Team:UNIK Copenhagen/Experiments

Media recipes

LB medium and LB agar

  1. Add 20g of LB broth low salt to a 1L bottle
  2. Fill the bottle with distilled water
  3. Adjust the pH to 7,2
  4. Autoclave the bottle
  5. For LB agar, add 15g of micro agar to the bottle before autoclaving.

 

ATCC medium: 1502 Medium E for Bacillus subtilis

  1. Make the Trace Salts Solution by mixing the following in a 1L bottle

1g

EDTA

3g

MnSO4·H2O

0,1g

FeSO4·7H2O

0,1g

CaCl2·2H2O

0,1g

CoCl2·6H2O

0,1g

ZnSO4·7H2O

0,01g

CuSO4·5H2O

0,01g

AlK(SO4)2·12H2O

0,01g

H3BO3

0,01g

Na2MoO4·2H2O

Up to 1L

Distilled water

 

  1. Mix the following in a 1L bottle to make the ATCC medium

10g

Sucrose

8,7g

NaCl

1g

(NH4)2SO4

10mL

Trace Salts Solution

10,6g

K2HPO4

5,3g

KH2PO4

Up to 1L

Distilled water

 

  1. Autoclave the medium
  2. Add 2,1mL 1M MgSO4 to the medium using filter sterilization

 

BG11 media

  1. Make the following stock solutions

Stock #

Content

Amount

1

NaNO3

15g in 1L distilled water

2

K2HPO4

2g in 500mL distilled water

3

MgSO4·7H2O

3,75g in 500mL distilled water

4

CaCl2·2H2O

1,8g in 500mL distilled water

5

Citric acid

0,3g in 500mL distilled water

6

Ammonium ferric citrate brown

0,3g in 500mL distilled water

7

EDTANa2

0,05g in 500mL distilled water

8

Na2CO3

1g in 500mL distilled water

 

  1. Make the trace salt elements solution, by adding the following to a 1L bottle.

2,86g

H3BO3

1,81g

MnCl2·4H2O

0,22g

ZnSO4·7H2O

0,39g

Na2MoO4·2H2O

0,08g

CuSO4·5H2O

0,05g

Co(NO3)2·6H2O

Up to 1L

Distilled water

 

  1. Make the final BG11 medium by adding the following in a 1L bottle

100mL

Stock 1

10mL

Stock 2-8

1mL

Trace salt elements solution

Up to 1L

Distilled water

 

  1. Adjust the pH to 7,2 and autoclave the medium.

 

BGH5 media

  1. Make the stock solution by mixing the following in a 1L bottle

0,1g

Na2EDTA

0,6g

Ferric ammonium citrate

0,6g

Citric acid·H2O

3,6g

CaCl2·2H2O  

Up to 1L

Distilled water

 

  1. Make a magnesium sulfate solution by taking 7,5g of MgSO4·7H2O and dissolve in 1L distilled water.
  2. Make a dipotassium phosphate solution by dissolve 3,05g K2HPO4 in 1L distilled water.
  3. Make the final medium by adding the following solutions in a 1L bottle

10mL

Stock solution

10mL

Magnesium sulfate solution

10mL

Dipotassium phosphate solution

1mL

Trace elements solution from BG11 medium

0,02g

Na2CO3

1,5g

NaNO3

10g

Bacto agar

1,1g

HEPES

Up to 1L

Distilled water

 

  1. Adjust the pH to 7.2 and autoclave the medium before using.

 

2xYT medium and 2xYT agar

For 1L mix the following and autoclave

16g

Tryptone

10g

Yeast extract

5g

NaCl

Up to 1L

Distilled water

For 2xYT agar, add 15g micro agar before autoclaving

 

HS medium

  1. Make the Spizizen’s salt solution by mixing the following

2g

(NH4)2SO4

14g

K2HPO4

6g

KH2PO4

1g

Sodium citrate

Up to 100mL

Distilled water

 

  1. Autoclave the solution
  2. Add 0,1mL 1M MgSO4 to the solution using filter sterilization
  3. Mix the following in a sterile bench

66,5mL

Sterile distilled water

10mL

Sterile Spizizen’s salt solution

2,5mL

Sterile 20% glucose solution

1mL

Sterile 2% casein solution

5mL

Sterile 10% yeast extract solution

10mL

Sterile 8% arginine solution

10mL

Sterile 0,4% histidine solution

 

  1. In the sterile bench, add 5mL 0,1% L-tryptophan to the medium using filter sterilization.

 

LS medium

  1. Mix the following in a sterile bench

80mL

Sterile distilled water

10mL

Sterile Spizizen’s salt solution

2,5mL

Sterile 20% glucose solution

0,5mL

Sterile 2% casein solution

5mL

Sterile 2% yeast extract solution

0,25mL

Sterile 1M MgCl2

0,05mL

Sterile 1M CaCl2

 

  1. In the sterile bench, add 0,5mL 0,1% L-tryptophan to the medium using filter sterilization.

 

0,1M EGTA

  1. Dissolve 3,8g EGTA in 50mL distilled water
  2. Adjust the pH to 7,2 using NaOH
  3. Add Distilled water to 100mL in total
  4. Autoclave the solution

 

Growth experiments

Materials needed

  • Liquid medium (In our case: LB, ATCC or BG11)
  • Inoculation loops
  • Plates with the respective organism (In our case: Escherichia coli, Bacillus subtilis or Synechococcus elongatus)
  • Falcon tubes or baffled flasks
  • Sterile bench
  • Spectrophotometer
  • Incubator (In our case: 37°C or 30°C)

 

Procedure

Step 1-3 and 5 is carried out in a sterile bench.

  1. Pour medium into either a falcon tube or baffled flask, so it is one-third from its maximum volume
  2. Inoculate the respective organism into the tube or flask using an inoculation loop that contains cells from the plate
  3. Take 1mL out for OD measurements at 600nm or 730nm (The latter: only if it is S. elongatus) to find the cell density at time point zero
  4. Incubate the tube or the baffled flask in the tested conditions. If falcon tubes are used, loosen the lid before incubation
  5. Each day until satisfactory measure OD by taking 1mL out of the incubated tubes or flasks and measure OD at 600nm or 730nm (The  latter: only if it is S. elongatus).
  6. Record the growth curve

 

Transformation of Escherichia coli using heat-shock

Materials needed

  • Competent E. coli cells (In our case: NEB21 or NEB10 cells)
  • 5-50ng of DNA to be transformed into E. coli (In our case: 48ng DNA was used)
  • Water-bath or heating block at 42°C
  • LB medium
  • Selection plates (In our case: LB plates containing 50µg/mL-100µg/mL ampicillin)
  • 37°C heating block

 

Procedure

  1. Thaw the competent cells on ice until the very last ice-crystal
  2. Add 5-50ng of DNA to the cells (however, no more than 5µL). Mix the cells and the DNA by tapping on the Eppendorf tube. Do not mix by pipetting.
  3. Incubate the cells on ice for 30 minutes
  4. Heat-shock the cells in a water-bath or heating block at 42°C for exactly 30 seconds!
  5. Incubate immediately on ice for 5 minutes.
  6. Add 400µL-950µL (depends on how diluted you want the cells) LB medium or recovery medium to the transformed cells.
  7. Incubate the Eppendorf tubes for up to two hours at 37 degrees shaker.
  8. Plate out on selection plates using different volumes (In our case: between 100µL and 200µL).
  9. Incubate the plates in 37°C overnight.

 

Preparation of competent Bacillus subtilis cells

Materials needed

  • B. subtilis cells
  • HS medium
  • 37°C incubator
  • Sterile 87% glycerol
  • Liquid Nitrogen

 

Procedure

Step 1-2 and 5-6 is carried out in a sterile bench.

  1. Make an overnight culture of appropriate recipient B. subtilis cells in 5mL HS medium
  2. Inoculate 0,5mL of the overnight culture in 50mL HS medium and incubate at 37°C
  3. Record the growth curve every half an hour
  4. When the sample reaches the beginning of the stationary phase (should be after 3-4 hours), take out 10mL every 15 minutes.
  5. On ice, add 1mL of sterile 87% glycerol to the sample, mix and leave on ice for 15 minutes
  6. Fractionate to 1mL aliquots and freeze in liquid nitrogen
  7. Store the samples at -80°C until use.
  8. [Optional] Test each time point for competency and discard the time points that do not show any competency.

 

Transformation of Bacillus subtilis

Materials needed

  • Competent B. subtilis cells
  • Up to 20ng DNA to be transformed into B. subtilis (In our case: 18ng DNA was used)
  • LS medium
  • 0,1M EGTA
  • 30°C and 37°C water-bath or heating block
  • One centrifuge
  • Selection plates (In our case: 2xYT plates containing 5µg/mL chloramphenicol)

 

Procedure

  1. Thaw one aliquot of competent B. subtilis cells at 37°C
  2. Inoculate the thawed cells in 20mL LS medium
  3. Incubate the cells at 30°C in water-bath or heating block for 1,5 hours
  4. Take 1mL aliquots in 2mL Eppendorf tube and add 10µL of 0,1M EGTA
  5. Incubate for 5 minutes at room temperature
  6. Add DNA and incubate for 2 hours at 37°C while shaking
  7. Centrifuge the cells at 7500rpm for 5 minutes and discard the supernatant carefully
  8. Resuspend the pellet in 200µL LB medium
  9. Plate out on selective 2xYT plates using different volumes (In our case: Between 50µL and 100µL)
  10. Incubate the plates overnight at 37°C

 

PCR amplification of the individual genes

The reaction mix for each of the genes was made by using the following protocol.

2µL

X7 Buffer

0,5µL

dNTPs

0,1µL

X7 polymerase

1µL

10mM forward primer

1µL

10mM reverse primer

11,4µL

Distilled water

4µL

Template

The PCR program used for the genes varied and can be found below for each of the genes.

 

Lactate Polymerizing Enzyme (LPE)

Step

Temperature

Time

Cycles

Initial denaturation

95°C

3 minutes

 

Denaturation

95°C

30 seconds

35

Annealing

60°C

30 seconds

35

Elongation

70°C

1 minute

35

Final elongation

70°C

10 minutes

 

Hold

12°C

 

After the PCR the DNA was checked on a 1% agarose gel and the gene was purified by PCR purification.

 

Green Fluorescence Protein (GFP)  

Step

Temperature

Time

Cycles

Initial denaturation

95°C

3 minutes

 

Denaturation

95°C

30 seconds

35

Annealing

55°C

30 seconds

35

Elongation

70°C

30 seconds

35

Final elongation

70°C

10 minutes

 

Hold

12°C

 

After the PCR the DNA was checked on a 1% agarose gel and the gene was purified by PCR purification.

 

Propionate-CoA transferase (PCT)

Step

Temperature

Time

Cycles

Initial denaturation

95°C

3 minutes

 

Denaturation

95°C

30 seconds

35

Annealing

60°C

30 seconds

35

Elongation

70°C

1 minute

35

Final elongation

70°C

10 minutes

 

Hold

12°C

 

After the PCR the DNA was checked on a 1% agarose gel and the gene was purified by PCR purification.

 

Yellow Fluorescence Protein (YFP)

Step

Temperature

Time

Cycles

Initial denaturation

95°C

3 minutes

 

Denaturation

95°C

30 seconds

35

Annealing

56°C

30 seconds

35

Elongation

68°C

30 seconds

35

Final elongation

68°C

10 minutes

 

Hold

12°C

 

After the PCR the DNA was checked on a 1% agarose gel and the gene was purified by PCR purification.

 

Beta-ketothiolase fused to Cyan Fluorescence Protein (PhaA-CFP)

Step

Temperature

Time

Cycles

Initial denaturation

95°C

3 minutes

 

Denaturation

95°C

30 seconds

35

Annealing

55°C

30 seconds

35

Elongation

68°C

1 minute

35

Final elongation

68°C

10 minutes

 

Hold

12°C

 

After the PCR the DNA was checked on a 1% agarose gel and the gene was purified by PCR purification.

 

NADPH-dependent acetoacetyl-CoA reductase fused to Red Fluorescence Protein and Terminator (PhaB-RFP-Term)

Step

Temperature

Time

Cycles

Initial denaturation

95°C

3 minutes

 

Denaturation

95°C

30 seconds

35

Annealing

58°C

45 seconds

35

Elongation

70°C

1 minute

35

Final elongation

68°C

10 minutes

 

Hold

12°C

 

After the PCR the DNA was checked on a 1% agarose gel and the gene was purified by PCR purification.

 

Fusion PCRS

To make the gene construct PCT-YFP fusion PCR were carried out using the following reaction mix and the following PCR program.

5µL

X7 Buffer

6µL

dNTPs

1,25µL

X7 polymerase

5µL

10mM PCT forward primer

5µL

10mM YFP reverse primer

19,5µL

Distilled water

5µL

GC Buffer

2µL

YFP template

1µL

PCT template

 

Step

Temperature

Time

Cycles

Initial denaturation

95°C

3 minutes

 

Denaturation

95°C

45 seconds

35

Annealing

60°C

35 seconds

35

Elongation

72°C

3 minute

35

Final elongation

72°C

10 minutes

 

Hold

12°C

 

 

Restriction digestion of the vector pHT254, PhaA-CFP and PhaB-RFP-Terminator

Materials needed

  • 1µg of DNA
  • The restriction enzyme(s) (In our case: XmaI, BamHI and XbaI, depending on the situation)
  • The appropriate buffer (In our case: CutSmart from NEB)
  • 37°C heating block
  • 75°C heating block

 

Procedure

  1. Amplify the vector in an appropriate organism (In our case: E. coli) by making an overnight culture
  2. Purify the plasmid from the overnight culture and measure the concentration
  3. On ice, mix the following in an Eppendorf tube in the given order:

1µg

DNA

3µL

CutSmart buffer

10,9µL

Water

1µL

Restriction enzyme

1µL

Restriction enzyme

 

  1. Incubate the Eppendorf tube at 37°C for 2 hours
  2. Inactivate the restriction enzymes by incubating the Eppendorf tube at 75°C for 20 minutes (Does not apply for BamHI)
  3. Store the digestion at room temperature overnight
  4. [Optional] Check the quality of the digestion by loading 1µL of the digestion on a 1% agarose gel.

 

Ligation of digested constructs

Materials needed

  • Up to 50μg of digested constructs
  • T4 DNA Ligase
  • 10x T4 DNA ligase buffer
  • 16°C incubator

 

Procedure

  1. Mix the digested constructs, 10x T4 DNA ligase buffer and T4 DNA ligase in the following order, while on ice
  2. Incubate the mixture at 16°C overnight

 

Gibson Assembly

Materials needed

  • The gene fragments used in the Gibson Assembly
  • The Gibson Assembly MasterMix
  • 50°C heating block

 

Procedure

To insert the gene constructs into the vector a Gibson Assembly was carried out according to the protocol below.

  1. Mix the following in an Eppendorf tube

1µL

117,1ng/µL LPE

0,5µL

103ng/µL GFP

6,5µL

21,2ng/µL PCT-YFP

2µL

67,1ng/µL PhaB-CFP-Term

2µL

70,9ng/µL PhaA-RFP

2µL

95ng/µL pHT254

14µL

Gibson MasterMix

 

  1. Incubate the Gibson Assembly at 50°C for 1 hour
  2. Put on ice until transformation
  3. Use 2µL for transformation

 

Colony PCR

Material needed

  • PCR tubes
  • Plates with colonies that need to be investigated
  • Inoculation loops
  • LB medium (with selection marker)
  • Cell culture tubes
  • Pipette tips
  • 95°C incubator for PCR tubes (a PCR machine can be used)
  • 37°C incubator

 

Procedure

After a transformation, one is going to test the colonies for the correct insert.  This is done by doing a colony PCR

  1. Prepare the cell culture tubes with LB medium (one third of its total volume) and selection marker (In our case: 50μg/mL ampicillin for E. coli and 10μg/mL chloramphenicol for B. subtilis)
  2. Take the plates with the transformed cells and choose the colonies that is going to be investigated
  3. Use a pipette tip to take up the colony and squeeze it into one of the PCR tubes. Hereafter, put the pipette tip into one of the marked cell culture tubes.
  4. Store the cell culture tubes at 37°C under shaking overnight
  5. Add 20μL water to the PCR tubes
  6. Incubate the PCR tubes at 95°C for 10 minutes
  7. Centrifuge the PCR tubes for 10 minutes at maximum speed.
  8. The supernatant is used to do the PCR reaction, which can be seen above.

 

Co-culture set-up

Materials needed

  • Liquid medium (BG11 and ATCC)
  • Inoculation loops
  • 2,5 L Baffled flask
  • Plates with cyanobacteria and Bacillus subtilis
  • Salt
  • IPTG
  • One <2000Da dialysis bag
  • 20% and 70% ethanol
  • 30°C incubator with light all day
  • Spectrum-Pore Dialysis Tubing Closure

 

Procedure

  1. Pour in approximately 1L of BG11 medium mixed with ATCC in a 1:1 ratio in the baffled flask. We used chloramphenicol resistant organisms, and the final concentration of chloramphenicol of 10μL/mg
  2. Inoculate some cyanobacteria into the baffled flask. The flask should be greenish after the inoculation
  3. Let the cyanobacteria grow a couple of days. The OD at 730nm of the culture should be above 1,2 before induction
  4. Induce the cyanobacterium culture with IPTG and salt to a final concentrations of 0,1mM and 150mM, respectively.
  5. Incubate the cyanobacteria for a week, preferably more.
  6. Sterilize a <2000Da dialysis bag emerged in 20% ethanol using UV light (20 minutes on every side)
  7. Close the dialysis-bag with a Spectrum-Pore Dialysis Tubing Closure in one of the ends.
  8. Add desired media and inoculate B. subtilis with inoculation loop directly into the media. Limit contact with the dialysis bag membrane.
  9. Close the top with Spectrum-Pore Dialysis Tubing Closure end and sterilize with 70% Ethanol by washing the remaining tube inside and outside, twice.

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