iGEM 2015 — Team NTNU_Trondheim

# Team:NTNU Trondheim/Notebook

![](https://static.igem.org/mediawiki/2015/b/bb/NTNU_Trondheim_logo.png)

* [Home](https://2015.igem.org/Team%3ANTNU_Trondheim)
* Project
  + [Description](https://2015.igem.org/Team%3ANTNU_Trondheim/Description)
  + [Notebook](https://2015.igem.org/Team%3ANTNU_Trondheim/Notebook)
  + [Experiments and Protocols](https://2015.igem.org/Team%3ANTNU_Trondheim/Experiments)
  + [Results](https://2015.igem.org/Team%3ANTNU_Trondheim/Results)
  + [Modeling](https://2015.igem.org/Team%3ANTNU_Trondheim/Modeling)
* [Parts](https://2015.igem.org/Team%3ANTNU_Trondheim/Parts)
* Software
  + [Overview](https://2015.igem.org/Team%3ANTNU_Trondheim/Software)
  + [IGEM Matchmaker](https://2015.igem.org/Team%3ANTNU_Trondheim/Software/iGEM%20Matchmaker)
  + [Quantitative Analysis Tool](https://2015.igem.org/Team%3ANTNU_Trondheim/Software/Quantitative_Analysis_Tool)
  + [Brixells Modeling Tool](https://2015.igem.org/Team%3ANTNU_Trondheim/Software/Brixells_Tool)
* [InterLab study](https://2015.igem.org/Team%3ANTNU_Trondheim/InterLab_Study)
* About
  + [Team](https://2015.igem.org/Team%3ANTNU_Trondheim/Team)
  + [Attributions](https://2015.igem.org/Team%3ANTNU_Trondheim/Attributions)
  + [Collaborations](https://2015.igem.org/Team%3ANTNU_Trondheim/Collaborations)
  + [Human Practices](https://2015.igem.org/Team%3ANTNU_Trondheim/Practices)
  + [Safety](https://2015.igem.org/Team%3ANTNU_Trondheim/Safety)
  + [Achievements](https://2015.igem.org/Team%3ANTNU_Trondheim/Achievements)

* ###### Jump to:
* June
* [Week 23](#1)
* [Week 24](#24)
* [Week 25](#25)
* [Week 26](#26)
* July
* [Week 27](#27)
* [Week 28](#28)
* [Week 29](#29)
* [Week 30](#30)
* [Week 31](#31)
* August
* [Week 32](#32)
* [Week 33](#33)
* [Week 34](#34)
* [Week 35](#35)
* September
* [Week 36](#36)
* [Week 37](#37)
* [Week 38](#38)
* [Week 39](#39)

## Notebook

##### Filter by category: show all categories

"\_"

Genetics

Encapsulation

Interlab Study

Modeling

Practices

Wiki

![](https://static.igem.org/mediawiki/2015/7/74/NTNU_Trondheim_plasmids_on.png)

only

![](https://static.igem.org/mediawiki/2015/b/b2/NTNU_Trondheim_techniques_on.png)

only

![](https://static.igem.org/mediawiki/2015/6/6b/NTNU_Trondheim_media_on.png)

only

![](https://static.igem.org/mediawiki/2015/4/41/NTNU_Trondheim_calculations_on.png)

only

![](https://static.igem.org/mediawiki/2015/9/9b/Team_NTNU_Trondheim_nb-hprac-sc.png)

only

![](https://static.igem.org/mediawiki/2015/9/98/Team_NTNU_Trondheim_nb-wiki-sc.png)

only

### Week 23

###### (01/06 - 07/06)

###### June 01

The iGEM Matchmaker (V2.0) is deployed for 2015.

### Week 24

###### (08/06 - 14/06)

###### June 11

Discussed *Escherichia coli* glucose uptake and possible promoters in *E. coli*.

### Week 25

###### (15/06 - 21/06)

###### June 15

Discussed *Pseudomonas putida* as a candidate microorganism.
Discussion of a system for glucose uptake in *P. putida*.

###### June 15

Interviews.

###### June 17

Design of promoter regions for *P. putida*.
Ordered promoter regions for *P. putida*.

###### June 18

Discussion of InterLab Study.
Preparations for the InterLab Study

###### June 19

Prepared and autoclaved LB medium 300 ml, LA medium 3 x 500 ml, Eppendorf tubes, distilled water and pipette tips.
Prepared antibiotic stock solutions (Ampicillin, Chloramphenicol and Kanamycin).

###### June 20

Created survey for students and employees at NTNU.
Interviews with diabetic patients.

###### June 21

Inoculation of DH5α in 3 ml, incubation over night at 37 0C.

### Week 26

###### (22/06 - 28/06)

###### June 22

Making *E. coli*electrocompetent (after 3h incubation, OD600 = 0.44).
Electroporation of E. coli.

###### June 23

Transformation.

###### June 24

Introduction to alginate encapsulation + demo on how to operate the electrostatic capsule generator.

###### June 24

Inoculation of a colony from plate in 3 ml LB.

###### June 25

Playing around with the electrostatic bead generator, making capsules of different sizes by varying the different parameters of the instrument.

###### June 25

Miniprep of transformed E. coli.
Nanodrop measurement of DNA concentration.

###### June 26

Enzyme digestion.
Ligation.
Transformation.
Prepared LA plates.

###### June 27

Put plates from incubator in the fridge (there were also colonies on the control plate, so we have to validate that the colonies we picked for inoculation really have the desired plasmid).

###### June 28

Inoculation of 3 colonies from each sample from the 50 μl plate, and 3 colonies from the positive and negative control plates (from June 23rd).

### Week 27

###### (29/06 - 05/07)

###### June 29

Moved the cell encapsulation equipment into our lab, so it was ready for the GM bacteria.

###### July 01

Miniprep.
Nanodrop measurement after miniprep (58.38 + 64.98 + 70.48 ~ 64.63).

###### July 01

Preparation of alginate solution, gelling solution and washing solution.

###### July 02

Inoculation of *Pseudomonas putida* pHH+GFP from -80 0C in 3 ml LB + Kan medium. Incubation at 30 0C.

###### July 03

5 % inoculation of overnight culture in fresh LB + Kan.
Incubation at 30 0C for two hours. Induction of *P. putida* pHH+GFP with m-Toluic acid.

###### July 03

Introduction to confocal microscopy.

###### July 05

Created the website design using Bootstrap, and set up the Notebook and Protocols pages.

### Week 28

###### (06/07 - 12/07)

###### July 06

PCR (1 μl template DNA = miniprepped from *P. putida* pHH+GFP, annealing temperature = 60 0C).
Prepared LB (200 ml with Kan, 100 ml with Chl).
GelRed to test the PCR. It did not work.
PCR repeated but with Rahmi’s DNA, did not work either.
PCR repeated again and gel shows the right band (but also an unknown second band).

###### July 06

Inoculation of *Pseudomonas putida* pHH+GFP from -80 0C in 3 ml LB + kan medium. Incubation at 30 0C.

###### July 07

Repeated PCR because of the unknown second band. This time annealing temperature is 61 °C, annealing time 10 sec.
OD measurement on devices (to make a standard curve with the plate reader, but the plate reader did not work).
2h incubation of InterLab samples at 37 °C, 1h at room temperature, then measurement of samples C by spectrophotometry and plate reader (Since that failed, I would also delete that point).

###### July 07

Preparation of LB + kan medium.
1 % inoculation of overnight culture in fresh LB + kan. Incubation at 30 0C for 6 hours. Measurement of the OD every 2h and observation of the bacteria number to the microscopy.
Results: 2 hours OD = 0.047, 4 hours OD = 0.3, 6 hours, OD = 0.5. Observation: 2 hours, microscopy shows the culture is not dense enough. 4 hours, microscopy shows the culture may be suitable for encapsulation. 6 hours, microscopy shows the culture may be a little too dense for encapsulation.

###### July 08

Inoculation of InterLab samples in 3 ml LB (+ Kan/Chl).
Prepared 400 ml PBS.
Plate reader measurement of InterLab samples (undiluted from ON culture).

###### July 09

1 % inoculation of samples Dev 1A – Dev 3B in 3 ml LB + Kan for flow cytometry.
1 % inoculation of samples Dev 3C – NCC in 3 ml LB (+ Kan/Chl) in the same way.
Purification of backbone PCR.
Nanodrop measurement of concentrations. 1: (9.0 + 9.3 + 9.6) ng/μl : 3 = 9.3 ng/μl. 2: (17.2 + 16.8 + 16.9) ng/μl : 3 = 17.0 ng/μl.

###### July 09

Preparation of alginate solution, and put this in the 4 0C fridge for future use.

###### July 10

Prepared TE buffer.
Added TE buffer to DNA.
HIFI (0.5 μl backbone DNA, 1 μl insert DNA Edd promoter), once with each of the purified backbones.

###### July 11

Heat transformation of *E. coli* DH5α competent cells with Edd promoter (HIFI).
Electroporation of *P. putida* with cells with Edd promoter.
Plating *P. putida* for overnight incubation.

###### July 11

Plate reader measurement of InterLab samples.

###### July 12

Checked plates, they did not grow.
Inoculation of *P. putida*.

###### July 12

Inoculation of InterLab samples in 3 ml LB (+Kan/+Chl).
Plate reader calibration.
Plate reader measurement of InterLab samples for 5 hours.

### Week 29

###### (13/07 - 19/07)

###### July 13

HIFI (0.5 μl backbone DNA, 1 μl insert DNA KguE1 and KguE2 promoter parts), once with each of the purified backbones.
Heat transformation of *E. coli* DH5α competent cell with KguE promoter (HIFI).
HIFI (0.5 μl backbone DNA, 1 μl Insert DNA edd promoter), once with each of the purified backbones, again.
Heat transformation of *E. coli* DH5α competent cell with Edd promoter (HIFI).
Preparation of LA plates (+ Kan).
Plating *E. coli* DH5α and ET12567 competent cells for overnight incubation.

###### July 13

Production of alginate capsules (first attempt). Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 5000 V, 15 ml/h, distance needle to gelling bath 1 cm.
Microscopy confirmed many capsules, and the diameter of nine of them was measured and was ranging from 214 and 275 micron, with an average of 249 micron. We will attempt to produce smaller capsules with a narrower size distribution.

###### July 14

Checked plates, they didn’t grow (only few colonies of edd promoter cells on some plates).
Inoculations of these colonies in LB for few hours and plating on LA for overnight.
Since we received very low amount of ordered DNA we decide to order primers to amplify it.
Design of primer sequence for KguE1, KguE2 and Kdd promoters.
Left yesterday’s plates for another day.

###### July 14

Production of alginate capsules (second attempt). Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 4000V, 15 ml/h, distance needle to gelling bath 1 cm.
Microscopy showed many capsules, the diameter of eight of them was measured and was ranging from 206 and 347 micron, with an average of 235 micron. We will attempt to produce smaller capsules with a narrower size distribution.
Production of alginate capsules (third attempt). Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 4000V, 15 ml/h, distance needle to gelling bath 1 cm.
Only big capsules, around 400 micron in diameter. We will attempt to produce smaller capsules with a narrower size distribution. Find out if keeping all parameters constant really makes capsules with the same size properties, or if something else in the procedure affects size and shape.

###### July 15

Yesterday’s plates grew, but very few like before.
2 days inoculated cells grew more.
No control plates did grow as expected.
ET12567 competent cells grew better than *E. coli* DH5α cells.
Inoculation of a single (doubt of taking only one) colony of edd1 and edd2 to LB + Kan medium.

###### July 15

Changed the medium of the Devices.

###### July 15

Written first version of the image analysis tool.

###### July 15

Production of alginate capsules. Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm.
The capsules are bigger than expected with diameters such as 286, 283, 263 microns.
Inoculation of a *P. putida* pHH+GFP that was stored in the 4 0C fridge since last week, 1 % in LB + kan medium. Incubation at 30 0C for 3 hours and OD check. After 3 hours of incubation, OD = 0.308.
Encapsulation of *P. putida* pHH+GFP in alginate. Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 4000V, 15 ml/h, distance needle to gelling bath 1 cm. Induction of the bacteria with m-Toluic acid.
Confocal microscopy, observation of encapsulated cells. Induction of GFP with m-Toluic acid did not work. The inducer should diffuse readily into the encapsulated cells through the alginate gel matrix, the lack of fluorescence is most likely due to using an old culture. Will attempt encapsulated cells from a fresh culture next time.
Inoculation of *P. putida* PHH+GFP from -80 0C in 3 ml LB + kan medium. Incubation at 30 0C.

###### July 16

Overnight inoculated cultures grew.
OD measurement: 0.3 to 0.5.
Plated them again to obtain single colonies.

###### July 16

1 % inoculation of overnight culture in fresh LB + kan. Incubation at 30 0C for 2h15. OD = 0.132.
Encapsulation of *P. putida* pHH+GFP in alginate. Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 4000V, 15 ml/h, distance needle to gelling bath 1 cm. Induction of the bacteria with m-Toluic acid.
Confocal microscopy, observation of encapsulated cells. Induction of GFP with m-Toluic acid did work. Presence of capsule with the fluorescent bacteria. The size of the capsules was ranging from 238 to 286 microns with an average of 272 micron.

###### July 17

Plates grew.
Single colony inoculation in LB + Kan.
Overnight incubation 37 0C.

###### July 17

Confocal microscopy of the encapsulated bacteria of the previous batch that did not work. This time the bacteria were induced with 5 mM of m-toluic acid. Fluorescence was observed.

###### July 18

Cells grew well in LB + Kan.
OD measurement: 0.2344.
2 % inoculation of cells.

###### July 19

Gel results were not clear.
Cells grew well in LB + Kan.
OD measurement: 0.2344.
Miniprep of Edd1 and Edd2.
Gel run to see sizes.
1 % inoculation of cells with Edd.

###### July 19

Inoculation of *P. putida* pHH+GFP from -80 0C in 3 ml LB + kan medium. Incubation at 30 0C.

### Week 30

###### (20/07 - 26/07)

###### July 20

Gel run to see sizes – results were not clear.
Gel run again – bands clearly visible, but ladder was not clear.
OD measurement: Edd1 – 1.9858, Edd2 – 1.8782.
HIFI (1 μl Backbone DNA, 2 μl Insert DNA KguE1 and KguE2 promoter parts), once with each of the purified backbones.
Heat transformation of *E. coli* DH5α competent cells with KguE promoter (HIFI).
Plating cells with KguE promoter for overnight incubation.

###### July 20

Preparation of gelling solution, washing solution + MQ water with NaCl for tomorrow’s alginate solution.

###### July 21

Checked plates, they didn’t grow.
PCR for KguE promoter, because our new primers arrived.
Gel run.
Inoculation of wild type *P. putida* from -80 0C and overnight incubation.

###### July 21

Preparation of alginate solution. Reduced the alginate concentration from 4.0% to 3.6% (1.8% in the finished encapsulation mixture).

###### July 22

Inoculation of wild type *P. putida* from overnight incubation.
Incubation for 2h.
OD measurement: 0.3 to 0.4.
Electrocompetent cells preparation.
Electroporation of *P. putida* with cells with Edd promoter.
Plating *P. putida* for overnight incubation.
Gel run to see sizes of Edd again – results were better this time, the band was between 3500 - 4000 bp.

###### July 23

Checked the electroporation plates, they didn’t grow.
Electrocompetent cells preparation, again.
Electroporation of *P. putida* with cells with KguE promoter (Last time something was wrong with volt shock).
Plating *P. putida* for overnight incubation.

###### July 24

Checked the electroporation plates, they didn’t grow.
PCR of KguE.
Gel run.

###### July 25

Checked plates, they didn’t grow.

###### July 26

Heat transformation of *P. putida* cells with Edd promoter.
Plating *P. putida* cells with Edd promoter for overnight incubation.

### Week 31

###### (27/07 - 02/08)

###### July 27

Checked plates, they didn’t grow.
Inoculation of wild type *P. putida* from -80 0C and overnight incubation.

###### July 28

Enzyme digest of KguE (EcoR1 and Pst1).
Backbone preparation.

###### July 29

PCR of KguE without primers.
Enzyme digestion.
Gel run.
Electrocompetent cells preparation.
Electroporation of *P. putida* with cells with Edd promoter.
Plating *P. putida* for overnight incubation.

###### July 30

Checked electroporation plates, they grew.
Overnight single colony inoculation (*P. putida*).
Enzyme digestion of KguE (EcoR1 and Pst1) again with 4x concentration.
Preparation of new LA plates (+ Kan).
Gel run, results in no band.
Ligation of backbone and KguE insert.
Heat transformation of DH5α with KguE.
Plating cells with KguE promoter for overnight incubation.

###### July 31

Checked plates, they didn’t grow.
Gel preparation (agarose).

###### August 01

Glucose preparation for medium.
*P. putida* incubation with new glucose medium.
Plate reading of *P. putida* with and without glucose in medium.
Results showed no fluorescence (mCherry).
HIFI and heat transformation for KguE promoter with DH5α competent cells.
Plating cells with KguE promoter for overnight incubation.

###### August 01

Prepared LA + kan plates for capsule leakage tests.

###### August 02

Checked plates, they didn’t grow.
Inoculation of *P. putida* with Edd promoter in new medium.

###### August 02

Inoculation of *P. putida* pHH+GFP in LB + kan.
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.

### Week 32

###### (03/08 - 09/08)

###### August 03

PCR of KguE with different annealing temperature.
Enzyme digest of KguE.
Miniprep of DH5α Edd and KguE.
Gel run.

###### August 03

5 % Inoculation of overnight culture in LB + kan.
Encapsulation of *P. putida* pHH+GFP in alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm.
Capsule leakage tests (encapsulated *P. putida* pHH+GFP stored in LB).

###### August 04

PCR of edd promoter.
Gel run.

###### August 04

Checked plates from leakage tests, substantial leakage observed.

###### August 05

Confocal microscopy after 1 day incubation of capsules.

### Week 33

###### (10/08 - 16/08)

###### August 10

Inoculation of pHH+mCherry DH5α cells to new medium + Kan.
Miniprep.
Gel run.

###### August 10

Prepared LA + kan plates.

###### August 11

Inoculation of pHH+mCherry DH5α cells to new medium + Kan.
Ordered new primers.

###### August 11

Inoculation of *P. putida* PHH+GFP in LB + kan.
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.

###### August 12

Cell encapsulation in alginate + poly-L-lysine HBr coating. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm.
Capsule leakage tests (encapsulated *P. putida* pHH+GFP with poly-L-lysine HBr coating stored in LB).

###### August 13

Looked at BioBrick protocol and preparations.
New primers arrived.
PCR of all promoters: Edd, KguE, Zwf and Gad.

###### August 13

Checked plates from leakage tests, substantial leakage observed. No improvement with poly-L-lysine HBr coating.
Analysis of results.

###### August 14

Measurements of PCR products and miniprep (backbone plasmid) concentrations on Nanodrop.
Digestion of backbone with Nde1 and Bste2 enzymes.
Digestion of PCR products (promoters).
PCR purification of all promoters (from PCR) and backbone.
Gel run to check sizes – results showed multiple bands (we needed only 2 bands).
Gel cut of highest 3 bands (ladder on gel was not so clear).
Gel cut DNA extraction.
Backbone plasmid separately digested with Nde1 and Bste2 enzymes.
Gel run again with all digested products and 2nd digestions.
Gel results showed that the Bste2 enzyme was cutting in many places.
Ligation of three gel cut DNA and one insert (Gad promoter).
Heat transformation of Dh5α competent cells with Gad promoter.
Plated cells on LA with Gad promoter for overnight incubation.

###### August 14

Prepared LA + kan plates for capsule leakage tests.

###### August 15

Checked plates, they didn’t grow.
Inoculation of new pHH+mCherry Dh5α cells to medium + Kan from -80 0C.

###### August 16

Miniprep of overnight inoculation pHH+mCherry Dh5α cells (backbone plasmid).
Measurements of miniprep (backbone plasmid) concentrations on Nanodrop.
Digestion of backbone with Nde1 and Bste2 enzymes (backbone preparation).
Gel run with digested plasmid backbone to see size.
Gel results showed Bste2 was cutting in many places again but this time less.
Ligation of plasmid backbone and one insert (Zwf promoter).
Heat transformation of Dh5α competent cells with Zwf promoter.
Plating cells on LA with Zwf promoter for overnight incubation.

### Week 34

###### (17/08 - 23/08)

###### August 17

Checked plates, they grew.
Single colony inoculation.
Digestion of all promoters (PCR products) with Nde1 and Bste2 enzymes (insert preparation).
PCR purification of digested products.
Ligation of backbone and other promoters.
Heat transformation of Dh5α competent cells with other promoter.
Plating cells on LA with other promoter for overnight incubation.

###### August 17

Inoculation of *P. putida* pHH+GFP in LB + kan.
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.

###### August 18

Checked plates, they grew (Edd, Kgu and Zwf).
Single colony inoculation (5 colonies from each plate).
Miniprep of overnight single inoculation (Zwf promoter plasmid).
Measurements of miniprep (Zwf promoter plasmid) concentrations on Nanodrop.
PCR it with our primers to check presence of promoter sequence.
Gel run to see PCR product (confirmation).
Gel results confirmed.
Electrocompetent cells preparation (Wild type *P. putida* with no plasmid).
Electroporation of WT *P. putida*.

###### August 18

Cell encapsulation in Alginate + poly-L-lysine HCl coating. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm.
Capsule leakage tests (encapsulated *P. putida* pHH+GFP + poly-L-lysine HCl coating stored in LB).

###### August 19

Checked plates: *P. putida* didn’t grow, electroporation didn’t work.
Miniprep of overnight single inoculation (Kgu and Edd).
Measurements of miniprep (Kgu and Edd) concentrations on Nanodrop.
PCR it with our primers to check presence of promoter sequence.
Gel run to see PCR product (confirmation).

###### August 19

Checked plates from capsule leakage tests, substantial leakage observed. No improvement with poly-L-lysine HCl coating.
Analysis of results.

###### August 22

Blunt ligation of Gad overnight.
Lig1 - PshA1, Lig2 - Pml1 and Lig3 - HPA1.

###### August 23

Heat transformation of Dh5α competent cells with overnight ligated plasmids.
Plating cells on LA for overnight incubation.

### Week 35

###### (24/08 - 30/08)

###### August 24

*P. putida* with Kgu and Zwf promoters are ready.
Checked plates, they grew (Gad promoter).
Single colony inoculation.

###### August 24

Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Inoculation of *P. putida* pHH+GFP in LB + kan.

###### August 25

Miniprep of overnight single inoculation (Gad).
Measurements of miniprep (Gad) concentrations on Nanodrop.
PCR it with our primers to check presence of promoter sequence.
Gel run to see PCR product (confirmation). Result of gel: was not confirmed.
Glucose medium preparation.
Incubation of *P. putida* with Kgu and Zwf promoters with glucose medium.
Digestion of Edd and Gad PCR products with PshA1, Pml1 and HPA1.
Gel run to see digestion result.
Gel extraction.

###### August 25

5 % inoculation of overnight culture in LB + kan + m-toluic acid (inducer).
Cell encapsulation in Alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm.
Capsule leakage tests (encapsulated *P. putida* pHH+GFP in PBS).

###### August 26

OD measurement of overnight incubated *P. putida*.
Plate reader (Spectrophotometry) of *P. putida* with Kgu and Zwf promoters.
Enzyme digestion.
Blunt ligation overnight.
Analysis of results.

###### August 26

Overnight inoculation of samples 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B and 3C.

###### August 26

Checked plates from leakage tests, much better results in PBS compared to LB.
Analysis of results.

###### August 27

Heat transformation of DH5α competent cells.
Plating cells on LA for overnight incubation.
Inoculation of *P. putida* in glucose medium.
2 hour incubation.
OD measurement of overnight incubated *P. putida*.
Plate reader (Spectrophotometry) of *P. putida* with Kgu and Zwf promoters.

###### August 27

Acquisition of images by confocal microscopy, devices 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B and 3C (each in technical triplicates).

###### August 28

Checked plates, they didn’t grow.
PCR amplification of all four promoters.
Inoculation of *P. putida* in different glucose media.
OD measurement of overnight incubated *P. putida*.
2 hour incubation.
Plate reader (Spectrophotometry) of *P. putida* with Kgu and Zwf promoters.
PCR of miniprep DH5α Kgu and Zwf plasmids with Prefix/Suffix primers.

###### August 28

Prepared LA + kan + 2mM m-Toluic acid plates for leakage tests.

###### August 28

Acquisition of images by confocal microscopy, control samples NCA, NCB, NCC, PCA, PCB and PCC (each in technical triplicates).
Analysis of images using quantitative image analysis based on ImageJ and MATLAB.

###### August 29

Gad digestion with Bste2 and Nde1 enzymes.
PCR amplification.
Nanodrop measurement of concentration.
Sticky end ligation.
Gel run for confirmation.
Heat transformation of DH5α competent cells.
Plating cells on LA for overnight incubation.
OD measurement of overnight incubated *P. putida*.
Plate reader (Spectrophotometry) of *P. putida* with Kgu and Zwf promoters.

###### August 30

Checked plates, they didn’t grow.

###### August 30

### Week 36

###### (31/08 - 06/09)

###### August 31

PCR of stock DNA (sent by company) with new Prefix/Suffix primers.
Digestion of backbone and insert with Pml1 and PshA1.
Gel run to check PCR products and digestion.
Measurement of PCR products and miniprep (GFP+pHH and mCherry+pHH) concentration on Nanodrop.

###### August 31

Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Inoculation of *P. putida* pHH+GFP in LB + kan.

###### September 01

Blunt ligation for Kgu and Zwf.
Heat transformation of DH5α competent cells.
Plating cells on LA for overnight incubation.
Enzyme digestion of new plasmid backbone and all four promoters.
Blunt ligation overnight.

###### September 01

5 % inoculation of overnight culture in LB + kan + 2mM m-toluic acid (inducer).
Cell encapsulation in Alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm.
Capsule leakage tests (encapsulated *P. putida* pHH+GFP in PBS).

###### September 02

Checked plates, they didn’t grow.
Heat transformation of DH5α competent cells with overnight ligation.
Plating cells on LA for overnight incubation.
New construction of two promoters on one plasmid.
Enzyme digestion with Xba1 and Spe1 of Zwf promoter plasmid as backbone and Kgu promoter as insert.
Blunt ligation overnight incubation.
M9 medium preparation.

###### September 02

Checked plates from leakage tests, similar results to last time.
Analysis of results.

###### September 03

Checked plates, they grew.
Single colony inoculation (5 colonies).
Inoculation of Kgu and Zwf *P. putida* in M9 medium.
Incubation for 2 hours.
Plate reader measurements.
Flow cytometry.

###### September 04

Proposed information theoretical modelling to Team Warwick for predicting the probability of bonding of Brixells.

###### September 04

Miniprep of all single colony inoculations.
PCR with prefix/suffix primers.
Gel run to confirm presence: confirmed.
Electrocompetent cells preparation *P. putida*.
Electroporation of wild type *P. putida*.
Plating *P. putida* cells on LA plates.

###### September 05

Check on plates: they all grew!
Single colony inoculation.

###### September 05

Preparation LA + kan + m-toluic acid plates for leakage tests.

###### September 06

Standard assembly for new constructs Kgu as backbone and Zwf as insert.
Digestion with EcoR1, Spe1 and Xba1 enzymes.
Run on gel to confirm digestion: confirmed.
Gel extraction.
Ligation.
Heat transformation of DH5α competent cells.
Plating cells on LA for overnight incubation.

###### September 06

Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Inoculation of *P. putida* pHH+GFP in LB + kan.

### Week 37

###### (07/09 - 13/09)

###### September 07

Checked plates, they didn’t grow.
Miniprep plasmids preparation for sequencing.
Inoculation of *P. putida* with all promoters in different media.

###### September 07

Cell encapsulation in Alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1.5 cm.
Capsule leakage tests (encapsulated *P. putida* pHH+GFP in PBS).

###### September 08

Miniprep preparation for sequencing.
Check on wild type *P. putida* and new *P. putida* with pHH-100 mCherry plasmid.
Inoculation of wild type for control.
Plate reader of overnight incubation in LB and LB + Glucose medium.

###### September 08

Checked plates from leakage tests, results were similar to first and second experiments in PBS.
Analysis of results.

###### September 09

Controls of wild type and *P. putida* with pHH-100 mCherry plasmid were confirmed.
Inoculation of *P. putida* in LB, LB + glucose and different concentrations of M9 medium.
Overnight incubation.

###### September 10

Plate reader of overnight samples.
Inoculation of *P. putida* in LB and different concentrations of M9 medium.
Overnight incubation.

###### September 11

Plate reader of overnight inoculations.
Inoculation of *P. putida* in LB and different concentrations of M9 medium
Overnight incubation.

###### September 12

Plate reader of of overnight inoculations.
Inoculation of *P. putida* in LB and different concentrations of M9 medium.
Overnight incubation.
RNA isolation of *P. putida* with our promoters for rtPCR.

###### September 13

Plate reader of Edd, Kgu, Zwf, Gad samples (3 biological and 3 technical replicates each) of overnight inoculations in M9 + 5%, 10%, 20%.
Inoculation of *P. putida* in LB in different concentrations of M9 medium.
Incubation of 3 biological replicates of Edd, Kgu, Zwf, Gad in LB.
Measured RNA concentration.
DNA free kit procedure.

### Week 38

###### (14/09 - 20/09)

###### September 14

Digest and Ligation of biobrick parts (BBa\_K184005, BBa\_K1840006, BBa\_K1840007, BBa\_K1840008) and pSB1C3
Heat transformation to competent E.coli

###### September 14

Deployed new iGEM Matchmaker version with keyword analysis and machine learning.

###### September 15

Plate reader of Edd, Kgu, Zwf and Gad samples in different M9 media incubated for 12 hours (3 biological and 3 technical replicates each) in LB with 0%, 5%, 10%, or 20%
Flow cytometry of pHH+mCherry as positive control
Inoculation of one colony from each transformation in LB+Chloramphenicol

###### September 16

Proposed and simulated Brixells based on Poisson-Boltzmann theory.

###### September 16

Inoculation of *P. putida* pHH + mCherry.
Inoculation of biological replicate A of Edd, Gad, Zwf + Kgu samples in M9 medium + glucose (1, 5, 10 + 20 %).
Miniprep of inoculated samples for biobrick part sending, digest with EcoR1 and Pst1, gelelectrophoresis shows only one band
Digest and Ligation of biobrick parts (BBa\_K184005, BBa\_K1840006, BBa\_K1840007, BBa\_K1840008) and pSB1C3
Heat transformation to competent E.coli

###### September 17

Plate reader measurement for 6 hours after 6 hours inoculation, and additionally after 12 hours inoculation of Edd, Kgu, Zwf and Gad samples in M9 media with 1%, 5%, 10%, and 20% glucose
Flow cytometry of Edd, Kgu, Zwf and Gad samples in M9 media with 1%, 5%, 10%, and 20% glucose
Acquisition of images of promoters Edd, Gad, Zwf + Kgu in M9 medium + glucose (1, 5, 10 + 20 %) by confocal microscopy.
Colony PCR with prefix/suffix primers to confirm the right contruct for biobrick part sending

###### September 17

Encapsulation of *P. putida* pHH+mCherry in alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1.5 cm.
Acquisition of z-stack images by confocal microscopy.

###### September 18

Miniprep of transformed cells with our Biobrick parts in the pSB1C3 backbone, preparation for sending

###### September 18

Analysis of survey responses.

###### September 18

3D Rendering of z-stack images obtained by confocal microscopy.

###### September 18

Quantitative analysis of confocal microscopy images of *P. putida* pHH+mCherry.

Retrieved from "[https://2013.igem.org/Team:Cornell/notebook](https://2013.igem.org/Team%3ACornell/notebook)" and "[https://2014.igem.org/Team:NTNU\_Trondheim/notebook](https://2014.igem.org/Team%3ANTNU_Trondheim/notebook)"

Template by [W3layouts](http://w3layouts.com/)