Construct Assembly
Our sequences of interest were either drawn from the corresponding publications (true for glnAP2, prpB, prpR and pchA)1, 2, 3 or from Addgene (true for GFP, BFP and TagRFP). The Glowcoli Tag is an invented sequence by our team which acts as a spacer between the promoter and the coding sequence. The sequences were ordered from IDT as “gBlocks Gene Fragments in tubes” containing 250 ng of DNA. We centrifuged the unopened tubes (to make sure everything was at the bottom to prevent loss due to it sticking to the sides of the tube) and diluted the fragments in TE-Buffer as instructed by IDT.
Materials
- Agarose
- 1x TAE Buffer
- Midori Green
Methods
- For a 2% agarose add 2g of agarose powder to 100ml 1x TAE buffer and heat in the microwave until the agarose is completely dissolved.
- Let the agarose cool down to around ~60°C (when comfortable to hold in hands) and add 7µl Midori Green.
- Pour the agarose in a gel chamber, remove air bubbles if needed, insert combs as needed and let the gel dry for 20-30 min.
- Run gels on 400V, 130mA for approximately 20-40 minutes (dependent on your sequence) and use DNA ladder appropriate for your sequences.
In order to insert the sequences of interest via Gibson Assembly the plasmid backbone first had to be linearized.
Materials
- 5µL plasmid (pBS KS(+), 500µg/µL)
- 1µL HindIII-HF (20.000 U/ml)
- 3 µL CutSmart Buffer
- 16 ddH20
- 2,5 µl Alkaline Phosphatase
- 5µl Phosphatase buffer
Methods
- 5µl plasmid, 1µl HindIII-HF, 3µl CutSmart Buffer and 16µl water were mixed and incubated for 1h at 37°C.
- 5µl phosphatase buffer and 2,5µl alkaline phosphatase were added to prevent religation of the vector and incubated for further 30 min.
- Reaction was ianctivated at 80°C for 2 min.
Linearization was verified via gel electrophoresis on a 2% agarose gel (as described previously), together with the undigested pBS KS(+) as a negative control.
After the DNA mass has been determined via Nanodrop, the needed amount of DNA for the Gibson Assembly was quantified using following equation:
With the target amount for the vector being set to 0,02 pmol and the target amount for the insert being set to 0,04 pmol, the following results were obtained:
Gibson Assembly is a method that enables cloning of two or more fragments in the same reaction. For that, the sequences have to contain overlapping ends to allow seemless joining. A T5 exonuclease chews back the 5’ ends of the sequences and subsequently leads to the annealing of the complementary DNA fragments. A DNA polymerase fills up gaps creating a continous doublestranded DNA. Finally, the DNA ligase seals any remaining nicks, making the assembly permanent.
Materials
- Ice box (work on ice!)
- Heating block at 50°C
- Linearized vector (pBS KS(+))
- Inserts: “Acetate promoter”, “Propionate promoter”, “Butyrate promoter”, “GFP”, “BFP”, “TagRFP”
- Gibson Assembly Master Mix
Methods
Each of the promoter sequences (acetate-inducible, propionate-inducible or butyrate-inducible) was joined with each of the reporter genes (GFP, BFP or TagRFP), resulting in 9 constructs. For the negative control, water was used instead of an insert. The positive control was provided by the Gibson Assembly Kit to verify that the reagents work.
- The reactions (including the desired promoter, reporter gene, deionized water and the Gibson Assembly MM (2x)) were prepared as indicated in the figure and pipetting scheme (on ice!).
- The reactions were incubated at 50°C for 15 min.
- The samples were stored at -20°C until the transformation.
Bacterial Transformation
Materials
- Chemical competent bacteria
- Assembly product
- LB agar plates containing Carbenicillin
- LB medium
Methods
- Chemically competent bacteria were thawed on ice.
- 2µl of the assembly product was added per 50µl of bacteria and slowly mixed by pipetting up and down.
- Bacteria–Assembly Mix was incubated on ice for 30 min.
- Heat shock was performed for 30 seconds at 40°C; then bacteria were put on ice for 2 min.
- Bacteria were rescued in 950µl LB medium and incubated for 1h at 37°C in a shaker at 250rpm.
- Prior plating out the bacteria, the LB agar plates were pre-warmed at 37°C.
- After plating out 100µl of transformed bacteria on agar plates containing antibiotics, plates were incubated at 37°C overnight.
- On the next day, 5 individual colonies were picked of the assembly plates and the negative control and each transferred to 5ml LB medium containing carbenicillin (1:1000 dilution). The liquid culture was incubated at 37°C overnight in a shaker at 110 rpm.
Control digestion
To validate the transformation, we performed a control digestion using HindIII-HF as described previously. After assembly the HindIII restriction site was still retained and could be used for a control digestion, conferring an insert of the right size was cloned into the plasmid backbone.
The GLOW.coli System
Materials
- 96 well plates
- PBS
- Transformed Bactera as overnight culture, OD=0.5
- SCFA dissolved on PBS at different concentrations
Methods
- Determine OD of ON liquid cultures in LB
- Add the same ammount of bacteria (calculated via meassured OD) to each well in the 96 well plate.
- Spind the plates down at for 10 minutes.
- Carefully discrad the supernatant and resuspend the bacteria in 200µL of the corresponding SCFA dilution. Carefully resuspend the bacteria.
- Incubate the plates at 37°C and shaking, aquire fluorescence every hour.
