Team:UGent2 Belgium/Protocols

Protocols

PCR

Polymerase Chain Reaction (PCR) is used extensively during both the assembly and verification of new constructs. For assembly work, we use the high-fidelity DNA polymerases: PrimeSTAR HS, PrimeSTAR GXL (TaKaRa Bio) or Q5 (New England Biolabs). The manufacturer's instructions are followed unless mentioned otherwise. To create linear fragments suitable for assembly, BsaI restriction sites or overlapping sequences can be added to the 5' end of the primers during the in silico design for the naringenin pathway constructs. To create linear fragments suitable for assembly of the P22 promoter, complementary primers were used. The length of each fragment is verified using agarose gel electrophoresis, reaction mixtures are purified using a PCR purification kit (Analytik Jena) and DNA concentration is determined using a NanoDrop device (Thermo Scientific).

After transformation, verification of construct assembly is mostly performed using Colony PCR with Taq DNA Polymerase. Colonies are randomly selected and picked by hand using sterile pipette tips, which are then briefly touched to a backup plate and placed in a PCR tube. We slightly modified the manufacturer's protocol by extending the initial denaturation time to two minutes, to ensure lysis of the colonies. The length of each fragment is again determined by agarose gel electrophoresis.

Circular Polymerase Extension Cloning (CPEC)

Shorter constructs such as the naringenin pathway that was split up, was assembled using Circular Polymerase Extension Cloning (CPEC) (Quan & Tian, 2009). Our version of the protocol makes use of Q5 polymerase in the following reaction mixture:

 

Component

Amount

Vector backbone

100-200 ng

Insert

3:1 molar ratio to the vector

Q5 buffer

5.0 uL

dNTPs (2mM)

5.0 uL

DMSO

0.75 uL

Q5 DNA polymerase

0.5 uL

dH2O (add first)

to 15uL total volume

The tubes are incubated for 30 sec. at 98℃, 15 cycles of (10 sec. at 98℃; 30 sec. at 55℃; construct length(kb) x 15 sec. at 72℃) and 10 min. at 72 ℃. 1-2uL is then used directly for transformation, being careful not to add more as this increases the risk of arcing.

Golden Gate Cloning

Golden Gate cloning (Engler et al, 2009) was used to efficiently and seamlessly assemble and clone linear fragments into our expression vector (pBBR1MCS2). 15µL reactions are set up using NEB enzymes according to the table below. The tubes are then incubated for 25 cycles of (3 min. at 37℃; 4 min. at 16℃), 10 min. At 50℃ and 10 min. at 80℃, after which µL is used for transformation, without purification.

 

Component

Amount

Vector DNA

100 ng

Linear pieces

3:1 molar ratio to the vector

T4 buffer

1.5 uL

BSA (100x)

2.0 uL

BsaI-HFv2

1.0 uL

T4 DNA ligase

1.0 uL (400 units)

dH2O (add first)

to 15uL total volume


Transformation

Assembly mixes are transformed by electroporation using in home-made electrocompetent cells of the E. coli  Top10 genotype (Thermo Fisher) using 0.1 mm cuvettes at 1.80 kV. We create electrocompetent cells using the glycerol/mannitol density gradient wash method (Warren, 2011). It is of vital importance to keep the cells cold during the washing steps. One cryobox containing competent cells is made as follows:

The day before:

Make SOB medium: to 500 mL of dH2O add 10 g of tryptone, 2.5 g of yeast extract, 0.25 g of NaCl, 5 mL of a 250 mM solution of KCl. Set to pH 7.0 with 1 M NaOH and sterilize by autoclaving. Just before use add 2.5 mL of a sterile 2 M solution of MgCl2.

Make SOC medium: to 100 mL of the SOB medium add 2 mL of a sterile 1 M solution of glucose.

Make GM washing solution: 20 mL of glycerol, 1.5 g of mannitol, ultrapure water (Milli-Q) to total volume of 100 mL. Mix thoroughly, sterilize by filtration and refrigerate.

Sterilise 0.5 L of ultrapure water by autoclaving, refrigerate.

Autoclave a 2 L Erlenmeyer flask as well as about 100 1.5 mL Eppendorf tubes.

Start an E. coli Top10 preculture in a 5 mL LB culture tube.

In the morning:

Pour ~350 mL of SOB medium in the 2 L culture flask and inoculate with 3.5 mL of the overnight preculture.

Incubate at 37℃ with shaking until the OD measures 0.6-0.8 (roughly 2 h).

Distribute 300 mL of the OD 0.6-0.8 culture over 6x 50 mL falcons, incubate on ice for at least 30 min.

After lunch:

Centrifuge for 15 min. at 5000 g, 4℃, discard supernatant by decanting.

Resuspend each pellet in 20 mL cold ultrapure dH2O, vortex very briefly.

Carefully pipette a layer of 10 mL cold GM solution underneath cell suspension, forming 2 layers.

Centrifuge for 15 min. at 2000 g, 4℃, discard supernatant by pipetting from top to bottom.

Resuspend each pellet in 400 µL cold GM solution and distribute into pre-chilled sterile Eppendorf tubes as 50 µL aliquots, immediately store at -80℃

Plasmid isolation

Colonies that tested positive by colony PCR are grown in 5 mL LB culture tubes, after which plasmid DNA is isolated using QIAprep Spin Miniprep kit (Qiagen). To send a sample for sequencing by Macrogen: mix 7.5 µL plasmid DNA with 2.5 µL primer.

Transformation to E. coli K12 and C. necator

Verified plasmids are transformed by electroporation using in home-made electrocompetent cells of the E. coli  K12 MG1655 using 0.1 mm cuvettes at 1.80 kV. We create electrocompetent cells using the glycerol/mannitol density gradient wash method (Warren, 2011), as described above.
Verified plasmids are transformed by electroporation using in home-made electrocompetent cells of the C. necator PHB-4 using 0.3 mm cuvettes at 2.30 kV. Add plasmid DNA (2 µL) (~400 ng) to 100 µL competent cells, mix gently, transfer into a pre-chilled 2-mm electroporation cuvette and incubate on ice for 5 minutes and electroporate at 2.3 kV. Add 1 mL LB to the cells immediately after electroporation, transfer the cells to a sterile 15 mL tube and incubate for 2 hours at 30 °C
Spread 20 µL of the cells on LB agar plates with 50 µg/mL kanamycin, 10 µg/mL gentamicin and incubate for 48 h at 30 °C.

We create C. necator electrocompetent cells as follows:
Inoculate a C. necator colony in 5 mL LB and incubate for 38-42 h at 30 °C. Inoculate 100 mL fresh LB with the preculture at a 1:50 dilution and cultivate at 30 °C until an OD600 of 0.6-0.8 is reached
Transfer the cells to the ice for 5-10 minutes. Aliquot the cells (50 mL each) and centrifuge at 3000 x g at 4 °C for 5 minutes. Wash the cells trice with 25 mL ice-cold sterile 10 % glycerol. Resuspend the cell pellet in 0.3 mL 10 % glycerol and aliquot into sterile 1.5 mL centrifugation tubes (100 µL/tube)
The competent cells can be used immediately or frozen in liquid nitrogen and preserved at -80 °C. Both procedures are adapted from (Xiong et al, 2018).

Growth trial of pBBR1MCS2_P22_RBS_mKate2 in E. coli K12

Preparation of minimal medium (or defined medium).

Take two autoclaved bottles, 1L and 250 mL

Each litre of medium contains:

2 g NH4Cl

5 g (NH4)2SO4

3 g KH2PO4

7.3 g K2HPO4

8.4 g MOPS

0.5 g NaCl

0.5 g MgSO4.7H2O

16.5 g glucose.H2O

1mL trace element solution

100 µL molybdate solution

All except the glucose, MgSO4, trace element solution, and molybdate is dissolved in 800 mL H2O and set to pH 7 with KOH. After autoclaving, the sterile trace element and molybdate solutions are added.

Glucose and MgSO4 were dissolved in H2O and autoclaved separately to avoid Maillard reaction.

All reagents are from Sigma Aldrich.

Isolate cultures (pBBR1MCS2_P22_RBS_mKate2 and pBBR1MCS2_AarI in E. coli K12 MG1655) are grown on LB + Kanamycin overnight. Single colonies are picked in triplicates with a sterile pipet tip. The sterile tips with the colonies are put into 150µL of LB medium in a multiter plate (MTP) with the lid closed, to grow overnight at 37°C, 800 RPM on a rotary shaker. Exact 24 hours later, the colonies are 1% diluted as follows:

Take 20 µL from the preculture plate and put it in 140 µL minimal medium.

Mix well by pipetting up and down.

10 µL of the dilution is added to the final plate. This is a black MTP plate to make sure there is no interference of fluorescence of neighbouring wells.

The black MTP plate is put into the Tecan microplate reader and the following settings are used:

Measurement Wavelength: 600nm

Fluorescence Bottom Reading

Excitation Wavelength: 588nm

Emission Wavelength: 633nm

Gain: 110Manual

Part of Plate: D1-D9

A small deviation from the protocol for C. necator: Stock solution of LB + Kan + Gent (10 mL LB + 10 µL Kan + 10µL Gentamicin) in 15 mL falcon. In the middle row of the MTP => Inoculation of the strains grown on a plate in a growth plate (transparent 96-well plate) using LB + antibiotics (150 µL in total). Pick colonies, for colony PCR and put it in the MTP. MTP plate is the backup plate of the colony PCR.

Data processing of growth trial of pBBR1MCS2_P22_RBS_mKate2 in E. coli K12

Collecting data points every 10 minutes during 24 hours requires clever data management. A script in python (Van Rossum, 1995) was used for handling the massive amount of data. First, the data is corrected for background interferences. Blank values for OD600, only the growth media, are subtracted from the OD600 values obtained from the colonies. The RFP is corrected by subtracting fluorescence obtained by the empty vector (pBBR-MCS2_AarI) from the RFP values obtained by the vector containing mKate2 (pBBR1-MCS2_P22_RBS_mKate2. Once the values have been corrected, they can be used to calculate the normalized RFP. Normalizing RFP is executed by dividing the RFP values at a certain time point by the OD600 at a certain time point. Note that these time points need to be the same.

References

Engler C, Gruetzner R, Kandzia R & Marillonnet S (2009) Golden gate shuffling: A one-pot DNA shuffling method based on type ils restriction enzymes. PLoS One 4: e5553 Available at: https://dx.plos.org/10.1371/journal.pone.0005553 [Accessed October 26, 2020]

Quan J & Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS One 4: e6441 Available at: https://dx.plos.org/10.1371/journal.pone.0006441 [Accessed October 26, 2020]

Van Rossum G (1995) Centrum voor Wiskunde en Informatica Python tutorial

Warren DJ (2011) Preparation of highly efficient electrocompetent Escherichia coli using glycerol/mannitol density step centrifugation. Anal. Biochem. 413: 206–207 Available at: https://pubmed.ncbi.nlm.nih.gov/21362398/ [Accessed October 26, 2020]

Xiong B, Li Z, Liu L, Zhao D, Zhang X & Bi C (2018) Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique. Biotechnol. Biofuels 11: 172 Available at: https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-018-1170-4 [Accessed October 26, 2020]