Team:Marburg/Experiments

E X P E R I M E N T S

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"When you're experimenting you have to try so many things before you choose what you want, and you may go days getting nothing but exhaustion."- Fred Astaire

P R O T O C O L S

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All the protocols used in our project are listed here.

Protocols

X

Cultivation

BG media

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5x BGM media:

Stock 1 (100x): filter sterilize or autoclave

Na2Mg EDTA	4.839 g/L
Ferric ammonium citrate	6.287 g/L
CaCl2 x 2 H2O	17.936 g/L
citric acid x 1 H2O	2.997 g/L

Stock 2 (100x): filter sterilize or autoclave

MgSO4 x 7 H2O

37.464 g/L

Stock 3 (100x): filter sterilize or autoclave

K2HPO4	10.451 g/L
KH2PO4	8.165 g/L

Stock 4 (500x microelements): autoclave

H3BO3	7.139 g/L
MnCl2 x 4 H2O	4.522 g/L
ZnSO4 x 7 H2O	311.599 mg/L
CuSO4 x 5 H2O	126.092 mg/L
NaMoO4	122.233 mg/L
CoCl2 x 6 H2O <- solve first in 30 ml water and add to stock 4.	975.059 mg/L

Combine stock solutions and add from 1 M HEPES/NaOH buffer (pH 8.0) stock to a final concentration of 20 mM.

Add 8.159 g of NaNO₃ per liter of medium.

For solid media add 1.4 % Agar.

Autoclave. After autoclaving add filter sterilized Na₂S₂O₃ to a final concentration of 1 mM to 5x BGM-Agar.

This recipe originated from the publication Włodarczyk et. al., 2019 and has been adapted by the iGEM Team Marburg 2019.

BG11 medium from Uni Marburg

Stock 1 (100x): filter sterilize or autoclave

Na2Mg EDTA	0.1 g/L
Ferric ammonium citrate	0.6 g/L
CaCl2 x 2 H2O	3.6 g/L
citric acid x 1 H2O	0.6 g/L

Stock 2 (100x): filter sterilize or autoclave

MgSO4 x 7 H2O

7.5 g/L

Stock 3 (100x): filter sterilize or autoclave

K2HPO4 or K2HPO4 x 3 H2O

3.05 g/L 4.0 g/L

Stock 4 (1000x microelements): autoclave

H3BO3	2.88 g/L
MnCl2 x 4 H2O	1.81 g/L
ZnSO4 x 7 H2O	0.222 g/L
CuSO4 x 5 H2O	0.079 g/L
CoCl2 x 6 H2O	0.05 g/L
NaMoO4	0.391 g/L

Combine stock solutions and add from 1 M HEPES/NaOH buffer (pH 8.0) stock to a final concentration of 20 mM.

Add 1.5 g of NaNO₃ per liter of medium.

For solid media add 1.4 % Agar.

Autoclave.

After autoclaving add filter sterilized Na₂S₂O₃ to a final concentration of 1 mM to BG11-Agar.

BG11 medium from Düsseldorf

Final concentration of medium:

Compound	Concentration
CaCl2 x 2 H2O	0.036 g/L
citric acid	0.0006 g/L
NaNO3	1.4958 g/L
MgSO4 x 7 H2O	0.0749 g/L
0.25 M Na2EDTA (pH 8)	0.0056 mL/L
Na2CO3	20 µg/L
Fe(III) ammonium citrate	6 µg/L
K2HPO4 x 3 H2O	30 µg/L
HEPES (pH 8)	10 mM
H3BO3	2.86 mg/L
MnCl2 x 4 H2O	1.81 mg/L
ZnSO4 x 7 H2O	0.222 mg/L
Na2MoO4 x 2 H2O	0.390 mg/L
Co(NO3)2 x 6 H2O	0.049 mg/L
(CuSO4 x 5 H2O	0.079 mg/L if required)

Guidelines:
Always work under sterile conditions when handling sterile media or stocks. Work under the clean bench.

Safety warnings:
Wear gloves when preparing stocks!
Heavy metals are toxic for the environment and need to be discarded accordingly.

Before starting:
For plates: Thaw antibiotic stocks before pouring plates.

1. 100x BG11 stock:

   CaCl2 x 2 H2O	3.6 g/L
   citric acid	0.6 g/L
   NaNO3	149.58 g/L
   MgSO4 x 7 H2O	7.49 g/L
   0.25 M Na2EDTA, pH 8	0.56 mL/L

   
   For 100x BG11 Stock-N:
   Omit NaNO₃
   


2. Supplemental stocks for standard media:

   1000x Na2CO3	20 mg/mL
   100x TES-buffer, pH 8.0 (1M)	adjust with KOH
   1000x K2HPO4 x 3 H2O	30 mg/mL
   1000x Fe(III) ammonium citrate	6 mg/mL
   5000x CuSO4 x 5 H2O	395 ng/mL



3. Trace metal mix (1000x concentration):

   H3BO3	2.86 g/L
   MnCl2 x 4 H2O	1.81 g/L
   ZnSO4 x 7 H2O	0.222 g/L
   Na2MoO4 x 2 H2O	0.390 g/L
   Co(NO3)2 x 6 H2O	0.049 g/L

   
   For BG11 lacking certain metals (e.g. for working with metal inducible promoters P_petE, P_coaT, P_ziaA etc., trace metal mix can be prepared lacking these chemicals and used instead of standard trace metal mix.


4. Standard 1x BG11:
   Fill 1 L bottle with 500 mL ultra pure water. Add stock solutions as shown below.
   

   Stock solution	Volume
   100x BG11 Stock	10 mL
   1000x Na2CO3	1 mL
   1000x K2HPO4 x 3 H2O 100x TES-buffer	1 mL 10 mL
   100x TES-buffer	10 mL
   1000x Trace metal Mix	1 mL

   
   Add ultra pure water to 1 L.
   Autoclave.
   After autoclaving, add 1 mL 1000x Fe(III) ammonium citrate.
   Optional: After autoclaving, add 200 µL 5000x CuSO₄
   


5. Standard 1x BG11-N:
   Fill 1 L bottle with 500 mL ultra pure water. Add stock solutions as shown below.
   

   Stock solution	volume
   100x BG11 Stock-N	10 mL
   1000x Na2CO3	1 mL
   1000x K2HPO4 x 3 H2O 100x TES-buffer	1 mL 10 mL
   100x TES-buffer	10 mL
   1000x Trace metal Mix	1 mL

   
   Add ultra pure water to 1 L.
   Autoclave.
   After autoclaving, add 1 mL sterile 1000x Fe(III) ammonium citrate.
   Optional: After autoclaving, add 200 µL 5000x CuSO₄


6. Standard 2x BG11 for agar plates:
   Fill 500 mL bottle with 250 mL ultra pure water. Add stock solutions as shown below.
   

   Stock solution	Volume
   100x BG11 Stock-N	10 mL
   1000x Na2CO3	1 mL
   1000x K2HPO4 x 3 H2O 100x TES-buffer	1 mL 10 mL
   100x TES-buffer, pH = 8.0	10 mL
   1000x Trace metal Mix	1 mL

   
   Add ultra pure water to 500 mL.
   Autoclave.
   After autoclaving, add 1 mL sterile 1000x Fe(III) ammonium citrate.
   Optional: After autoclaving, add 200 µL 5000x CuSO₄


7. BG11 plates:
   Prepare 1.5 % agar: Weigh 4.5 g Bacto Agar. Fill up to 300 mL. Autoclave.
   Microwave agar until liquid. Let cool.
   In a 50 mL Falcon, add 1 vol 2x BG11 and 1 vol liquid 1.5 % agar. (Note: Usually, one plate requires 30-40 mL total volume.)
   When mixture is hand warm, add appropriate antibiotics, if required. Quickly pour late, avoiding air bubbles.

BG11 medium from Tübingen

For 1 L n mL of the stock solutions are used:
stock solution 1-7: 5 mL
stock solutin 8: 5 mL
stock solution of trace elements: 1 mL

BG11-media stock solutions (200x)	m (for 200 mL)	final concentration
1.   NaNO3	60.00 g	17.65 mM
2.   K2HPO4	1.25 g	0.18 mM
3.   MgSO4 x 7 H2O	2.96 g	0.30 mM
4.   CaCl2 x 2 H2O	1.47 g	0.25 mM
5.   Na2EDTA 2 x H2O	0.04 g	0.003 mM
6.   Na2CO3	1.61 g	0.38 mM
7.   Fe(III) citrate citric acid	0.29 g 0.23 g	0.03 mM 0.03 mM
8.   NaHCO3	16.80 g	5 mM

Trace elements stock solution (1000x)	m (for 100 mL)	final concentration
H3BO4	286 mg	46.26 µM
MnCl2x 4 H2O	181 mg	9.15 µM
ZnSO4 x 7 H2O	22.2 mg	0.77 µM
Na2MO4 x 2 H2O	39 mg	1.61 µM
CuSO4 5 x H2O	7.9 mg	0.32 µM
Co(NO3)2 x 6 H2O	4.94 mg	0.17 µM

Preparation of stock solutions:

stock solutions 1-7: weigh out substances and fill up with 200 mL MilliQ-H₂O.
Note: Fe(III) citrate and citric acid are weighed in together and have to be protected from light.
Autoclave stock solutions and store them at room temperature. Stock solution 7 should be wrapped up in aluminium foil.

Preparation of BG11-medium:

give 900 mL MilliQ H₂O in a 1 L measuring cylinder.
5 mL of each 200x stock solution are added (7 stock solutions)
1 mL trace element stock solution (1000x) is added.
Fill the cylinder with MilliQ H₂ O up to 1000 mL or 995 mL.
Autoclave. There will be a precipitate so shake before use.
Store at room temperature.
Add 5 mL 1 M NaHCO₃ stock solution (8) (200x) before use.

agar plates:

end concentration 10 g/L agar (Use Bacto-Agar for cyanobacteria!)
Mix agar and BG11-medium after autoclaving.
For 1L agar plates: Autoclave 10 g agar in 500 mL MilliQ-H₂O and 500 mL 2x BG11 (5 mL of each stock solution + 1 mL of the trace element stock solution) seperately.
Mix them when handwarm and add 5 mL NaHCO₃ (+ Glc + Casaminoacids)
500 mL last for approximately one bag of petri dishes.
Pour thick agar plates (30-40 mL) as cyanobacteria have long generation times.
Verify that there are no contaminations: (Rippka et al., 1979)
BG11 agar plates + 2 % glucose + 0.02 % Casaminoacids (for 500 mL agar plates):
20 mL glucose (50 % stock solution, 55 g glucose monohydrate on 100 mL (warm up to dissolve) --> sterile filtration)
1 mL casaminoacids (100 g/L stock solution).

Competent cells

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Competent cells (E. coli)

1. HK was inoculated with 2.00 mL of the overnight culture
2. Incubation until the optical density is 0.6 – 0.8 (37°C, 220 rpm)
3. Transfer into 50 mL falcons and centrifugation (5000 rpm, 10 min)
4. Remove supernatant
5. Resuspension in cold 0.1 M CaCl2 (15.0 mL)
6. Incubation on ice for 30 min
7. Centrifugation (5000 rpm, 10 min)
8. Remove supernatant
9. Resuspension in 0.1 M CaCl2 (3.25 mL) + 80% glycerin (0.75 mL)
10. Aliquot in sterile eppies
11. Blast-freeze in N2 (l)
12. Store in freezer (-80°C)

Transformation

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Transformation

1. x µL of DNA in either top10 or DH5α
   (x = 1.00 µL for Retransformation; 5.00 µL of GG lvl 1 construct)
2. incubation on ice for 30 minutes
3. heatshock (42°C, 90 sec)
4. incubation in ice for 5 minutes
5. addition of 500 µL LB medium
6. incubation (37°C, x h)
   (x = 1 h when antibiotic resistance kanamycin, chloramphenicol, ampicillin; 2 h when spectinomycin)
7. centrifugation (5000 rpm, 2 min)
   (when doing a retransformation this step was not executed, 100 µL of liquid were then plated on an agar plate with antibiotic resistance)
8. remove supernatant
9. plate on agar plate with antibiotic resistance
10. incubation (37°C, overnight)

Transformation protocol for Synechococcus 7942

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Transformation protocol for Synechococcus 7942

1. Start a fresh culture (50 mL) in BG-11
2. Measure OD_730nm to use cells when the OD is between 0.5 and 1.0
3. Spin down cells in sterile tube at 6000 × g at room temperature.
4. Concentrate the cells by re-suspending them in smaller amount of fresh growth medium so to get a final OD_730nm of 2.5 – 3.5
5. Place 0.4 mL of re-suspended cells in sterile culture tubes.
6. Add 50 ng – 2 µg plasmid DNA to each tube and gently mix. (Leave one tube as a control w/o DNA added).
7. Wrap tubes in aluminum foil (perhaps make hole in Eppie lid for gas exchange).
8. Place the tubes in the growth chamber at 30° (if high CO2 requiring phenotype is expected place plates in 3 % CO2) for 4 – 24 h.
9. Spot 200 μL (about 10-15 μL drops) on a sterile filter (Whatman Nuclepore Track-Etch Membrane #111107, 47mm diameter; 0.4 μM pore size) that has been placed on a BG-11 agar plate + antibiotics.

Triparental conjugation

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Triparental conjugation

• UTEX 2973 culture was inoculated at OD 0.1
• When UTEX 2973 culture was at OD ~0.3, cultures of prK2013 and HB101 were inoculated from “overnight cultures” to OD 0.1
• When UTEX 2973 culture was at OD 0.6-0.8 the conjugation was started:

1. Centrifugation of 2.00 mL of each culture (4000 x g, 2min)
2. Washing (2x) with 2.00 mL of respective media
3. Pipet-mixing and inverting (no vortexing!)
4. Resuspension in media (UTEX 2973: 200 µL; E.coli: 100 µL)
5. Mixing of all three strains
6. Incubation (37°C, 30 minutes, 100-150 µE)
7. Blotting 5 µL on sterile filters on LB/BG11 (95:5) plates without antibiotics
8. Incubation (37°C, overnight, 150 µE)
9. Moving sterile filters on LB/BG11 (95:5) plates containing spectinomycin
10. Incubation (37°C, few days, 150 µE)
11. Streaking cyano blots on new BG11 plates containing spectinomycin
12. Colony PCR to verify the conjugation

Well-plate cultivation

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Well-plate cultivation

1. inoculate colony of UDAR 4787 into liquid media in Volume of 1 mL + 0.5 μL Spec. incubate (24-48h, 42 ˙C, 130 rpm, 5% CO₂, 500 μE)
2. inoculate row A from 24-wellplate with preculture to OD₇₃₀= 0.1
3. inoculate B6 with UDAR as blank
4. when OD reaches 0.6 inoculate row A to row C and D to OD = 0.1.
5. well B6 to well B4+5

Measurement

NanoLuc measurement

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NanoLuc measurement

1. Inoculation of E. coli cultures (~0.1 OD 600 nm)
2. Let culture grow until OD ~ 0.8.
3. Preparation of Nano-Glo: mixing Nano-Glo buffer with NanoLuc substrate (1:50)
4. Add 50 µL of culture and 50 µL of substrate per well (96 well plate inoculation).
5. Wait 3 minuntes.
6. Measure luminescence

Parts measurement

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Parts measurement

after the well-plate cultivation (see above) the part measurement follows

1. when row C and D reach OD = 0.6 measure OD with plate reader
   Settings: 730 nm, 3 measuring points (circle)
2. transfer 200 μL of blanks, C1-6 and D1-6 into 96 well plate black
3. measure fluorescence in a plate reader
4. Setting: Excitation 488 nm, Emission 518 nm, size 2x2 (circle), frame 1200 μm, strengthener:optimal

Cloning

Colony PCR protocol for Synechococcus elongatus UTEX 2973

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Colony PCR protocol for Synechococcus elongatus UTEX 2973

1. Pick colony you want to use as template
2. Resuspend in 50µl ddH₂O
3. Boil at 100°C for 10 minutes
4. Afterwards prepare the reaction as follows:
• 2.5 µl forward primer
• 2.5 µl reversed primer
• 25 µl Thermo Scientific™DreamTaq Green PCR Master Mix (2X)
• 20 µl boiled cells
5. Perform PCR using the following conditions:



step	Temperature	Time	cycle number
Initial Denaturation	95°C	180 s	1
Denaturation	95	30 s	25
Annealing	Tm	30 s	25
Elongation	72	60 s	25
Terminal elongation	72	15 min	1


For PCR products longer than 2kb, the elongation time should be prolonged by 1min/kb.

6. Load your gel

Digestion

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Digestion

1. measurement of concentration of DNA
2. mix components listed in table below




component	volume /µL
DNA (1 µg)	x
H2O	44.0-x
CutSmart Buffer or NEBuffer	5.00
Enzyme e.g. BsmbI or BSaI	1.00
Σ in tube	50.0



3. 2h, 37°C in Mastercycler
4. Agarose gel with Ethidium bromide (EtBr) (3 µL EtBr in 60 mL agarose gel), 70-120 V, 30-60 minutes

DNA plasmid purification with the Macherey-Nagel kit

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DNA plasmid purification with the Macherey-Nagel Kit:

Before starting:

Add 1 mL of Buffer A1 to the RNase A vial and vortex. Transfer the solution back into the Buffer A1 bottle and mix thoroughly. Indicate date of RNase A addition. Store Buffer A1 containing RNase A at 4 °C. The solution will be stable at this temperature for at least six months.

Add the indicated volume of 96-100 % ethanol to Buffer A4 and Buffer AQ.

1. Cultivate and harvest bacterial cells

   Use 1-5 mL of a saturated E.coli LB culture, pellet cells in a standard benchtop microcentrifuge for 30 s at 11,000 x g. Discard the supernatant and remove as much of the liquid as possible.

   Note: For isolation of low-copy plasmids refer to section 5.2.

   ~Personal note: We centrifuged in 15 mL falcons at 4000 rpm for 10 minutes at 4 °C.~



2. Cell lysis

   Add 250 µL Buffer A1. Resuspend the cell pellet completely by vortexing or pipetting up and down. Make sure no cell clumps remain before addition of Buffer A2!

   Attention: Check Buffer A2 for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer several minutes at 30-40 °C until precipitate is dissolved completely. mix thoroughly and cool buffer down to room temperature (18-25 °C).

   Add 250 µL Buffer A2. Mix gently by inverting the tube 6-8 times. Do not vortex to avoid shearing of genomic DNA. incubate at room temperature for up to 5 min or until lysate appears clear.

   Add 300 µL Buffer A3. Mix thoroughly by inverting the tube 6-8 times until blue samples turn colorless completely! Do not vortex to avoid shearing of genomic DNA!

   Make sure to neutralize completely to precipitate all protein and chromosomal DNA. LyseControl should turn completely colorless without any traces of blue.



3. Clarification of lysate

   Centrifuge for 5 min at 11,000 x g at room temperature.

   Repeat this step in case the supernatant is not clear!



4. Wash silica membrane

   Place a NucleoSpin® Plasmid / Plasmid (NoLid) Column in a Collection Tube (2 mL) and decant the supernatant from step 3 or pipette a maximum of 750 µL of the supernatant onto the column. Centrifuge for 1 min at 11,000 x g. Discard flowthrough and place the NucleoSpin® Plasmid / Plasmid (NoLid) Column back into the collection tube.

   Repeat this step to load the remaining lysate.



5. Wash silica membrane

   Recommended: If plasmid DNA is prepared from host strains containing high levels of nucleases (e.g., HB101 or strains of the JM series), it is strongly recommended performing an additional washing step with 500 µL Buffer AW, optionally preheated to 50 °C, and centrifuge for 1 min at 11,000 x g before proceeding with Buffer A4. Additional washing with Buffer AW will also increase the reading length of DNA sequencing reactions and improve the performance of critical enzymatic reactions.

   Add 600 µL Buffer A4 (supplemented with ethanol, see section 3). Centrifuge for 1 min at 11,000 x g. Discard flowthrough and place the NucleoSpin® Plasmid/Plasmid (NoLid) Column back into the empty collection tube.



6. Dry silica membrane

   Centrifuge for 2 min at 11,000 x g and discard the collection tube.

   Note: Residual ethanolic wash buffer might inhibit enzymatic reactions.



7. Elute DNA

   Place the NucleoSpin® Plasmid/Plasmid (NoLid) Column in a 1.5 mL microcentrifuge tube (not provided) and add 50 µL Buffer AE. Incubate for 1 min at room temperature. Centrifuge for 1 min at room temperature. Centrifuge for 1 min at 11,000 x g.

   Note: For more efficient elution procedures and alternative elution buffer (e.g., TE buffer or water) see section 2.5.

   ~Personal note: We heated Buffer AE to 80 °C and used 30-40 µL of it.~

DpnI digest

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DpnI digest

1. add 2 µL of DpnI to sample
2. incubate at 37°C for 2h
3. Agarosegel with Ethidium bromide (EtBr) (3 µL EtBr in 60 mL agarose gel), 70-120 V, 30-60 minutes

Gel Elektrophoresis

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Gel Elektrophoresis

1. Preparation of the buffer and agarose gel



TAE-buffer (50x):




Tris	EDTA-Na2-salt	acetic acid
242.3 g/L	18.6 g/L	60.05 g/L





1.2 % agarose gel:




4.8 g agarose in 400 mL TAE-buffer (1x)



2. On 100 mL 1.2 % Agarose gel 5 µL EtBr are added, stirred, filled into the box and then let it solidify.
3. Add 10 µL of loading dye to 50 µL of the sample.
4. Fill the pockets of the gel: 3 µL of gene ladder in one pocket and 15-20 µL of sample in the other pockets.
5. Apply a voltage of 70-100 V and let gel run for 30-60 minutes.

Gel extraction with the QIAquick® Gel Extraction kit

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Gel extraction with the QIAquick® Gel Extraction kit

Notes before starting

• This protocoll is for the purification of up to 10 µg DNA (70 bp to 10 kb).
• The yellow colour of Buffer QG indicates a pH = 7.5. DNA adsorption to the membrane is only efficient at pH ≤ 7.5.
• Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).
• Isopropanol (100%) and a heating block or water bath at 50°C are required.
• All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top centrifuge.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colourless tube. Add 3 volumes of Buffer QG to 1 volume gel (100 mg gel ~ 100 µL). The maximum amount of gel per spin column is 400 mg. For > 2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the colour of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
4. Add 1 gel volume isopropanol to the sample and mix.
5. Place a QIAquick spin column in a provided 2 mL collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard the flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 µL, load and spin/ apply vacuum again.
6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 µL Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
7. To wash, add 750 µL Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
   Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt-ended ligation), let the column stand 2-5 min after addition of Buffer PE.
   Centrifuge the QIAquick column in the provided 2 mL collection tube for 1 min to remove residual wash buffer.
   
8. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.
9. To elute DNA, add 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the centre of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL Buffer EB to the centre of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time up to 4 min can increase the yield of purified DNA.
10. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Golden Gate level 0

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Golden Gate level 0

1. Mix components listed in table below



component	volume /µL
Entry vector (60 ng/µL)	1.00
TF buffer	1.00
T4 ligase	1.00
BsmbI	1.00
Insert (180 ng/µL)	x
H2O	6.00-x
Σ in tube	10.0



2. Golden Gate program in Mastercycler (Eppendorf)

Note: all the parts, the buffer and enzymes were kept on ice consistently
GoldenGate standard program:
Step 1: 37°C, 2 min
Step 2: 16°C, 5 min
Repetition (30x) of step 1 and 2
Step 3: 60°C, 10 min
Step 4: 80°C, 10 min
Step 5: 4°C, hold until lid is opened

The protocol Golden Gate level 0 can also be found following this link.

Golden Gate level 1

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Golden Gate level 1

1. Measurement of concentration of parts used
2. Dilution of parts to 20 fmol
3. Preparation of Mastermix (consisting of parts) in PCR-tube (1.00 µL of each part)
4. Addition of 1.00 µL level 1 Ori part
5. Addition of 1.00 µL T4 Lig buffer
6. Addition of 1.00 µL T4 Ligase
7. Addition of 1.00 µL BsaI
8. Pipet-mixing
9. GoldenGate program in Mastercycler (Eppendorf)

Note: all the parts, the buffer and enzymes were kept on ice consistently
GoldenGate standard program:
Step 1: 37°C, 2 min
Step 2: 16°C, 5 min
Repetition (30x) of step 1 and 2
Step 3: 60°C, 10 min
Step 4: 80°C, 10 min
Step 5: 4°C, hold until lid is opened
GoldenGate spaceholder constructs program:
Step 1: 37°C, 2 min
Step 2: 16°C, 5 min
Repetition (50x) of step 1 and 2
Step 3: 4°C, hold until lid is opened

The protocol Golden Gate level 1 can also be found following this link.

Ligation Protocol with T4 DNA Ligase (M0202) (New England BioLabs Inc.)

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Ligation Protocol with T4 DNA Ligase (M0202) (New England BioLabs Inc.)

1. Set up the following reaction in a microcentrifuge tube on ice.(T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios



component	20 µL reaction
T4 DNA Ligase Buffer (10X)*	2 µL
Vector DNA (4 kb)	50 ng (0.020 pmol)
Insert DNA (1 kb)	37.5 ng (0.060 pmol)
Nuclease-free water	to 20 μl
T4 DNA Ligase	1 µL


* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
5. Heat inactivate at 65°C for 10 minutes.
6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

PCR Purification with the QIAquick® PCR Purification Kit

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PCR Purification with the QIAquick® PCR Purification Kit

Notes before starting

• This protocol is for the purification of up to 10 µg PCR products (100 bp to 10 kb in size).
• Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).
• All centrifugation steps are carried out at 17.900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature.
• Add 1:250 volume pH indicator I to Buffer PB. The yellow colour of Buffer PB with pH indicator I indicates a pH ≤ 7.5. The adsorption of DNA to the membrane is only efficient at pH ≤ 7.5. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I; do not add pH indicator I to buffer aliquots.
• Symbols: ● centrifuge processing; ▴ vacuum processing

1. Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the colour of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The colour of the mixture will turn yellow.
2. Place a QIAquick column in ● a provided 2 mL collection tube or into▴a vacuum manifold. For details on how to set up a vacuum manifold, refer to the QIAquick Spin Handbook.
3. To bind DNA, apply the sample to the QIAquick column and ● centrifuge for 30-60 s or▴apply vacuum to the manifold until all the samples have passed through the column. ● Discard flow-through and place the QIAquick column back in the same tube.
4. To wash, add 750 µL Buffer PE to the QIAquick column ● centrifuge for 30-60 s or▴apply vacuum. ● Discard flow-through and place the QIAquick column back in the same tube.
5. Centrifuge the QIAquick column once more in the provided 2 mL collection tube for 1 min to remove residual wash buffer.
6. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.
7. To elute DNA, add 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0-8.5) to the centre of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL elution buffer to the centre of the QIAquick membrane, let the column stand for 1 min and then centrifuge.
8. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

PCR Using Q5® High-Fidelity DNA Polymerase (M0491)

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PCR Using Q5® High-Fidelity DNA Polymerase (M0491)

1. Please note that protocols with Q5 High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
   Reaction Setup:
   We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors.



component	25 µL reaction	50 µL reaction	final concentration
5X Q5 reaction Buffer	5 µL	10 µL	1X
10 mM dNTPs	0.5 µL	1 µL	200 µM
10 µM Forward Primer	1.25 µL	2.5 µL	0.5 µM
10 µM Reverse Primer	1.25 μl	2.5 µL	0.5 µM
Template DNA	variable	variable	< 1,000 ng
Q5 High-Fidelity DNA Polymerase	0.25 µL	0.5 µL	0.02 U/µL
5X Q5 High GC Enhancer (optional)	(5 µL)	(10 µL)	1X
Nuclease-Free Water	to 25 μl	to 50 µL


Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
Transfer PCR tubes to a PCR machine and begin thermocycling.
Thermocycling Conditions for a Routine PCR:


step	temp	time
Initial Denaturation	98°C	30 seconds
25-35 cycles	98°C *50-72°C 72°C	5-10 seconds 10-30 seconds 20-30 seconds/kb
Final Extension	72°C	2 minutes
Hold	4-10°C


*Use of the NEBTm Calculator is highly recommended.

2. General Guidelines:
   Template:Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows:



DNA	amount
DNA Genomic	1 ng - 1 µg
Plasmid or Viral	1 pg - 10 ng



3. Primers:
   Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.
4. Mg⁺⁺ and additives:
   Mg⁺⁺ concentration of 2.0 mM is optimal for most PCR products generated with Q5 High-Fidelity DNA Polymerase. When used at a final concentration of 1X, the Q5 Reaction Buffer provides the optimal Mg⁺⁺ concentration.
   Amplification of some difficult targets, like GC-rich sequences, may be improved by the addition of 1X Q5 High GC Enhancer. The Q5 High GC Enhancer is not a buffer and should not be used alone. It should be added only to reactions with the Q5 Reaction Buffer when other conditions have failed.
5. Deoxynucleotides:
   The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates.
6. Q5 High-Fidelity DNA Polymerase concentration:
   We generally recommend using Q5 High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the optimal concentration of Q5 High-Fidelity DNA Polymerase may vary from 10–40 units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and difficulty. Do not exceed 2 units/50 μl reaction, especially for amplicons longer than 5 kb.
7. Buffers:
   The 5X Q5 Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such as GC-rich templates or those with secondary structure, the addition of the Q5 High GC Enhancer can improve reaction performance. The 5X Q5 Reaction Buffer is detergent-free and contains 2.0 mM Mg⁺⁺ at the final (1X) concentration.
8. Denaturation:
   An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it.
   During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates.
9. Annealing:
   Optimal annealing temperatures for Q5 High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. The NEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. Typically, use a 10–30 second annealing step at 3°C above the T_m of the lower T_m primer. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.
   For high T_m primer pairs, two-step cycling without a separate annealing step can be used (see note 12).
10. Extension:
    The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.
    A final extension of 2 minutes at 72°C is recommended.
11. Cycle number:
    Generally, 25–35 cycles yield sufficient product. For genomic amplicons, 30-35 cycles are recommended.
12. 2-step PCR:
    When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.
13. Amplification of long products:
    When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb.
14. PCR product:
    The PCR products generated using Q5 High-Fidelity DNA Polymerase have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated.
    Addition of an untemplated -dA can be done with Taq DNA Polymerase (NEB #M0267 ) or Klenow exo^- (NEB #M0212 ).

qPCR

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qPCR

1. Mix the components listed below



component	Volume
template	1 µL
Primer-F	1 µL
Primer-R	1 µL
2x master mix (provided)	10 µL
H2O	7 µL
total volume	20 µL



2. Start program in Mastercycler:

1. 50°C, 2 minutes

2. 2.95°C, 2 minutes

3. 95°C, 15 seconds

4. 60°C, 60 seconds

cycles: 40(cycle 2 to 4)

Quick electroporation of E. coli

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Quick electroporation of E. coli

This quick electroporation protocol was explained to us by Dr. Alberto Sánchez-Pascuala Jerez from the research group of Prof. Dr. Tobias J. Erb in the Max-Planck-Institute for terrestrial microbiology in Marburg. It was adapted by us and might not be exactly how Dr. Alberto Sánchez-Pascuala Jerez does it.

Optional:

1. Restreak cells from glycerol stock on LB.
2. Inoculate a single colony in liquid LB.

Standard:

1. Inoculate your culture at OD₆₀₀=0.05 from an overnight culture
2. Grow the culture until an OD₆₀₀=0.5 is reached. Note: If the culture reaches OD600= ~0.6 it can still be used, but reinoculating is recommended if higher values are reached.
3. Directly put the culture on ice for 10-15 min.
4. Transfer the culture into a falcon and spin down in a cooled centrifuge at 2500 rpm, 4°C for 10-15 min.Heraeus™ Multifuge™ X1 is used for higher volumes.
5. Wash the cells 2-3 times in dd H2O (or other sterile water).
6. Resuspend in 0.5-2.0 mL water, depending on how many aliquots you want and how big the pellet is.
7. Make 50 µL – 100 µL aliquots.
8. Add DNA to your aliquot on ice.
9. Transfer cell/ DNA mix into an electroporation cuvette on ice. Gene Pulser®/MicroPulser™ Electroporation Cuvettes (green cap) from Bio-Rad are used.
10. Wipe the cuvette with a paper towel to remove any liquid that might cause an arc and place it in the electroporation chamber.
11. Electroporate the sample, directly add recovery medium and transfer the cells into a reaction tube. The following settings are used: 2500 V, 25 µF, 200 Ω, 2 mm. As medium 500 µL SOB medium are added.
12. Incubate the cells at 37°C and 250 rpm for 1h (amp resistance) or 2h (other resistances; kan, cam, spec..)
13. Plate and incubate at 37°C over night.

Sequencing

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Sequencing

substrate	volume
Primer	3.00 µL
DNA (500-1200 ng) + H2O	12.0 µL
∑ in eppie	15.0 µL

1. Add Primer, DNA (500-1200 ng) and water
2. Attach sequencing label on eppie
3. Register the label number online on Microsynth website
4. send registered eppies to Microsynth

L A B B O O K S

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Our labbook entrys.

Labbooks

X

• Cultivation of UTEX
• Light measurement
• Building placeholder constructs lvl 1
• Level 1 assembly method
• Toolbox: Building parts
• Synechococcus elongatus PCC 7942
• Toolbox: General
• Toolbox Marburg Collection 1.0
• Triparental conjugation
• aNSo integration into the genome
• Schönas lvl 1 GG
• pANS domestication
• Natural Competence
• Growth Curves
• Strain Engineering: General
• Cpf1 system
• NanoLuc + YFP (codonoptimised)
• Farnesen-Synthase
• MetabolicEngineering: General
• Metabolic Engineering: General
• Metabolic Engineering
• Limonen-Synthase
• MEP-Pathway
• New parts/ improve parts
• qPCR

A U T O M A T I O N
P R O T O C O L S

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The protocols of the Automation Lab can be found here.

Automation Lab protcols

X

Hardware

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Follow this link to see all the hardware designs of the Automation Lab.

Plasmid Purification

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Follow this link to see the plasmid purification protocols of the Automation Lab.

Nuclear Integration Site Model

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Follow this link to see the nuclear integration site model of the Automation Lab.

Faster R-CNN Implementation

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Follow this link to see the faster R-CNN implementation protocol of the Automation Lab.