Team:UFRGS Brazil/Protocol

Plasmid Minipreparation - Alkaline Lysis

1. Inoculate one colony of bacteria in 5 mL of LB medium, incubate at 37 oC overnight;

2. Prepare solution 1 and 3. (Solution 1: Glucose 50 mM; Tris-Cl pH 8.0 25 mM; EDTA pH 8.0 10 mM) (Solution 3: Potassium acetate 5M - 60 mL ; Glacial acetic acid - 11.5 mL ; H2O - 28.5 mL)

3. Add 2 mL of grown culture in microtubes, centrifuge at 14,000 g for 10 minutes; Discard supernatant. Repeat until all cells are pelleted;

4. Freshly prepare Solution 2 (NaOH 0.2 N; SDS 1% (w/v))

5. Add 250 μL of Solution 1.

6. Add 250 μL of Solution 2. Vortex intensely to resuspend cells;

7. Add 250 μL of Solution 3. Gently invert tubes 5 times;

8. Centrifuge at 14,000 g for 10 minutes;

9. Collect supernatant to new 2 mL tubes;

10. Add 0.5V of phenol (pH 8.0) and 0.5V of chloroform. Vortex intensely until all solution becomes a white cloud;

11. Centrifuge at 14,000 g for 10 minutes.

12. Carefully collect the upper phase to new 2 mL tubes. Do not collect the middle and lower phases;

13. Add 0.5V chloroform. Vortex intensely until all solution becomes a white cloud.

14. Carefully collect the upper phase to new 1.5 mL tubes. Do not collect the middle and lower phases;

15. Add 1V of cold Isopropanol and 0.2M of NaCl. Incubate at -20 oC for at least 20 minutes;

16. Centrifuge 14,000 g for 10 minutes. Discard the supernatant.

17. Wash with cold 70% ethanol. Centrifuge at maximum rotation for 2-3 minutes. Discard the supernatant.

18. Dry pellet to completely evaporate ethanol (speed-vac at 60 oC for 15 minutes).

19. Resuspend pellet in Mili-Q sterile water.

20. Treat DNA with RNAse.

Medium preparation

Luria-Bertani medium (LB)
LB medium was used in almost all steps requiring growth of E. coli. We used a ready-to-use medium composition, 20 g/L.
Minimal Salts medium (M9)
M9 medium is a minimal medium suitable for selection. As it is a well-known medium for E. coli, we decided to use it to test glyphosate usage as a phosphorus source. For that, we did different phosphorus concentrations as listed below.

	M9 Complete	M9 1/2 phosphate	M9 1/5 phosphate
Na2HPO4	6.78 g/L	3.395g/L	1.35g/L
KH2PO4	3 g/L	1.5 g/L	0.6 g/L
NH4Cl	1 g/L	1 g/L	1 g/L
NaCl	0.5 g/L	0.5 g/L	0.5 g/L
Glucose	10 g/L	10 g/L	10 g/L

Tris-Glucose medium (TG)
As we did not have good results using M9 medium, and salts were difficult to weight with precision, we decided to use a medium without any phosphorus at all, in order to possibilitate glyphosate addition as a sole phosphorus source.

Tris-Glucose Composition	Concentration
Tris	12 g/L
Glucose	10 g/L
NaCl	0.5 g/L
KCl	0.5 g/L
NH4SO4	2 g/L
MgSO4.7H2O	0.2 g/L
CaCl2	0.01 g/L
FeSO4.7H2O*	0.001 g/L

* 0.5 g of FeSO₄ . 7H₂O was diluted in 1 mL of distilled water to make a stock solution. It was stored at -4 ^oC.

PCR

Recombinant taq polymerase – Used for screening

	25 µL reaction
H2O	Up to 25 µL
10x buffer	2.5 µL
MgClH2	1µL
dNTP	5 µL(1mM)
Forward primer	1.5 µL(10 pmol)
Reverse primer	1.5 µL(10 pmol)
Taq Polymerase	0.1µL
Template	1~30ng

Recombinant taq polymerase – Program

Temperature	Time
95oC	2 min
*95oC (Denaturation)	30 sec
*55-64 oC (Annealing, depending of primer Tm)	30 sec
*72oC (Extension)	1kb/min
72oC	7 min
14oC	---

* 30 - 35 cycles

Q5 polymerase – Used for high fidelity amplification

	25 µL reaction
H2O	Up to 25 µL
Q5 Master Mix	12.5 µL
Forward primer	1.25 µL(10 pmol)
Reverse primer	1.25 µL(10 pmol)
Template	0.5~30ng

Q5 polymerase - Program

Temperature	Time
98oC	30 sec
*98oC (Denaturation)	10 sec
*64-72 oC (Annealing, depending of primer Tm)	10 sec
*72oC (Extension)	20 sec/kb (1sec for &lt 200bp amplification)
72oC	2 min
14oC	---

*10 cycles

Sinfle-joint PCR (Primerless) - Program

Q5 mix without primers - Template -1:1 ratio of DNA fragments to be fused.

Temperature	Time
94oC	3 min
*98oC (Denaturation)	30 sec
*58 oC (Annealing)	10 min
*72oC (Extension)	5 min
72oC	10 min
14oC	---

*10 cycles

Chemically Competent Cell Preparation

1. Inoculate one colony in 5 mL of LB medium without antibiotic and incubate at 37 oC overnight;

2. Inoculate 1 mL of bacteria grown overnight in 100 mL of LB medium and incubate at 37 oC until OD600 reaches 0.3 – 0.6;

3. Centrifuge culture at 3200 g for 10 minutes at 4 oC;

4. Discard supernatant and put tubes at ice immediately. Add 7.5 mL of CaCL2 100 mM and vortex briefly to resuspend cells;

5. Incubate at ice for 20 minutes;

6. Centrifuge at 3200 g for 10 minutes at 4 oC;

7. Discard supernatant and put tubes at ice immediately. Add 5 mL of CaCL2 100 mM and vortex briefly to resuspend cells;

8. Incubate at ice for 1 hour;

9. Centrifuge at 3200 g for 10 minutes at 4 oC;

10. Discard supernatant and resuspend pellet in 1 mL of CaCL2 100 mM + 1 mL of 15% Glycerol;

11. Sample 100 μL in sterile tubes and store at -80 oC.

Alginate Sphere Production

• Dilute 0.5, 1.0, 1.5 and 2.0% of alginate in distilled water
• Warm in water bath at 80ºC
• Drop the alginate into CaCl2 2% solution
• Wait 15 minutes for reticulation

For electron microscopy:

• Pour alginate spheres in liquid nitrogen for 10 minutes



• Lyophilize overnight

Electrocompetent Cells

• Dilute overnight culture in 1,4ml of LB and culture with agitation at 37ºC.
• After 2 hours when the OD at 600nm reaches approximately 0,6 centrifuge at 4000G for 10 minutes at room temperature (24ºC).
• Discard the supernatant, resuspend cells in 1ml of dH2O and centrifuge at 4000G for 10 minutes at room temperature (24ºC), repeat.
• Resuspend cells in 30ul of dH2O and proceed to electroporation.

Electroporation

• Add 300ng of plasmid or PCR fragment to the electrocompetent cells.
• Transfer the Cells and DNA mixture to a 1mm cuvette and electroporate 1800 volts.
• Immediately transfer the electroporate cells to 1ml of SOC medium without antibiotics and let it recover in agitation at 37ºC (30ºC for cells containing temperature sensible plasmid).
• Plate different volumes (ex. 100ul and 500ul) in agar LB containing the antibiotics for selection of transformants.




Arabinose-induced Lambda-Red Recombinase




• Dilute (1:100) overnight culture of cells containing the PKD46, plasmid that encodes the lambda-red recombinases, in 30ml of LB medium with ampicilin and arabinose 0,1M. Incubate with agitation at 30ºC.
• When the culture reaches OD₅₅₀ between 0,5 and 0,6 add arabinose 0,1M.
• When the culture reaches OD₅₅₀ between 0,7 and 0,8 centrifuge at 4000G for 10 minutes at room temperature (24ºC).
• Discard the supernatant, resuspend cells in 1ml of dH2O and centrifuge at 4000G for 10 minutes at room temperature (24ºC), repeat.
• Resuspend cells in 30ul of dH2O and proceed to electroporation




LC-MS/MS Method



An Agilent 1260 LC system (Waldbronn, Germany) coupled to ABSciex 5500 QTRAP triple quadrupole mass spectrometer (Toronto, Canada) was employed. The chromatographic separation was carried out on Acclaim Trinity Q1 column (3.0 µm, 100mm×3 mm; Thermo Scientific, Waltham, MA, USA). The mobile phase gradient elution was 100 mM ammonium formate 1% formic acid (A) and acetonitrile (B). The flow rate was 500µL.min-1 and a 4 minutes equilibrate time was applied. The gradient started with 100% of A, kept for 3 min and decreasing to 0% in 3 min and maintained until 8 min returning to initial condition. The column temperature was maintained at 30 °C. The injection volume was 5 µL.

Mass spectrometer resolution in multiple reactions monitoring (MRM) was unitary and dwell time applied was 50 ms for all transitions. The mass spectrometer was operated in negative electrospray ionization mode and precursor ions monitored were [M-H]-.

Nitrogen was used as nebulizer gas, curtain gas, heater gas and collision gas (psi units). Collision gas (CAD) was set at 4 psi, nebulizer gas (GS1) and dryer Gas (GS2) were set at 50 psi. Curtain gas and temperature was set at 20 psi and 600◦°C, respectively. Electrospray capillary voltage was set at -4.5kV.



Sample preparation:

500 µL of sample added 500 µL of 10 mL methanol, 1% formic acid followed by centrifugation at 3,500xg for 10 minutes.


Q1	Q3	Dwell Time (ms)	ID	DP(V)	EP(V)	CE(V)	CXP(V)
110.027	62.9	100	AMPA1	-15	-10	-26	-15
110.027	79	100	AMPA2	-15	-10	-38	-15
110.027	80.9	100	AMPA3	-15	-10	-18	-15
110.027	80	100	AMPA4	-15	-10	-24	-15
168	63	100	Glif 1	-30	-10	-26	-15
168	150	100	Glif 2	-30	-10	-14	-15
168	124	100	Glif 3	-30	-10	-16	-15
168	81	100	Glif 4	-30	-10	-20	-15





DP – Declustering Potential

EP – Entrance Potential

CE – Collision Energy

CXP – Exit Collision Cell Potential