iGEM 2015 — Team Hamburg

# Team:Hamburg/Notebook

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  + [microRNA 2911](https://2015.igem.org/Team%3AHamburg/Project/miRNA)
  + [Killgene PezT](https://2015.igem.org/Team%3AHamburg/Project/PezT)
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## Notebook

### Monday, 22nd June 2015

Henning, Nikolas

* Notion: linearized plasmid backbones have been stored at room temperature for eight weeks. Maybe need to resupply.
* P4 mg/ml preparation of Chloramphenicol Stock (3 in EtOH)
  + dissolve 34 mg of Chloramphenicol in 1 ml 100% ethanol
  + filter through a 0.22 μm filter to sterilize
  + use at 1:1000 dilution in LB or LB-Agar
  + comment: maybe prepare bigger stock next time (10 ml). It’s not very exact working with 34 mg CAmp
  + marked as CAmp (top) and CAmp / 22.06.15 / 34 mg/ml (side) in Antibiotics
  + stored at -20 °C
  + (<http://www.cytographica.com/lab/solutions/Chlor.htm>)
* Preparation of LB-Agar plates with Chloramphenicol (CAmp)
  + heat 250 ml LB-Agar, cool down to ca. 50 °C and add 250 μl Chloramphenicol
  + Preparation of 20 plates under sterile conditions
  + stored at 4 °C, marked as CAmp Plates iGEM, 22.06.2015

### Tuesday, 23rd June 2015

Dana, Nikolas

* ‘Competent Cell Test Kit’
  + spin down DNA (0.5, 5, 10, 20 & 50 pg/µL RFP Construct BBa\_J04450, pSB1C3) from Competent Cell Test Kit ( 30 sec, 10.000 rpm)
  + thaw competent cells XL1 blue Tetracyclin resistent) on ice, label one 2 ml tube for each concentration ( 6 (five concentrations plus 1 control) + 1 tube for control of the backbone ‘’) and pre-chill
  + 1 µl of DNA in each tube, respectively
  + add 50 µl of competent cells to each tube, incubate for 30 min on ice
  + pre-heat water bath (42 °C), heat shock for 1 min
  + incubate for 5 min on ice
  + add 200 µl LB media, incubate for 1 h at 37 °C
  + pipet 20 µl on each plate (triplets, control only 1, 2 for backbone), respectively (add 50 µl LB media for easier distributing)
  + incubate overnight at 37 °CThe 50 pg/µL sample was forgotten on ice and the trafo will be repeated on wednesday

### Wednesday, 24th June 2015

Heiko, Jannis, Julian, Nikolas

* The trafo failed: there are no colonies on all plates -> repetition necessary
* ‘Competent Cell Test Kit’
  + repetition of the trafo using the 50 pg/µL solution. the 3 plates are stored at 37 °C in room 1.069
  + Use of DH5α instead of XL1 Blue in the Transformation, following the protocol used on tuesday
* Preparation of 10 mL Annealing buffer (10mM Tris, pH7.5-8.0, 50mM NaCL, 1mM EDTA)
* Preparation of 26 LB-Agar-CAmp plates (34µg/mL CAmp)
* to do on Wednesday (left over of protocol)
  + Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in
    the calculation.
    - (colonies on plate) / ng of DNA plated x 1000 ng/µg
    - Note: The measurement "ng of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:
    - 1 µL x concentration of DNA (refer to vial) x (volume plated / total reaction volume)

### Thursday, 25th June 2015

Heiko, Maurice, Vincent

* Counting the colonies in the trafos from Wednesday and calculating the transformation efficiency
  + Max. efficiency: ~ 3e+7 cfu / μg DNA
  + around one order of magnitude lower than iGEM standard
  + sealed the plates and put into 4 °C fridge
* Picked one colony from each plate and inoculated each into 10 mL of liquid LB-CAmp-medium
  + remaining LB medium in the 4 °C fridge
  + tubes in the 37 °C incubation room
* Annealing of the miRNA2911 oligos
  + got the amount of DNA from the delivery note
  + diluted both oligos to 100 μM
  + took 500 μL each and annealed in water bath at 94 °C for 10 min
  + cooling to room temperature in the water over 60 min
  + stored the solution at 4 °C in the fridge

### Friday, 26th June 2015

Henning, Nikolas, Sonja

* Mini-Prep of 10 ml cultures from 25.06. → Trafo of Backbones (Trafo efficiancy-kit)
  + → Protocol used: High speed plasmid medi kit (geneaid)
  + 1C3-50A: 0.133 µg/µl, V = 48 µl
  + 1C3-50A: 0.133 µg/µl, V = 49 µl
  + 1C3-BBc: 0.225 µg/µl, V = 49 µl
  + 1C3-BBc: 0.198 µg/µl, V = 49 µl
  + stored in freezer
* Annealing of GroEL oligos (old and new oligos)
  + total of 4 samples
    - note: the old GroEL-oligos were diluted with a (very) wrong amount of buffer and shouldn’t be used anymore
  + <https://www.addgene.org/plasmid-protocols/annealed-oligo-cloning/>
  + used 2 g each in 50µl Buffer total
  + Heatblock at 95 °C for 3 min and cool down slowly for approx 1 h
  + Stored as “GroEL Insert” in freezer
* Preparation of Agar-Plates with Kanamycin
  + 16 Plates
  + Kan (50 µg/ml)
  + Stored in Fridge (“26.06 LB Agar Kan”)

### Tuesday, 30th June 2015

Artur, Dana, Henning, Kira, Nikolas

* miRNA
  + Control of annealing → c=19.55 ng/µL
* Agarplates
  + without resistance for DH5ɑ
* DH5ɑ
  + distribution on agarplates without antibiotic → 37 °C
* GroEL
  + first annealing, no DS-DNA → repeated with waterbath (95°C)
  + 0.092 µg/µL → 4.6 µg (after 50x)

### Wednesday, 1st July 2015

Artur, Henning, Julian, Nikolas

* control of miRNA2911 and old GroEL-Annealing in Agarose-gel
  + both positive
* New annealing of GroEl-Promotor with full volume (300 µl each)
* Prepare start culture of DH5α for main culture on thursday
  + stored in 37°C room
  + three tubes stored in 37°C room
* Preparation of BW29655 Coli on agar plates for start culture on thursday
* Restriction of vector backbone (1C3) with EcoRI and PstI
* Ligation of Vector (1C3) with GroEL-Insert (first annealing from last week)
* protocols are in the hardcopy labbook

### Thursday, 2nd July 2015

Artur, Dana, Henning, Kira, Nikolas

![](https://static.igem.org/mediawiki/2015/7/75/Hamburg_notebook_giessen.JPG)

* Repetition of annealing control in gel
  + miRNA2911 positive, GroEL negative (false sample)
* Ligation of GroEL in 1C3-Backbone
* Repetition of annealing control in gel
* Preparation of main culture DH5α
* storage of BW29655 plates in fridge for miniprep on monday

### Friday, 3rd July 2015

Heiko, Maurice, Vincent

* measured the OD600 of the competent DH5α cells => 0,44
* prepared new Annealing Buffer
  + 0.5 M EDTA (Shini)
  + 5 M NaCl (Shini)
  + 1 M Tris (self-made:made in 10 mL ddH2O, pH 8.04)
* Annealing Buffer:
  + EDTA 20 µL
  + Tris 100 µL
  + NaCl 100 µL
  + + H2O
  + - → 10 mL
* diluted new FW miRNA 573 µL (73 µL still in the freezer, orange box)
* diluted new REV miRNA 769 µL (269 µL still in the freezer, orange box)
* Annealing (94°C) (water quench, 10 min, then cooling at RT)
  + → product in freezer
* OD600 = 0.5
* gone on working with cells from 02/07/15
  + protocol “Kompetente Zellen” (Shini)
  + end: 13:50h, cells at -80°C

### Monday, 6th July 2015

Dana, Heiko, Henning, Sonja

* measured the concentration of the miRNA-Annealing from friday → 2.2 µg/µl
* Ligation of the miRNA-Insert in pSB-1C3 (cut with EcoRI & PstI)
* forgot the ligation control again -.-
* efficiency kit used for trafo-control
  + competent DH5α + 0.5 - 50 ng RFP construct
* Trafo of GroEL (2x) and miRNA in DH5α
* Preparation of start culture with BW29655
  + 4x 5 ml without any AB!
  + Stored at 37°C in shaker

### Tuesday, 7th July 2015

Artur, Heiko, Vincent

* Preparation of SOC-medium (instruction “Roche, Lab FAQ”, by Dagmar)
  + Bacto-Tryptone 2.000 g
  + Bacto-Hefe-Extract 0.500 g
  + NaCl 0.050 g
  + KCl 0.019 g
  + MgCl2 \* (H2O) 6 0.203 g (without (H2O)6 -> 0.095 g)
  + Glucose 0.036 g
  + pH 7.0 with pH-Meter
  + 10 mL (fridge)
* Transformation with Competent Cell Test Kit (5 diff. conc.: 1.5, 5, 10, 25, 50 pg/µL) in 5 aliquots of DH5α
  + each with 50 µL DH5α + 1 µL DNA
  + Incubation on ice (30 min)
  + Heat shock (water, 42°C)
  + 5 min on ice (rather 15 min)
  + 200 µL SOC in tubes
  + Incubation for 2 h at 37°C with rotation
  + Plating (2 plates each: 20 µL + 200 µL) → 10 plates
  + Incubation 37°C over night
* 2 Glycerol stocks from BW29655 liquid cultures
  + 500 µL (50% Glycerol, 50% H2O)
  + 500 µL BW29655
  + → - 80°C
* Pooling of the liquid cultures of BW29655 in 200 mL LB medium (sterile)
  + → 37°C

### Wednesday, 8th July 2015

Heiko, Henning, Nikolas, Sonja

* Preparation of Agar plates with Chloramphenicol (CAMP, 8.7.2015)
* Transformation of miRNA-Ligation and GroEL-Ligation product as well as PezT-Plasmid (Kan resistance!!!) in DH5α
  + each with 50 µL DH5α + 1 µL DNA
  + Incubation on ice (30 min)
  + Heat shock (water, 42°C)
  + 5 min on ice (rather 15 min)
  + 200 µL SOC in tubes
  + Incubation for 2 h at 37°C with rotation
  + Plating (2 plates each: 20 µL + 200 µL) → 10 plates (using kan resistance for PezT-Plasmid)
  + Incubation 37 °C over night
* Preparation of new start cultures of BW29655
  + 3x 5 ml “iGEM a,b, c BW”
  + stored at 37°C over night

### Thursday, 9th July 2015

Artur

* Sealing of the plates with the transformation and storing in the fridge
* Storing the BW29655 liquid cultures in the fridge

### Tuesday, 14th July 2015

Maurice, Vincent

![](https://static.igem.org/mediawiki/2015/4/41/Hamburg_notebook_150714.JPG)

Picked liquid cultures of miRNA and pezT

* Evaluation of the transformation from July 8th
  + colony counts in the physical labjournal
Preparation of ca. 170 mL LB+Kan medium (30 µg/mL Kan)* + stored in the fridge
* Picked one miRNA colony and 3 pezT colonies
  + transferred into 5 mL of LB (+CAmp/Kan) each
  + incubation at 37 °C over night
* Transfer of 500 µL BW29655 culture into 50 mL LB for competent cell protocol
  + incubation at RT and 200 rpm over night

### Wednesday, 15th July 2015

Dana, Julian, Kira

* OD of BW29655 culture was too high (4), stored in the freezer ; again 500 µL BW29655 culture were transferred into 50 mL LB for competent cell protocol
* over night culture of miRNA and 3 pezT colonies were prepped, stored in freezer in the second box, blue label on top and blue label on each eppi

- First cell cultures checked again at 5 pm OD600: 5
- We stopped the growth of the new cells via freezing (in the Freezer, with the date of today),
- please restart growing step in the 37°C room.

### Thursday, 16th July 2015

Henning, Nikolas, Heiko

* digestion control of prepped miRNA and pezT from wednesday. All controls were positive.
* Maybe we pipetted enzyme to all the controls

+ digested with EcoRV and XhoI

* Repetition of the digestion controls

+ digested with EcoRV and XhoI

* digest of vector plasmid (prepped from trafo in first week)

+ digested with EcoRI and SpeI
+ again the control was positive (3 fragments)
+ isolated and prepped the supposed fragment
+ control of the prepped fragment in gel together with repetition of the control
+ used two controls, one with alternative buffers and loading dye, one as before

### Friday, 17th July 2015

Henning, Nikolas, Heiko

* Glycerol-Stock (50%)

+ sterile filtration

* pezT-Backstock

+ for each (A,B,C), in -80°C

* New Pipes

### Monday, 20th July 2015

Nikolas, Sonja

* TB PIPES Buffer
* für 100 mL:

+ 1.865 g KCl
+ 0,22 g CaCl x 2 H20
+ 2 mL 0.5 M PIPES

* Auf pH 6.7 bringen

+ 0,889 g MnCl2 x 2H2O
+ ddH2O ad 100 ml

* Restriktion des 1C3 BB:

+ 4 µL pSB-1C3 Vector
+ 1 µL EcoRI
+ 1 µL PstI
+ 2 µL FD buffer
+ 2 µL ddH20

* 1 h 37° C stored in fridge

### Tuesday, 21st July 2015

Nikolas

* Ligation GroEl in 1C3:

+ 2 µL T4 Ligase buffer
+ 1 µL T4 Ligase
+ 9 µL 1C3 BB
+ 2 µL “i2” insert
+ 6 µL ddH20

* DNA concentration in GroEL tubes

+ GroEL 1: 2 ng/µL
+ i: 2.5 ng/µL
+ GroEL Insert: 171 ng/µL

* Kompetente Zell Kultur anlegen

+ OD600= 0.066 (14:30)

### Wednesday, 22nd July 2015

Nikolas, Henning, Sonja

* New GroEl ligation (see Tuesday)

### Thursday, 23rd July 2015

Dana, Henning

* Make BW29655 competent

+ OD measurement of BW29655 → OD600 = 0.644 → broke centrifuge :(

* Cut pSB\_1C3 with EcoRI & PstI

+ 20 µL pSB-1C3 Vector
+ 1 µL EcoRI
+ 1 µL PstI
+ 3 µL FD buffer
+ 5 µL ddH20

* 20 minutes at 37 °C, clean with Kit → measure concentration : 23 ng/µL (460 ng)
* Ligation

+ 1 µL pSB-1C3 (23 ng)
+ 1/ 1.5 µL GroEl-Insert (35 ng) / miRNA (33 ng)
+ 2 µL T4 Buffer
+ 1 µL T4 Ligase
+ 5 µL ddH2O

* 15 minutes at room temperature, then 10 min at 65 °C
* Transformation

+ 1 µL Ligation Product
+ 50 µL DH5α competent cells
+ 30 minutes on ice, 1 min heat shock (42 °C), 5 min on ice
+ add 200 µL SOC media, 2 h at 37 °C
+ plate out on C-amp-plates

### Friday, 24th July 2015

Heiko, Vincent

![](https://static.igem.org/mediawiki/2015/3/3c/Hamburg_notebook_nitrogen.JPG)

Freezing the competent cells in liquid nitrogen

* Trafo results:
* 2 measurements OD600 ( 11:00, 12:30) = 0.00
* use of the competent cells from 17/07 “BW 2. Versuch”OD600 = 0.546
* Protocol “Kompetente Zellen”
* use of the centrifuge in the 3rd floor (Rm. 3.004)
* N2 (l) (80°C) at 15:00

### Monday, 27th July 2015

Nikolas, Sonja

* miRNA-mini preparation

+ 3 tubes (source: 200 µL plate)
+ 37 °C

* GroEl ligation (NEW)

+ **1) cut pSB1C3 with EcoRI and PstI**
+ 1 µL EcoRI
+ 1 µL PstI
+ 2 µL pSB1C3
+ 1 µL Buffer
+ 5 µL a. bidest.

* → 37 °C for 15 minutes
* → 65 °C for 10 minutes heatshock
* **2) Backbone dephosphorylation**

+ 10 µL pSB1C3 (cut)
+ 1 µL FastAP
+ 4 µL FastAP Buffer

* → 37 °C for 30 minutes
* → purify with PCR clean up kit
* **3) Ligation**

+ 20 µL pSB1C3 (dephosphorylated, cut)
+ 10 µL Insert
+ 8 µL T4 Buffer
+ 4 µL T4 ligase

* Transformation Efficiency Kit für BW\_29965 (see iGEM protocol)

### Tuesday, 28th July 2015

Dana

* miRNA preparation:

+ OD was too low for preparation (Probes were not on the shaker)
+ → Julian will pick new colonies on wednesday
+ → Preparation on Thursday

* GroEl-Ligation → Trafo

- 10 µL Ligation + 50 µL DH5α
- 30 minutes on ice, 1 min heat shock (42 °C), 5 min on ice
- add 200 µL SOC media
- incubate for 2 h at 37 °C
- plate out on C-amp-plates
- Transformation Efficiency- BW29655

* → all but one were not turned upside down for incubation at 37 °C over night
* Explanation for DNA per Plate:
* (0.5;5;10;20;50) x (20 or 200 µL-volume plated/ 250 µL -total reaction volume (50 µL cells + 200 µL SOC)) x 1 ng/1000 pg

- Efficiency:

* (colonies on plate) / ng of DNA plated x 1000ng/µg
* for 20 µL-Probes
* 10 pg/µL → 1/0.0008 cfu/ng x 1000 ng/µg → 1.25 e+3 cfu/µg
* 20 pg/µL → 1/0.0016 cfu/ng x 1000 ng/µg → 6.25 e+5 cfu/µg
* 50 pg/µL → 3/0.004 cfu/ng x 1000 ng/µg → 7.5 e+2cfu/µg
* for 200 µL-Probes
* 5 pg/µL → 10/0.0004 cfu/ng x 1000 ng/µg → 2.5 e+7cfu/µg
* 10 pg/µL → 12/0.008 cfu/ng x 1000 ng/µg → 1.5 e+7 cfu/µg
* 20 pg/µL → 125/0.016 cfu/ng x 1000 ng/µg → 1.6 e+7 cfu/µg
* 50 pg/µL → 70/0.04 cfu/ng x 1000 ng/µg → 1.75 e+7 cfu/µg

### Wednesday, 29th July 2015

Julian

* Picked the colonies, named them and put them into 3 glasses each filled with 5mL LB+CAmp with a colony inside.
* Counted the colonies on the prepared agar plates: All were white.

+ Control: no colonies
+ 20µL : no colonies
+ 200µL, 1st pick: 23-26 colonies, and about 10 really little ones
+ 200µL, 2nd pick: 27-30 colonies, and about 10 really little ones

### Thursday, 30th July 2015

Nikolas, Sonja

* miRNA plasmid isolation
* NanoDrop

+ c(04)= 0.035 µg/µL
+ c(05)= 0.038 µg/µL
+ c(06)= 0.068 µg/µL

### Friday, 31st July 2015

Kira, Vincent

* Preparation of the GroEL-minis

+ stored in freezer
+ concentration: (1)= 118 ng/µL, (2)= 178 ng/µL, (3)= 233 ng/µL

### Monday, 3rd August 2015

Heiko, Julian

* GroEL (picked colonies)

+ instruction “Presto Mini Plasmid Kit”

- at step 7 method 2 used for higher DNA concentration
- each time 3 min absorption time
- last elution: centrifugation for 4 min at 20°C

* Results from the picked miRNA (29.07.15)

+ Resistance with BW29655 in 5µL each:

→ CAmp, Kan, Without
→ 37°C over night
* Preparation of new agar plates with CAmp resistance

### Tuesday, 4th August 2015

Henning

* Redistribution of some samples from 37°C to freezer and the other way round

### Wednesday, 5th August 2015

Henning, Heiko, Vincent

![](https://static.igem.org/mediawiki/2015/d/d7/Hamburg_notebook_picking.JPG)

Picking the colonies

* Plating of pezT (14.07.15) (l) A, B, C on plates (200 µL each)
  → 37 °C* Picking from plates: The 5 BBas, miRNA (23.07.15), GroEL
    + pipette-tips in 5 mL LB+CAmp each+ two preparations each
        → 37 °C* Mutagenesis-PCR
      + “PCR-Phusion protocol” (NEB)+ primers diluted to 10 µM+ charging the PCR (middle PCR device block 2, program 42 in directory “Katja”)* supporting information:
        + dNTPs from Katja, primers solved in Annealing buffer because lyophilized+ concentration with NanoDrop (volume: 4 µL) : 41 ng/µL

### Thursday, 6th August 2015

Dana

* PCR-Mutagenesis (1&2), check on gel, two different dilutions
* 10 µL: 9 µL sample + 1 µL loading dye
* 10 µL: 2 µL sample + 7 µL water + 1 µL loading dye
* new PCR (3)
* Nanodrop: PCR 1: 148 ng/µL
  PCR 2: 175 ng/µL
  PCR 3: 68 ng/µL
  * PCR-fragment 3 not detectable
  * light-inducable promotor:
  + 1 : BBa\_K801042
  + 2 : BBa\_K801041
  + 3 : BBa\_K801043
  + 4 : BBa\_K1017301
  + 5 : BBa\_K1017726* prepped samples (ng/µL)
  + 1A : 375 1B : 328
  + 2A : 275 2B : 275
  + 3A : 252 3B : 265
  + 4A : 178 4B : 157
  + 5A : 108 5B : 68

### Friday, 7th August 2015

Dana

* pezT-Mutagenesis
  + PCR-Fragments 1 + 2, gel clean up, cut out of gel+ Gel extraction with kit, probes are labelled as 1 + 2 in the second box in the freezer

### Monday, 10th August 2015

Heiko, Julian

![](https://static.igem.org/mediawiki/2015/9/9b/Hamburg_notebook_pcr.JPG)

Programming the PCR

* new pezT plates plated out (How to do IPTG induction?)* PCR (5x buffer, Phusion Polymerase, DMSO from Siegfried)* DNA concentration from purified PCR 1, 2
      + 1 230 ng/µL+ 2 100 ng/µL* Biometra Thermocycler (DNA under 1 kB) ??* At “Ulrike” number 15, IGEM* Again PCR 1 + 2* gel: PCR 3, 2, 1

- 9 µL product + 1 µL loading dye

### Tuesday, 11th August 2015

Henning

Extraction of Dna from gels isolated yesterday. Concentrations are too low.
Next time prepare full volume of PCR-product in Gel for the Extraction.

### Wednesday, 12th August 2015

Heiko, Julian, Dana

* pezT

+ repeated thinning streak to get single colony

* Mutagenesis-PCR

+ repeated PCR 1 & 2
+ load result onto gel
+ gelextraction
+ concentration PCR 1: 8 ng/µL PCR 2: 10 ng/µL

* Lightinduceable promotor:

+ trafo of BBa\_…(1A,2A,3A,4A,5A) into BW29655
+ 10 µL of BBa each

### Thursday, 13th August 2015

Heiko, Vincent

* Mutagenesis-PCR
  + gel extraction+ PCR 3 with cleaned PCR fragments+ PCR 4 (iGEM MUT4)* Lightinducable promotors
    + picked single colonies from trafo into 5 mL LB (+Camp)* pezT
      + picked single colonies into 5 mL LB (+Kan) (1,2,3)

### Friday, 14th August 2015

Dana

* Lightinducable Promotor:

+ Glycerolstocks (stored at -80°C)

* pezT-Mutagenesis

+ Restriction:

- 20 µL PCR4
- 1 µL EcoRI
- 1 µL PstI
- 1 µL FD Buffer
- 5 µL ddH20
→ 20 minutes at 37 °C

+ Clean up with PCR-Clean Up Kit
+ Concentration after clean up: 38 ng/µL
+ Ligation

- 1 µL Vektor (pSB-1C3, 23 ng)
- 1.2 µL pezT-Insert (38 ng)
- 2 µL T4 Buffer
- 1 µL T4 Ligase
- 5 µL ddH20 (6 µL for control)
→ 20 minutes at RT, 10 min 65°C

### Tuesday, 18th August 2015

Dana

* Constitutive promoter (BBa\_J23100, 35 bp) (for 3A-Assembly with miRNA)

+ Amp-Plates
+ Biobrick on Plate 4 of distribution kit;
   Well 17D, in Plasmid Backbone BBa\_61002, Amp-Resistance+ punch hole into foil of cover, add 10 µL of dH2O, pipette up and down,
     let sit for 5 minutes+ transfer into tube with competent cells (BWs), two seperate samples,
       5 µL into each, + one control, trafo

### Wednesday, 19th August 2015

Henning

* Constitutive promoter (BBa\_J23100, 35 bp) (for 3A-Assembly with miRNA)
  + All plates were positive including control. Maybe the BW are resistant against Amp?+ Inoculation of new Amp plates with competent BW (20 µl) for control+ other samples are not usable either (too much bacteria)* PezT
    + control gel of ligation and ligation control from monday.+ 2 µl sample + 7 µl water + 1 µl LD+ Ligation seems not to be successful due to two equal signals in sample and control.+ Repetition is necessary.

### Friday, 21st August 2015

Henning, Maurice, Vincent, Artur

![](https://static.igem.org/mediawiki/2015/4/42/Hamburg_notebook_loading.JPG)

Loading the samples into the gel

* Supplies

+ made approx. 20 new Amp-plates
+ stored in fridge

* Transformations

+ pezT ligation product into DH5α (two batches -> Ligation A and B)
+ BBa\_J23102 into DH5α (two batches -> Promoter A and B)
+ BBa\_K592009 into DH5α (two batches -> Pigment A and B)

- plated all and incubated at 37 °C

* PezT amplification PCR

+ PCR of the mutagenesis PCR product with the standard Fusion protocol

* control gel of PezT amplification PCR

+ measured the concentration of the PCR product

* control gel of PezT amplification PCR

+ measured the concentration of the PCR product
+ control gel with 1% agarose
+ discard the sample due to strong impurity

* Preparation of PezT containing 5 ml cultures for IPTG induction

+ made new liquid culture from glycerol stock in LB + Kan medium

- incubated at 37 °C

+ preparation of 9 different dillutions of 3 samples to get good OD on saturday
+ 1:5, 1:10 and 1:20 dilutions of samples with OD 0.8

### Saturday, 22nd August 2015

Nikolas, Sonja

* Picking Minis (Duplicates):

+ Pigment A (in CAMP LB)
+ Pigment B (in CAMP LB)
+ Ligation A (in CAMP LB)
+ Ligation B (in CAMP LB)
+ Promoter A (in Amp LB)
+ Promoter B (in Amp LB)

* Incubation at 37° C
* IPTG Induktion von pezT Colis:

+ OD measurement of prepared cultures
+ selecting culture with an Od of ~0,4 (OD=0,354)
+ Dilute selected culture in 20 ml LB
   and split those in 2 Erlenmeyerflasks (~12 mL each, control+sample)+ IPTG induction by adding 12 µl 1 M IPTG
  + OD measurement every 20 minutes

* We noticed a constant decline in the OD
   and decided to abort the experiment after 1 h

### Sunday, 23rd August 2015

Henning

* Glyercolstock of trafo samples from saturday

+ added 300 µl Glycerol (70%) to 700 µl bacterial culture
+ stored at -70 °C (one night at -20 °C)

* Preparation of mini-cultures from saturday

+ preparation of all mini cultures from saturday
+ Ligation, pigment and promoter
+ appropriate to the kit’s protocol
+ measured the concentration
+ stored at -20 °C

* Control digestion of the PezT ligations

+ 100 µg sample DNA
+ 1 µl EcoRI
+ 1 µl PstI
+ 1 µl FD Buffer
+ x µl ddH2O
+ 20 minutes at 37°C
+ loaded on 1% agarose gel

* Dilution of pezT cultures for IPTG induction

+ prepared two culture dilutions
+ approx. 125 ml LB with Kan each
+ added half of the control culture from Saturday
+ stored one in the fridge and the other down to the bench at RT

### Monday, 24th August 2015

Artur, Vincent

* Repetition of the control digestion

+ beside ligation sample volumes:

- 1µL EcoRI
- 1µL PstI
- 1µL FD Green Buffer
- ad 10 µL H2O

+ checked with control gel
+ suggestion: repeat again with positive control of unmutated PezT

* creation of miRNA probe

+ four single stranded PCRs with VF or VR primers and miRNA insert over night/ 100 cycles
+ suggestion for further steps: tag with 32P via 32P-ATP and PNK
+ (stored at -20° C, Tuesday)

* IPTG induction of PezT culture

+ first try: three samples with

- LB-Kan
- PezT cells + IPTG (1mM)
- PezT cells - IPTG (1mM)

+ second try:

- PezT culture growth over night

### Tuesday, 25th August 2015

Artur, Maurice

* Continuing IPTG induction of PezT culture

+ measured ODs
+ no recognition of sufficient OD decrease in both tries
+ suggestion: make new IPTG stock

* 3A-Assembly

+ of GroEL biobrick with pigment biobrick and constitutive promoter with miRNA
   following the iGEM protocol (restriction and ligation), both in pSB1K3 backbone (Kan resistance)+ protocol changes/used materials: fast digest restriction enzymes and buffer,
     no BSA, our samples (pigment A1 from 23.08., promoter A1 from 23.08, GroEL from 31.07 (233 ng/µL),
     miRNA (elution) from 30.07 (38 ng/µL)), 1:10 dilutions of samples (except miRNA)+ obtained two samples (1 GroEL + pigm 3A Assembly, 2 prom + miRNA 3A Assem) for transformation,
       stored at -20° C

### Thursday, 27th August 2015

Dana, Vincent

* pezT IPTG induction

+ plated liquid cultures from the 26th on Kan-plates (20 µL each)

- incubated at 37 °C

+ OD600 of liquid cultures with and without IPTG was the same
+ plates with and without IPTG are similarly covered with colonies

* GroEL+Pigment & Promoter+miRNA

+ picked colonies into liquid cultures

- incubated at 37 °C

* Colony PCR

+ picked colonies from miRNA, GroEL, GroEL+Pigment and Promoter+miRNA

- 8 colonies each

+ made reciprocal plate for picked colonies

- incubated at 37 °C

+ used DreamTaq-Mix
+ made control gel

### Friday, 28th August 2015

Nikolas, Sonja

* GroEL+Pigment & Promoter+miRNA

+ Miniprep, DNA stored at -20 °C
+ Glycerol Stock; stored at -80 °C

* pezT Colony PCR

+ Preparation of 2 plates from liquid culture pezT A

* Incubation at 37 °C

### Saturday, 29th August 2015

Julian

* pezT Colony PCR

+ Put the cultures in the fridge

### Monday, 31st August 2015

Artur, Nikolas

* pezT Colony PCR

+ Picked 2 colonies from pezT plates (stored in the fridge)
+ used DreamTaq MM
+ Made control gel

* GroEL+Pigment

+ Diluted cultures A and B to an OD of 0,45
+ incubated 20 min at 37 °C
+ Heatshock37 in water bath 1 min 42 ° C
+ incubated at 37 °C
+ Decided to repeat the heatshock for culture B to ensure sufficient stress level

### Tuesday, 1st September 2015

Heiko, Julian, Vincent

* GroEL+Pigment

+ cultures from 31st August did not express the pigment
+ diluted cultures in liquid medium and incubated at 42 °C over night

* pezT Colony PCR

+ repetition of colony PCR with DreamTaq-Mix

- Meinhart plasmid, the PezT preparations A, B and C,
   a colony from the dilution plating, negative control

+ control gel in 1 % agarose
  - two fragments in the Meinhart plasmid and the PezT preparations A and B at ca. 3000 nt and 2500 nt

### Wednesday, 2nd September 2015

Nikolas, Julian, Maurice

* pezT Colony PCR

+ Colony + pezT A, B, C
+ Important: Strip tubes/lids are leaky -> The water in a few samples evaporated just like yesterday -> no PCR

* Light Promoter 3A Assembly

+ 3A assembly of BBa\_K1017726 and BBa\_101301 according to the iGEM protocol
+ Ligation stored in freezer
+ Important: The BBs might be in bacteria, we inoculated a mini culture to test this possibility

### Thursday, 3rd September 2015

Heiko, Maurice

* analysis of ‘BBs in bacteria’ suspicion

+ no growth in Kan mini cultures and C mini cultures (BBa\_K1017726 and negative control),
   but growth of C mini culture with BBa\_101301+ nevertheless continuing the transformation protocol, could be false positive

* transformation of Light Promoter 3A Assembly ligation product

+ four samples: E/P restricted pSB1K3 BB (Kan plates), 3A assembly ligations product (Kan Plates),
   negative control with DH5-alpha only (Kan plate) and positive control of pSB1C3-RFP standard biobrick (brown eppi) (C plates)+ growth over night in 37° room

* repetition of pezT colony PCR

+ scheme like yesterday
+ gel with PCR products

### Friday, 4th September 2015

Nikolas, Sonja

* Annealing miRNA and GroEl

+ 10 µL miRNA fw + 10 µL miRNA rev
+ 10 µL GroEl fw + 10 µL GroEl rev

10 min at 94 °C
Cool down to RT slowly* pSB1C3 restriction

+ 10 µL pSB1C3 RFP
+ 1 µL EcoR1
+ 1 µL Pst1
+ 2 µL FD buffer
+ 6 µL ddH2O

30 min at 37 °C
Heat inactivation 80°C for 20 min* pSB1C3 dephosphorylation

+ 10 µL pSB1C3 (cut)
+ 1 µL FastAP
+ 2 µL FastAP buffer
+ 7 µL ddH2O

30 min at 37 °C
Cleaning of pSB1C3 with PCR Clean Up Kit* NanoDrop

+ pSB1C3 (dephosphorylated) 8 ng/µL

* Ligation

With miRNA old and new, with GroEl old and new+ 3 µL pSB1C3
+ 1 µL insert
+ 2 µL T4 buffer
+ 1 µL T4 ligase
+ 3 µL ddH2O

* Transformation

GroEl old and new, miRNA old and new in DH5 alpha+ DH5α 30 min on ice
+ 1 min heat shock 42.2 °C
+ 15 min on ice
+ ad 200 µL SOC-medium
1 h and 10 min, 37 °C plate rotator
Samples plated on cAmp plates:

- 200 µL
- 20 µL each
- 1 neg. control a 200 µL

Stored at 37 °C overnight.
* New SOC-medium

+ 200 µL aliquots in freezer

* GroEl+ pigment A, B mini

+ 5 mL overnight culture in LB Kan medium
Stored at 37 °C.

### Saturday, 5th September 2015

Nikolas, Sonja

No colonies after transformation on Friday.* NanoDrop

(For annealings)+ GroEl new 4.26 µg/µL + 50 µL annealing buffer
+ GroEl old 0.0 µg/µL
+ miRNA new 2.313 µg/µL + 50 µL annealing buffer
+ miRNA old 0.0 µg/µL

* Maxi for GroEl+ pigment A, B

+ 300 mL culture
Overnight 37 °C
(were not prepped)
* GroEl promotor test

+ 1.5 µL per sample
+ 10, 20, 30 min heat shock (42 °C)
samples transferred to 5 mL overnight cultures after heat shock, stored at 37 °C.
* Backbone restriction

200 ng plasmid of each sample:+ pSB1C3: 8 µL
+ BB 16.7.: 14.3 µL
+ BBC 26.6.: 1.2 µL
For each sample:

- 3 µL FD buffer
- 1 µL EcoR1
- 1 µL Pst1
final sample volume 20 µL
* Backbone dephosphorylation

+ 20 µL restriction product
+ 1.5 µL FastAP
+ 2.4 µL Fast AP buffer
30 min 37 °C
Stored in freezer at -20 °C.

### Sunday, 6th September 2015

Julian, Henning

* Preparation of new LB-Agar-Camp plates

+ Preparation of 36 new Chloramphenicol containing LB-Agar plates

* PCR-Clean-Up of dephosphorylated Ligations from Sep. 5th

+ using PCR-Clean-Up Kit
+ concentrations of eluted samples are noted in the hardcopy labbook

* Annealing of miRNA oligos

+ mixed 100 µl of miRNA and GroEL oligos (100 mM each)
+ heated up to 95°C (heatblock)
+ slowly cool down to RT (approx. 2h)
+ measured concentration (hardcopy labbook)

* Digestion of pSB-1C3 backbones

+ used different samples of backbone

- linearized backbone, provided by iGEM (50 µg total)
- backbone from stock preparation (very old)
- backbone from RFP-construct (very low concentration (pmol)??)

+ digested with EcoRI and PstI for 20 min (FD)
+ heat inactivation of enzymes at 65°C for 20 min

* Dephosphorylation of pSB-1C3 backbones

+ dephosphorylated with fast AP for 10 min at 37°C
+ PCR-Clean-Up of all samples

- Not necessary, fast AP ist heat sensitive!

+ concentrations are noted in the hardcopy labbook

* Ligation of miRNA and GroEL inserts with pSB-1C3 backbones

+ used insert samples from different steps of the hole experimental phase
   and from the annealing from the same day+ Prepared different combinations of Insert and backbone samples
  + Used different concentrations of insert (3:1 & 30:1)
  → 19 samples+ Ligated with T4 DNA ligase for 20 min at RT
  + Heat inactivation at 65°C for 10 min
  + chilled on ice

* Transformation of ligation constructs
  + used a total of 24 different samples (19 new + 5 old ligation constructs)
  + added 12 µl of ligation to 50µl of competent DH5a
  + incubation on ice for 30 min
  + Heatshock at 42°C for 30 sec
  + chilled on ice until use (approx. 5 min)
  + Added 950 µl LB medium (RT)
  + Incubation at 37°C for 30 min while shaking
  + spreaded 200 µl of cells and ligation mixture to LB-Agar plates (RT, Camp, very thin!)
  + Incubated over night at 37°C

### Monday, 7th September 2015

Dana

* Trafos from yesterday (sunday 6th)

+ no growth on any plate, put back into 37 °C→ on the table on the left

* New trafos

+ all ligations from sunday 6th were transformed into DH5a
+ 2 hours incubation with SOC-medium
+ labelled 2-18, on the table in 37°C room on the right

* pSB1C3-RFP-positive control

+ picked 3 colonies, minis in 37 °C

### Tuesday, 8th September 2015

Nikolas

* Mini preparation

+ picked 3, 6, 17 and 22 (duplicates for each plate)
+ 5 mL overnight cultures
stored at 37 °C

### Wednesday, 9th September 2015

Dana

* iGEM Plasmids

+ Kit Plate 1 1A BBa\_K314110
+ Kit Plate 1 3A BBa\_K873002
+ Kit Plate 1 12L BBa\_K517003

→ Trafo into DH5a from the group of Ignatova* Minis from yesterday:

+ 6+ 17: media were red, discarded them
+ 3+22: mini plasmid prepp with kit, taken them to get sequenced

Concentrations:- 3A: 165 ng/µL
- 3B: 208 ng/µL
- 22A: 230 ng/µL
- 22B: 180 ng/µL

### Thursday, 10th September 2015

Julian

* Picking minis:

+ Visual colonies had been picked.
   Due to the fact that only some colonies had been on the plates
   i have returned them to the 37°C storage room.

* Efficiency calculation:

+ Couldnt be calculated because i didn't find a control plate with 20µL.

### Friday, 11th September 2015

Dana

* Started with restriction of pSB1C3 backbone

+ restriction with PstI, 1h, 37 °C
+ PCR clean up
+ restriction with EcoRI, 1h 37 °C, stopped here, because data from sequencing came back

* Overview Sequencing:
  48DD03 GroEl 118 ng/µL
  48DD06 GroEl 178 ng/µL
  48DD09 GroEl 233 ng/µL
  48DD02 miRNA 35 ng/µL
  48DD05 miRNA 38 ng/µL
  48DD08 miRNA 68 ng/µL
  48DD24 3A 165 ng/µL miRNA Ligation from 06.09.15
  48DD23 3B 208 ng/µL miRNA Ligation from 06.09.15
  48DD20 22A 230 ng/µL miRNA Ligation from 23.07.15
  48DD21 22B 180 ng/µL miRNA Ligation from 23.07.15
  + As miRNA (48DD05) and 22a+b are postive, miRNA 05 and 22A were transformed into DH5a

### Sunday, 13th September 2015

Julian

* Picking of the minis

### Monday, 14th September 2015

Julian

* Prepping of the minis (plasmid kit)

### Tuesday, 15th September 2015

Julian

* Annealing of the oligos

+ 10 min at 94°C (Heatblock)
+ Temperature reduction to room temperature in one hour

* Restriction of the linear backbone

+ 60µL pSB-1C3 (25ng/µL)
+ 3µL EcoR1 (fast digest)
+ 3µL Pst1 (slow digest)
+ 9µL FD-Buffer
⇒ 1 hour at 37°C
⇒ PCR clean up kit

- 90µL restricted Backbone
- 450µL DF-Buffer
- rest as said in the protocol
⇒ concentration BB1 = 23,5 ng/µL
* Dephosphorilation of the restricted Backbone

+ 30µL pSB-1C3 (cut)
+ 3µL Fast AP
+ tip from Chao, not to use the Fast AP Buffer
+ PCR Clean up kit
⇒ Concetration restricted and dephosphorilated BB2= 5 ng/µL
* Ligation

+ Dilution of the annealed oligos up to 10 ng/µL
+ ratio of 1:3 vector to insert
+ Groel

- 10µL rest. cut BB (5ng/µL ⇒ 50ng)
- 2,2µL alligatet Groel oligos (10ng/µL ⇒ 22ng)
- 2µL T4- DNA Ligase Buffer
- 1µL T4 -DNA Ligase
- 5µL ddH2O

+ miRNA

- 10µL rest. cut BB (5ng/µL ⇒ 50ng)
- 1,5µL alligatet Groel oligos (10ng/µL ⇒ 15ng)
- 2µL T4- DNA Ligase Buffer
- 1µL T4 -DNA Ligase
- 5µL ddH2O

+ control

- 10µL rest. cut BB (5ng/µL ⇒ 50ng)
- 2µL T4- DNA Ligase Buffer
- 1µL T4 -DNA Ligase
- 7µL ddH2O

⇒ Overnight Ligation with 16°C

### Wednesday, 16th September 2015

Julian, Maurice

* Transformation of the ligated Oligos (as told as in the protocol written by Henning and Dana)
* Restriction of the miRNA with constitutive promoter to change the backbone
⇒ Ligation with new backbone (pSB-1C3)
⇒ Transformation with new backbone (37°C room)

### Thursday, 17th September 2015

Sonja, Julian, Maurice

* Restriction of new IDT-Products ( const. Prom. + miRNA)
   Ligation (into the vector)
   Preparation of new liquid and solid bacterial cultures

### Friday, 18th September 2015

Henning, Julian

* Prepping, drying and colony PCR of the minis (all made by official and already established protocols)* Unspecific validation of the heat induction promotor via an agarose gel* Finishing the Wiki-Page in particular by Dana, Vincent and Maurice, the rest of the team participated too