Standard Protocols Rice University 2018 iGEM Team

Protocol 1: Golden Gate Assembly 2 Table 1: Golden Gate reaction materials 2 Table 2: BsaI Golden Gate assembly thermocycling conditions 2 Table 3: Esp3I Golden Gate assembly thermocycling conditions 3 Procedure 3 Protocol 2: Phusion/Q5 PCR 5 Table 1: Phusion/Q5 PCR materials 5 Table 2: Phusion/Q5 thermocycling conditions 5 Procedure 5 Protocol 3: Colony PCR 6 Table 1: Colony PCR materials 6 Table 2: Phusion/Q5 thermocycling conditions 6 Procedure 6 Protocol 4: Electroporation Transformation 7 Table 1: Electroporation materials 7 Procedure 7 Protocol 5: Heat Shock Transformation 8 Table 1: Heat Shock Transformation materials 8 Procedure 8 Protocol 6: Preparing Heat Shock Competent Cells 9 Table 1: Preparing Heat Shock Comp Cells materials 9 Procedure 9 Protocol 7: Gel Electrophoresis 10 Procedure: Preparing agarose gel 10 Procedure: Loading samples and running agarose gel 10 Procedure: Analyzing Your Gel 11 Protocol 8: LB agar plates preparation 12 Materials 12 Procedure 12 Table 1: Antibiotic Concentrations 12 Protocol 9: Purification of Plasmid DNA 13 Protocol 11: F luorescence Time Course 13 Protocol 12: Preparing E . coli and P . putida electrocompetent cells 14 Protocol 13: Preparing S . oneidensis and S . meliloti electrocompetent cells 15 Protocol 13: Preparing L . lactis electrocompetent cells 16 Protocol 14: Preparing C . glutamicum electrocompetent cells 17 Protocol 15: Preparing B . subtilis chemically competent cells 18

Protocol 1: Golden Gate Assembly

Table 1: Golden Gate reaction materials Component Volume Notes BsaI/Esp3I/BbsI 0.5 µL 0.5–0.75µL range.

T DNA Ligase 0.5 µL 0.5–1 µL range 4

10× T Ligase Buffer 1.5 µL Titurate/vortex to dissolve solids. 4

10× BSA 1.5 µL Enables full BsaI activity at 37°C

DNAs 25 fmol each 10–40 fmol equimolar. 0.5 µL 50 nM 2–5-fold less vector to reduce background. Deionized Water up to 15 µL 10–20 µL range.

Table 2: BsaI Golden Gate assembly thermocycling conditions BsaI Golden Gate, Long, ≥6 parts: 142.5 min / 2:23 Basic*: 52.5–75 min Short, ≤5 parts: 97.5 min / 1:38

Step Temp Time Temp Time

Initial Digestion (opt. ) 37°C 10 37°C 15 min min

Repeat Digestion 37°C 1.5 Repeat 37°C 1.5 25× / 15× min 5–10× min

Annealing & Ligation 16°C 3 min 16°C 3 min

Digestion & Ligase 50°C 10 50°C 5 min Inact. min

Inactivation 80°C 10 80°C 10 min min

Storage 12°C ∞ 12°C ∞

Table 3: Esp3I Golden Gate assembly thermocycling conditions Esp3I Golden Gate, Long, ≥6 parts: 142.5 min / 2:23 Basic*: 52.5–75 min Short, ≤5 parts: 97.5 min / 1:38

Step Temp Time Temp Time

Initial Digestion (opt. ) 37°C 10 37°C 15 min min

Repeat Digestion 37°C 1.5 Repeat 37°C 1.5 25× / 15× min 5–10× min

Annealing & Ligation 16°C 3 min 16°C 3 min

Digestion 37°C 5 min 37°C 5 min

Digestion & Ligase 50°C 5 min 50°C 5 min Inact.

Inactivation 80°C 10 80°C 10 min min

Storage 12°C ∞ 12°C ∞

P rocedure

1. Prepare a mastermix by adding together enzyme, T 4 DNA Ligase, 10X T 4 Ligase Buffer, 10X BSA, and water 2. Pipette up and down to mix 3. Distribute mastermix into labelled pcr tubes 4. Add DNA components for each reaction to the concentrations specified in the Table 1 5. Flick to mix 6. Place the reaction tubes into the thermocycler and adjust the settings for a given enzyme as shown in Table 2 or Table 3

Protocol 2: Phusion/Q5 PCR

Source: h ttps://www.neb.com/protocols/1/01/01/pcr-protocol-m0530

Table 1: Phusion/Q5 PCR materials Component Volume Final Concentration H20 to 50 µL

5X Phusion HF or GC 10 µL 1X Buffer

10 mM dNTPs 1 µL 200 µM

10 µM forward primer 2.5 µL 0.5 µM

10 µM reverse primer 2.5 µL 0.5 µM

template DNA** variable 10 ng

phusion polymerase 0.5 µL 1.0 units per reaction

Table 2: Phusion/Q5 thermocycling conditions Step 1 2 3 4 5 6 7

98C 98C Tm 72C GO TO 72C 4C Temperature Step 2 0:30 0:10 0:30 30s/kb 34X 5:00 hold Time

Procedure

1. Prepare master mix by adding together water, GC/HF buffer, dNTPs, primers and phusion polymerase to the concentrations shown in Table 1 2. Add 10 ng of template DNA 3. Flick to mix 4. Place the reaction tubes into the thermocycler and adjust the settings for a given enzyme as shown in Table 2

Protocol 3: Colony PCR

Table 1: Colony PCR materials Component Volume Final Concentration H20 to 25 µL

5X Phusion HF or GC 5 µL 1X Buffer

10 mM dNTPs 0.5 µL 200 µM

10 µM forward primer 1.25 µL 0.5 µM

10 µM reverse primer 1.25 µL 0.5 µM

template DNA** 1 uL unknown

phusion polymerase 0.25 µL 1.0 units per reaction

Table 2: Phusion/Q5 thermocycling conditions Step 1 2 3 4 5 6 7

98C 98C Tm 72C GO TO 72C 4C Temperature Step 2 3:00 0:10 0:30 30s/kb 34X 5:00 hold Time

Procedure 1. Prepare LB with chloramphenicol(17) by adding 0.5uL chloramphenicol to 1mL of LB 2. For 5 PCR reactions per sample: a. Pipette 5 uL of LB solution into each of the 15 PCR tube b. Pick up five colonies per plate and inoculate a colony per PCR tube 3. Prepared a mastermix for the appropriate number of 25uL reactions 4. Add 10 uL of mastermix to each PCR tube 5. Add 1 uL of LB cultures to corresponding tubes 6. Flick the tubes to mix 7. Place the reaction tubes into the thermocycler and adjust the settings as shown in Table 2

Protocol 4: Electroporation Transformation Table 1: Electroporation materials Component Volume

Electrocompetent 50 uL cells

DNA 1uL

SOB 1 mL

Glucose (1M) 20 uL

Electroporation cuvette

LB agar plate with appropriate antibiotic

Procedure 1. Thaw electrocompetent cells on ice 2. Add 1uL of DNA to the tube containing electrocompetent cells 3. Pipet cells into a chilled cuvette and keep on ice 4. Prepare 1 mL of SOC medium by adding together 1 mL of SOB and 20 uL of 1M glucose 5. Turn on the Microcomputer and set to appropriate program (Ec1 for 0.1 cm cuvettes) 6. Insert cuvette into the slide 7. Pressed the "Pulse" button 8. Immediately remove the cuvette, add 1mL SOC, and gently mix by pipetting up and down 9. Transfer cells to the falcon tubes and place in shaking incubator at 37C for 1h 10. Retrieve the tubes from the incubator and plated 100 uL of cells 11. Place the plates into 37C incubator

Protocol 5: Heat Shock Transformation

Modified from: http://parts.igem.org/Help:2017_Competent_Cell_T est_Kit

Table 1: Heat Shock Transformation materials Component Volume

Electrocompetent 50 uL cells

DNA 1uL

SOB 1 mL

Glucose (1M) 20 uL

LB agar plate with appropriate antibiotic

Procedure 1. Thaw competent cells on ice. 2. Pipet 1 µL of DNA into each tube with cells. 3. Incubate on ice for 30 minutes. 4. Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too! 5. Heat-shock the cells by placing into the waterbath for 45 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by. 6. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. 7. Add 950 µL of SOC media per tube, and incubate at 37°C for 1.5 hours shaking at 200-300rpm. 8. Pipet 100 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 37°C overnight or approximately 16 hours. Position the plates with the agar side at the top, and the lid at the bottom.

Protocol 6: Preparing Heat Shock Competent Cells

Table 1: Preparing Heat Shock Comp Cells materials Component Volume

Cells glycerol stock

LB agar plate (no antibiotic)

LB 11 mL

TSS buffer 1.6 mL

Procedure 1. Streak cells from glycerol stock on LB agar plate and incubate in 37C incubator overnight 2. Pick up a colony and prepare liquid culture in 4 mL LB. Incubate overnight in 37 shaking incubator. 3. Retrieve liquid culture from the incubator. 4. Add 100uL of seed culture to 900uL LB media (previously prepared) in a cuvette 5. Blank the spectrophotometer with 1mL LB media 6. Measured the OD600 of the diluted culture 7. Start a new culture by adding 50ul of seed culture to 4.95mL of LB media for a total volume of 5mL to get 100 fold dilution 8. From the OD of the diluted culture and doubling time of the cells, calculate the time needed to reach the OD of about 0.2 and place the culture into 37C incubator. 9. Retrieve the culture from the incubator and place on ice to stop growth. 10. Centrifuge at 4 degrees C for 10 min at 2500 rpm 11. Discard supernatant 12. Resuspend cell pellet in 1.6mL pre-chilled TSS buffer (pre-prepared) 13. Distributed competent cell solution across (32) 1.5mL tubes to make 50uL aliquots 14. (optional) flash freeze the (32) aliquots in liquid nitrogen 15. Store the aliquots in the -80C freezer

Protocol 7: Gel Electrophoresis Procedure: Preparing agarose gel 1. Measure 1 g of agarose (for 1 00 mL of 1% gel:) 2. Mix agarose powder with 100 mL 1xTAE in a microwavable flask. 3. Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.) Note: Caution HOT! Be careful stirring, eruptive boiling can occur. Note: It is a good idea to microwave for 30-45 sec, stop and swirl, and then continue towards a boil. Keep an eye on it as the initial boil has a tendency to boil over. 4. Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins. 5. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light. Note: Caution EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical. Note: If you add EtBr to your gel, you will also want to add it to the running buffer when you run the gel. If you do not add EtBr to the gel and running buffer, you will need to soak the gel in EtBr solution and then rinse it in water before you can image the gel. 6. Pour the agarose into a gel tray with the well comb in place. Note: Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip. 7. Place newly poured gel at 4°C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.

Procedure: Loading samples and running agarose gel 1. Add loading buffer to each of your digest samples. Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and will also allows you to gauge how far the gel has run while you are running your gel; and 2) it contains a high percentage of glycerol, so it increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer. 2. Once solidified, place the agarose gel into the gel box (electrophoresis unit). 3. Fill gel box with 1x TAE (or TBE) until the gel is covered. 4. Carefully load a molecular weight ladder into the first lane of the gel. Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, push the pipettor to the second stop and carefully raising the pipette straight out of the buffer. 5. Carefully load your samples into the additional wells of the gel. 6. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel.

Note: Black is negative, red is positive. (The DNA is negatively charged and will run towards the positive electrode.) Always Run to Red. Note: A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage. 7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box. 8. (Optional) If you did not add EtBr to the gel and buffer, place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, replace EtBr solution with water and destain for 5 mins. 9. Using any device that has UV light, visualize your DNA fragments. Note: When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab coat. Note: If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA. Note: The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.

Procedure: Analyzing Your Gel Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can interpret the bands that you get in your sample lanes to determine if the resulting DNA bands that you see are as expected or not.

Protocol 8: LB agar plates preparation

Materials: ● 5 g NaCl ● 2.5 g yeast extract ● 50 uL NaOH ● 5 g tryptone ● agar 7.5 g ● 500 mL H20 ● 250 uL of 34 mg/ml chloramphenicol* *Note: this protocol is for preparing plates with 17 µg/mL of chloramphenicol antibiotics. We also prepared plates throughout the course of our work with the antibiotics and concentrations listed in Table 1 below.

Procedure: 1. Add all materials except antibiotic to a 1L flask, and use a stir bar/stir plate to dissolve all the solids. 2. Cover top of flask with loose foil and autoclave. Autoclaved on setting #3 (liquids) 3. Once autoclaved agar is cool enough to handle without gloves, add 250uL of 34mg/ml chloramphenicol for a final concentration of 17ug/ml chloramphenicol 4. Use a serological pipette in the biosafety cabinet to add 20mL of agar+antibiotic to each plate 5. Label plates 6. Allow plates to solidify and then transfer to 4C fridge

Table 1: Antibiotic Concentrations

Working Antibiotic Shorthand Concentration

Ampicillin 100 µg/mL Amp100

Chloramphenicol 17 or 34 µg/mL Chl17 / Chl34

Kanamycin 50 µg/mL Kan50

Protocol 9: Minipreparation of Plasmid DNA (“Miniprep” for short)

Procedure* 1. Pellet bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C). 2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. 3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue. 4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless. 5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. 6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through 7. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through. Transfer the QIAprep 2.0 spin column to the collection tube. 8. Centrifuge for 2 min to remove residual wash buffer. 9. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min. 10. Measure concentration of the purified plasmid DNA using nanodrop

*W e use the Qiagen Spin Miniprep Kit and reference the Qiagen protocols

Protocol 11: Fluorescence Time Course Measurements

Procedure

1. Prepare liquid cultures by picking a colony into 4 mL of LB agar (with appropriate antibiotic, if needed) a. Use 3 biological replicates per construct 2. Grow cultures to saturation in either 37C or 30C incubator depending on the optimal growth temperature for a given strain 3. Dilute cultures to OD600 of approximately 0.05 in LB with appropriate antibiotic 4. Grow cultures to OD of about 0.5 5. Dilute cultures to OD of of approximately 0.05 in LB with appropriate antibiotic in a 96-well flat bottom plate a. Final volume used was 100 uL 6. Remove the plastic lid and cover the plate with transparent membrane permeable to oxygen 7. Place the plate into TECAN a. TECAN settings: 37C for measurements with E . coli, 30C when measurements involved S . oneidensis a nd P . putida b. Excitation: 588; emission: 613 (for mKate2 fluorescent protein) 8. Take measurements over 15 hours, measuring fluorescence intensity and OD600

Protocol 12: Preparing E . coli and P. putida electrocompetent cells

From Bio-Rad MicroPulser Manual Based on Ausubel e t al( 1987) and Miller and Nickoloff (1995)

1. Inoculate 500mL of L-broth with 1/100 volume of a fresh overnight E . coli c ulture

2. Grow the cells @ 37C shaking @ 300RPM to an OD6 00 of approximately 0.5-0.7 3. Chill cells on ice for ~20min. For all subsequent steps, keep the cells as close to 4C as possible and chill all containers in ice before adding cells. To harvest, transfer the cells to a cold centrifuge bottle and spin @ 4000 x g for 15min @ 4C 4. Carefully pour off and discard the supernatant. It is better to sacrifice yield by pouring off a few cells than to leave any supernatant behind 5. Gently resuspend the pellet in 500mL of ice-cold 10% glycerol. Centrifuge @ 4000g for 15min @ 4C; carefully pour off supernatant 6. Resuspend the pellet in 250mL of ice-cold 10% glycerol. Centrifuge @ 4000 x g for 15min @ 4c; carefully pour off supernatant 7. Resuspend pellet in 25 mL of ice-cold 10% glycerol. Transfer to a 30 mL sterile Oakridge tube. Centrifuge @ 4000 x g for 15min @ 4C; carefully pour off supernatant 8. Resuspend pellet(s) in 10 mL of ice-cold 10% glycerol. Transfer to a 30 mL sterile Oakridge tube. Centrifuge @ 4000 x g for 15min @ 4C; carefully pour off supernatant. 9. Resuspend the cell pellet(s) in a final volume of 1-2 mL of ice-cold 10% glycerol. The cell concentration should be about 1-3 x 101 0 cells/mL

This suspension may be frozen in aliquots on dry ice and stored @ -70C. The cells are stable for at least 6 months under these conditions.

Shewanella Oneidensis MR-1 Electrocompetent

1. Inoculate1:100 from overnight culture in 20 ml LB for about 2-3 hours 2. Centrifuge at 4000g for 5 min, pellet is pink 3. Wash with 6 ml 1M D-sorbitol (sterile, pH= 7.5). Use 1.5 ml tube, 14000 rpm 1 min to pellet. 4. Repeat previous step 3 times. 5. Resuspend in 100 ul of 1M D-sorbitol

(You can do these steps on ice but it also works without it)

6. Add 200ng of DNA. 7. Electroporate at 1.8 kV -> 4.2 ms 8. Add 1 ml LB, recover for 90 min at 30 C shaker 9. Plate everything on LB+antibiotic, incubation at 30C. Colony formed the next day.

Sinorhizobium meliloti 1020 Electrocompetent

1. Sinorhizobium cells were grown in TY medium for about 6 h until the OD600 reached 0.6.

(It requires more time in my experience. Overnight at room temperature if using TY medium).

2. Cells were harvested by centrifugation at 8000 rpm for 10 min at 4ºC. 3. Pellet from a 500 ml culture was washed twice with an equal volume of sterile distilled water and once with an equal volume of sterile 10% glycerol. 4. Finally, re-suspend in 2 ml of 10% glycerol and stored at -80ºC.

5. Add 200ng of DNA. 6. Electroporate at 2.1 kV/cm with a time constant fixed at 5 ms.

(I use the EC2 setting (2.5kV) in our electroporator and it works fine).

7. Add 1 ml LB, recover for 3hrs at 30ºC shaker 8. Plate everything on LB+antibiotic, incubation at 30C. Colony formed after 2-3 days.

Lactococcus lactis

Protocol No. 4308 915.519 – 12/2001

Microorganism Lactococcus lactis MG1363 Cell type Bacteria, gram positive Molecules injected Plasmid DNA (pGK12)

Growth medium Complex medium with 1% glycine Washing solution 0.5 M sucrose, 10% glycerol Electroporation solution 0.5 M sucrose, 10% glycerol

Outgrowth medium Ice-cold complex medium with 0.5 M sucrose, 20 mM MgCl2, 2 mM CaCl2 Cuvette 1 mm gap width

Reference Dr. Horst Neve Œ Bundesanstalt für Milchforschung Œ Institut für Mikrobiologie Hermann-Weigmann Str. 1 Œ D-24103 Kiel Œ Phone +49 431 6091 Œ Fax +49 431 609222

Making electrocompetent cells:

1. Grow cells overnight at 30 °C to an O.D.620 of 0.7. 2. Wash twice with ice-cold washing solution. 3. Resuspend cells in 1/100 volume of electroporation solution. Keep on ice.

Electroporation of cells: 1. Add 0.25 µg plasmid DNA (in water) to 100 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into cuvette. 2. Wipe moisture from the cuvette and insert the cuvette into the device. 3. Electroporation: Mode Prokaryotes “{“ Voltage (V) 2,000 V Time constant (τ) 5 ms

4. Add 1 ml ice-cold complex medium, incubate 2 hours at 30 °C. 5. Plate diluted cells on selective chloramphenicol plates. Incubate 2 days at 30 °C.

Expected results: Transformation efficiency up to 1.4 x 106 transformants/µg of DNA.

Corynebacterium glutamicum

Protocol No. 4308 915.510 – 12/2001

Microorganism Corynebacterium glutamicum Cell type Bacteria, gram positive Molecules injected Plasmid DNA

Growth medium LBG (LB with 0.5% glucose) supplemented with 2.5% glycine and isonicotinic acid hydrazid (4 mg/ml) Washing solution 15% glycerol (v/v) Electroporation solution 15% glycerol (v/v) Outgrowth medium LBG medium (LB with 0.5% glucose) Cuvette 2 mm gap width

Reference Haynes J. and Britz M. Π1990 ΠJournal of General Microbiology 136 Π255-263

Making electrocompetent cells:

1. Grow cells in LBG medium at 30 °C and shaking at 200 rpm to an O.D.600 of 0.15 to 0.25. 2. Harvest by centrifugation. 3. Wash in one culture volume of 15% glycerol. 4. Resuspend cells in 0.002 culture volumes of 15% glycerol (number of cells: approx. 2.5 x 1010 cells/ml).

Electroporation of cells: 1. Add 1-2 µl plasmid DNA (in water) to a minimum of 40 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette. 2. Wipe moisture from the cuvette and insert the cuvette into the device. 3. Electroporation: Mode Prokaryotes “{“ Voltage (V) 2,500 V Time constant (τ) 5 ms

4. Immediately transfer cell suspension into LBG medium at a 1:25 dilution, incubate 1 hour at 30 °C. 5. Plate cells on selective plates; incubate for 4 days.

Expected results: Transformation efficiency up to 5 x 105 transformants/µg of DNA.

Competency Protocol 1. Grow a single colony of the strain on LB agar overnight (with antibiotic if necessary). 2. Inoculate single colony (or more colonies) in 20 ml SpC medium in 250 ml flask. Incubate in shaker at 37°C and follow growth by OD600 readings. Plot OD600 readings on a graph and draw a growth curve. 3. When OD600 reading does not change at all for 20­30 minutes (entry into stationary), dilute culture 1:10 (2 mls into 20 mls) into pre­warmed, SpII in 250 ml flask (the same flask for SpC can be reused). It should take 4­6 hours to get to this point if you started with single colony or small colonies. 4. Incubate for 90 minutes in shaker at 37°C. 5. Spin at room temperature (NOT 4°C; Bacillus will lyse) for 5 minutes to pellet cells. Pour off supernatant into sterile tube. Save this supernatant (competence factors are here). 6. Resuspend pellet (from 20 ml culture) in 1.6 ml saved supernatant and add 0.4 ml sterile 50% glycerol. Resuspending in fresh media will not work: supernatant contains secreted competence factors. 20ml culture: 1.6 ml supernatant + 0.4 ml 50% glycerol 40ml culture: 3.2 ml supernatant + 0.8 ml 50% glycerol 50ml culture: 4.0 ml supernatant + 1.0 ml 50% glycerol 60ml culture: 4.8 ml supernatant + 1.2 ml 50% glycerol 7. Distribute aliquot (~1 ml) into 1.5 ml tubes (x 2) and store at ­80°C. Transformation (B. Subtilis) 1. Thaw competent cells in 37°C water bath. 2. To 1.5 mL tube, add: 50 ul competent cell 200­1000ng of plasmid DNA. 50 ul SpII­EGTA 3. Mix gently and incubate in 37°C shaker for 40 min 4. Plate on selective media Media Recipes

SpC 20mL 50mL 60mL 200mL

AC'd H20 18mL 45mL 54ml 180mL

10x T­base 2mL 5mL 6ml 20mL

20% Glucose 500uL 2x625uL 2x750ul 5mL

12% MgSO4 (1M) 30uL 75uL 90ul 300uL

10% Yeast Extract 400uL 1000uL 2x600ul 4mL

1% Casamino Acids 500uL 2x625uL 2x750ul 5mL

SpII 20mL 30mL 200mL AC'd H20 18mL 27mL 180mL

10x T­base 2mL 3mL 20mL

20% Glucose 500uL 750uL 5mL

1M MgSO4 140uL 210uL 1.4mL

1M CaCl2 10uL 15uL 100uL

10% Yeast Extract 200uL 300uL 2mL

1% Casamino Acids 200uL 300uL 2mL

To make SpII­EGTA add 2% v/v 0.1M EGTA for a final concentration of 2mM

10x T­base (Autoclave)

Name Concentration MW Amount Parameter (g)

Concentrated HCl (for pH adj.)

Ammonium sulfate (NH4)2SO4 150 mM 132.14 9.91 g per bottle

K­phosphate (K2HPO4, dibasic) 800 mM 174.2 69.68 g per bottle

K­phosphate (KH2PO4, 440 mM 136.09 29.94 g per bottle monobasic)

Na­Citrate (Na3C6H5O7­2H2O) 35 mM 294.1 5.15 g per bottle

Filtered water 500 mL per bottle

0.1M EGTA

Name Concentration MW (g) Amount Parameter

Concentrated HCl (for pH adj.)

EGTA­4Na (C14H20N2O10Na4) 0.1M 468.28 4.682g g per bottle

Filtered water 100 mL per bottle

1. Weight the appropriate amount of EGTA­4Na. 2. Add 50mL H2O 3. pH will be around 12. Adjust to 7.5­8.0 with HCl 4. Fill up to 100mL with H2O. 5. Autoclave. SpC

Name Amount Parameter

1x T­base 20 mL per bottle

50% Glucose 0.2 mL per bottle

1.2% MgSO4 (100mM) 0.3 mL per bottle

10% Yeast Extract 0.4 mL per bottle

1% Casamino Acids 0.5 mL per bottle

SpII

Name Amount Parameter

1x T­base 20 mL per bottle

50% Glucose 0.2 mL per bottle

100mM (1.2%) MgSO4 1.4 mL per bottle

0.1M CaCl2 0.1 mL per bottle

10% Yeast Extract 0.2 mL per bottle

1% Casamino Acids 0.2 mL per bottle

SpII­EGTA 10mL

1x T­base 10mL

50% Glucose 0.1

100mM (1.2%) MgSO4 0.7

0.1M CaCl2 0.05

10% Yeast Extract 0.1 1% Casamino Acids 0.1

0.1M EGTA 0.2

Notes

20141031

The competency protocol was followed as described for bth a Bs168 culture and a Bs84, the following growth curves were obtained:

Bs168 Bs84

­120 0 0

0 0.444 0.076

33 0.796 0.339

61 1.087 0.641

91 1.433 0.962

125 1.56 1.21

155 1.66 1.47

185 1.768 1.55

220 1.85 1.507

100ng pSC120 was transformed without freezing the cells and gave ~500 colonies giving an efficiency of 5*10^3.