes in total. An additional readout was conducted 285 minutes post induction. The readout was done at 450 nm and 480 nm wavelength.

• Gibson assembly of K1319008
  • Template Backbone: I746909, Insert: K1319004
  • Transformation in E.coli NEB10β and BL21

• PCRs for Gibson assembly of K1319010 and K1319015
  • Template Backbone: I20260 for K1319010 and K1319015
  • Template Insert: K1319000 for K1319010 and K1319015 (but different primer)

• made precultures of K1319013 and K1319014 in pSB3K3

• K1319011 in pSB1C3 prepped for sequencing

• Gibson assembly of K1319017 (PCRs, Gibson assembly, restriction with DnpI, transformation into NEB10β)
  • Template Backbone: B0015
  • Template Insert 1: LasI synthesized gene
  • Template Insert 2: K660004

3rd

• made master plates of K1319008 in NEB10β and BL21 and precultures

• check PCR for K1319008 to validate the Gibson assembly check for potential I746909 residues

Check PCR K1319008 K1319008 was checked with the Primers K1319008_Check_R and I746909_Check_R (both with G00100 Alternative as froward primer) for presence of K1319008 or I746909. All tested clones were positive for K1319008 and negative for I746909.

• Florian and Stefan did a plasmid prep and made cryo stocks of K1319008 in NEB (clones #1, #2, #3) and BL21 (clones #1, #2)

• Plasmid prep of K1319013 and K1319014 in pSB3K3

• Gibson assembly for K1319010 and transformation into NEB10β

• made cryo stocks of K1319011 clone #6

• restriction of K1319012, k1319013 and K1319014 with EcoRI and PstI. Restriction of linearized plasmid backbone pSB1C3 with EcoRI and PstI. Ligation of K1319012, K1319013 and K1319014 into pSB1C3. Transformation into NEB10β.

• Gibson assembly of K1319015 and transformation into NEB10β

• colony PCR of K1319017

colony PCR K1319017 K1319017 was checked with a colony PCR for the right insert length. Clones #2 and #4 were correct and used forthwith.

• did plasmid prep and cryo of clones #2 and #4 of K1319017

• new plasmid backbone of pSB1C3 was made using the [http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones|standard protocol].

• transformation of K1319008 clone #1 (from BL21) and K1319013 into BL21 (two plasmids in one cell).

• transformation of K1319008 clone #1 (from BL21) and k1319014 into BL21 (two plasmids in one cell).

• OD measurements of three biological triplicates from E. coli BW21 113, P. putida and S. cerevisiae. Measurement as an analytic triplicate in the spectrophotometer (absorbance and transmission) and our own OD/F device.

• Gibson assembly of K1319021
  • Template Backbone: K1319008
  • Template Insert: LasI gene synthesis

• At 19:00 Patrick and Florian measured OD (our OD-device), absorption (spectrophotometer) and transmission (spectrophotometer) for 19 diltuions in the range of 2.5-100 % from yeast (Saccharomyces cerevisiae) and P. putida liquid cultures. Measurements were conducted in biological as well as technical triplicates. Aim of this experiment was the comparison of our OD-device to commonly used devices in terms of OD determination.

• Prepartations for sensor-chip production the following day (2014-10-04) were done accordingly to the sensor chip manufacturing protocol:
  • At 22:00 Patrick prepared over-night cultures from B0015, K1319017 and K131026 by inoculating 250 mL Erlenmeyer flasks each containing 50 mL LB medium for sensor-chip manufacturing the next day. The flasks were incubated for ~12 hours at 37 °C on a shaker.

• Our BioBrick K1319021 enables expression of the TEV protease inducible by the autoinducers of Pseumomonas aeruginosa. In order to construct this BioBrick, a Gibson assembly of K1319008 (IPTG-inducible expression of TEV protease) and the synthezised composite HSL-promotor J23101.B0032.C0079.B0015.J64010.B0034 activated by the respective autoinducers (HomoSerineLactones) was perfomed by René. Prior to this, René used two PCRs to linearize pSB1C3-K1319008, serving as backbone for Gibson assembly, and cutting out J23101.B0032.C0079.B0015.J64010.B0034 as well as adding adequate overlapping sequences.

4th

• colony PCR of K1319015 and Check PCR of K1319010
  • K1319010: clone #1 was positive
  • K1319015: in all clones the inserts were too short.

• new digestion of Gibson master mix of K1319015 with DnpI

• transformation of new Gibson master mix into NEB 10β

• restriction of K1319010, K1319012, K1319013 and K1319014 (all in pSB3K3) with EcoRI and PstI, cutting of pSB1C3 with EcoRI and PstI, and then ligation.

• made master plates and precultures of the transformations of K1319008 in BL21, K1319013 + K1319008 in BL21 and K1319014 + K1319008 in BL21

• colony PCR of the master plates with the double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008

• Sending the first BioBricks to the iGEM headquarters:
  • K1319000
  • K1319001
  • K1319002
  • K1319003
  • K1319004
  • K1319008
  • K1319011
  • K1319017
  • K1319020
  • K1319042

• shake flask experiments with K1319008 (clone #1), K1319013 + K1319008 (clone #2) and K1319014 + K1319008 (clone #2) in LB (2 flasks each). Inoculation with 50 µL preculture and inducing with iPTG at OD of 1.5.

• Production of sensor-chips was done accordingly to the sensor chip manufacturing protocol:
  • At 13:30 Patrick prepared sensor chips from pre-cultures of B0015, K1319017 and K131026.
  • Subsequently to 1 h icubation at 37 °C B0015, K1319017and K131026 were induced with 0.2 µL homoserinlacton stock solution (50 µM). The induced sensor-chips were read out every 30 minutes for 240 minutes in total. Readout was conducted at 450 nm and 480 nm wavelength. An additional readout was conducted after 12 hours.

• At 17:00 Patrick prepared 4 liquid cultures from the K1319010_pSB3K3 master plate (clone#1) in 5 mL LB-medium, each. The liquid cultures were prepared in order to create cryo stocks from K1319010-pSB3K3. Kanamycin was added to the liquid cultures as antibiotic at an concentration of 1 µL/mL.

• At 18:00 Patrick prepared a master plate (LB+C) and corresonding liquid cultures from 6 clones of E.coli NEB10B k1319021-psB1C3. Liquid cultures and master plate were incubated at 37 °C.

• At 23:30 Patrick prepared liquid cultures from K1319015-pSB3K3 clones #7, #8 and #9 in 5 mL LB-medium. Kanamycin was used as antibiotic and the cultures were incubated at 37 °C. Purpose of the cultures was cryo stock preparation and plasmid prep.

5th

• At 11:45 Patrick and Anna prepared cryo stocks from NEB K1319010-pSB3k3 #1, K1319015-pSB3K3 #7, K1319015-pSB3K3 #8 and K1319015-pSB3K3 #9 by mixing 750 µl lquid culture with 750 µl 50% (v/v) Glycerol-solution in 2 ml eppis.
  • Plasmid prep was also done for the cultures mentioned above.

• At 12:00 Patrick prepared liquid cultures from K139010-pSB3K3, K139011-pSB3K3, K139012-pSB3K3, K139013-pSB3K3, K139014-pSB3K3, K139015-pSB3K3 in 5 ml LB-medium each. Kanamycin was used as antibiotic. Purpose for the cultures was the characterization of constituitive expression and an additional plasmid prep of K139013-pSB3K3 and K139014-pSB3K3.

• Production of sensor-chips was done accordingly to the sensor chip manufacturing protocol:
  • At 13:00 Patrick and Anna prepared sensor chips from shake flask pre-cultures of BL21 pSB1C3-K1319008+pSB3K3-K1319014, BL21 pSB1C3-K1319008+pSB3K3-K1319013 and NEB pSB1C3-B0015.
  • Subsequently to 1 h incubation at 37 °C, BL21 pSB1C3-K131900+pSB3K3-K1319014, BL21 pSB1C3-K1319008+pSB3K3-K1319013 and NEB pSB1C3-B0015 were induced with 0.2 µL IPTG. The induced sensor-chips were read out every 30 minutes for 360 minutes in total. K1319013 was induced earlier and thus measurements were taken for 450 min in total. Readout was conducted at 480 nm wavelength. An additional readout was conducted next day at 11:00.

• At 15:30 Patrick prepared liquid cultures for the characterization of ITPG inducible expression:
  • I746909 in pSB1C3
  • I20260 in pSB3K3
  • K731520 in pSB1C3
  • K1319008 in pSB1C3
  • K1319013 in pSB3K3
  • K1319014 in pSB3K3
  • B0015 in pSB1C3
  • K1319013 in pSB3K3 + K1319008 in pSB1C3
  • B0015 in pSB1C3
  • K1319014 in pSB3K3 + K1319008 in pSB1C3

• Florian and Patrick prepared a colony PCR from K1319014-pSB1C3 clones #3, #4, #5 and#6. H20 and K1319014-pSB3K3 were used as controls.

• At 22:20 Michael and Vera prepared 1 L LB+C plates (2.5 g NaCl, 10 g Agar, 2.5 g yeast extract and 5 g Trypton). N-Z-amine (peptone from casein) was used instead of tryptone, because the tryptone stock was depleted.

• Philipp and René prepped multiple cultures K1319013 in pSB3K3 and K1319014 in pSB3K3. Now we have sufficient amount of both plasmids.

• Plates were made by Philipp and Michael for the following constructs:

• To determine whether the conducted Gibson assembly of K1319021 and subsequent transformation into E. coli NEB 10β were successful, a colony PCR on these cells was performed by René. Primers binding to each of the templates used for Gibson assembly were used: LasI_Insert_F binding in J23101.B0032.C0079.B0015.J64010.B0034 and K1319004_Check_R binding in pSB1C3-K1319008. Bands at 1341 bp would indicate the successful construction and transformation of K1319021.

Check PCR on K1319021 (Homoserinlactone-inducible expression of the TEV protease)

Since Bands >2000 bp were observed, another PCR was performed by René using primers binding upstream and downstream of the insert in pSB1C3: G00100_alternative and G00101_alternative. Here, bands with a length of 2210 bp would verify the correct length of K1319021.

6th

• Anna made a SDS page of K1319010-15 and B0015
• Nina made a master plate at 15:30 containing two clones of Vera's K1319015 plate from yesterday.

• At 22:00 Patrick inocculated two 500 ml flasks containing 50 ml LB-medium with 1 ml pre-culture of K1319017, which hab been prepared by Vera earlier this day. Chloramphinicol was used as antibiotic. The Flasks were incubated at 37 °C on a shaker until an OD of X was reached. The cultures were used for fluorescence characterization.