Team:CCA San Diego/protocols

protocols

Transformation with Digest

Materials

• DNA miniprrep
• LB agar plates, Cat No. Teknova, appropriate antibiotic
• DH5a competent cells, Invitrogen, Cat 18265-017
• SOC (Recovery Medium), Lucigen, Cat No. F98226
• 1.5 mL tube
• Vortex
• Pipet and tips
• Ice bucket and ice
• Water bath (42°C)
• Incubator (37°C) and shaker

Methods

1. Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
2. For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells
3. Turn on incubator-shaker at 37°C.
4. Turn on incubator for plates at 37°C.
5. Set up water bath at 42°C.
6. Bring to room temperature S.O.C medium.
7. Bring LB plates supplemented with appropriate antibiotic at room temperature.
8. Thaw competent cells on ice.
9. Aliquots competent cells in as many tubes as needed.
10. Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix
11. Incubate on ice for 20 minutes
12. Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.
13. Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube
14. Incubate in shaker at 37°C, 225 rpm for 30 min
15. Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)
16. Incubate plates at 37°C overnight
17. Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Digest Ligations

Materials

• T4 DNA Ligation Buffer. Invitrogen, Cat No. 46-0114
• T4 DNA Ligase, Thermo Scientific, Cat No. K1231
• Reagent grade water, NERL, Cat No. 98555
• 1.5 mL tube
• Vortex
• Pipet and tips
• Ice bucket and ice

Methods

1. Set ligation as shown below in 1.5 mL tube.
2. The tubes were incubated at room temperature for 1 hour.
3. Turn on incubator-shaker at 37°C.
4. The tubes were then transferred to ice.
5. Ligation Condition (with insert)

   Component	Volume (μl)
   Reagent grade water	4.5 μl
   10X T4 ligation buffer	1.0 μl
   EcoR1-Pst1 Linearized - pSB1C3	0.5 μl
   EcoR1/SpeI - Linearized –promoter (For us: BBa_J23100, BBa_J23101, BBa_J23110)	2.0 μl
   XbaI/Pst1 - Linearized -Insert (Catabolic pathway) (For us: Fluorene 1, or Fluorene 2, or Phenanthrene 1, or Phenanthrene 2)	1.5 μl
   T4 DNA Ligase (5Weiss/μl)	1.5 μl

6. Control Ligation Condition (no insert)

   Component	Volume (μl)
   Reagent grade water	8.0 μl
   10X T4 ligation buffer	1.0 μl
   EcoR1-Pst1 pSB1C3	0.5 μl
   T4 DNA Ligase (5Weiss/μl)	0.5 μl

OD Calibration

Materials

• 1 mL LUDOX (provided in kit)
• Water
• 96 well plate with flat with transparent bottom

Methods

1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
2. Add 100 μl of H 2 O into wells A2, B2, C2, D2 (or 1 mL H 2 O into cuvette)
3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
4. Record the data in the table below or in your notebook
5. Import data into Excel ( OD600 reference point tab )

Biotransformation Measurement

Materials

• Cultures of clones in transformed in Escherichia coli strain BL21(DE3)
• LB (Luria Bertani) media
• Minimal medium
• Testing substance (Fluorene and Phenanthrene)
• Appropriate Antibiotics (Chloramphenicol, Tetracycline and Ampicillin)
• IPTG
• Baffled flasks
• 50 mL Falcon tube with vented cap
• Incubator at 37℃
• 1.5mL Eppendorf tubes for sample storage
• Ice bucket with ice
• Pipettes
• 96 well plate with flat transparent bottom devices
• Shaker

Instrumental Methods

1. Set instrument to read OD600 (as OD calibration setting)
2. Measure OD600 of the overnight cultures
3. Record data in notebook
4. Import data into Excel Dilution Calculation
5. Dilute the cultures to a target OD 600 of 0.02 (see the volume of preloading culture and media in Excel) (Dilution Calculation Sheet_1) in 4 mL LB medium + correct antibiotic combinations (for us Chloramphenicol or tetracycline or both) in 14 mL falcon tube
6. Incubate the cultures at 37℃ and 220 rpm
7. Take 450 μL samples of the cultures at various time points. At each time point, take a sample from each clone, two colonies per clone
8. Place samples on ice.
9. At the end of sampling point, measure samples
10. Use the same instrument setting for all time point
11. Pipette 100 μL of each sample into each well.

DNA Preparations

Materials

• Promoters (100, 101, 110):
• Reagent grade water
• The promoter areas were designed by CCA-IGEM-Team 2017.
• The promoter areas were synthesized by IDT.
• The synthetic promoter areas were delivered to us lyophilized.
• Polycistronic codons (F1, F2, P1, P2):
  • Reagent grade water
  • The mini-genes were designed by CCA-IGEM-Team 2017.
  • The mini-genes were synthesized by Genscript.
  • The genes were delivered to us lyophilized.
• DH5a competent cells, Invitrogen, Cat 18265-017
• SOC (Recovery Medium), Lucigen, Cat No. F98226
• 1.5 mL tube
• Vortex
• Pipet and tips
• Ice bucket and ice
• Water bath (42°C)
• Incubator (37°C) and shaker

Methods

1. Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
2. For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells
3. Turn on incubator-shaker at 37°C.
4. Turn on incubator for plates at 37°C.
5. Set up water bath at 42°C.
6. The vials containing the lyophilized DNA(~4 µg) were spun down before opening the vials for the first time
7. 16 µL of 0.2µm filtered water was added to the lyophilized powder using a P20 pipet.
8. After closing the tubes, they were vortexed for 2-3 minutes and were allowed to sit at 60-65°C for 15 minutes to resuspend the DNA. The tubes were then spun.
9. Aliquots were taken to start cloning.
10. All stocks are stored at -20°C.

Gel

Materials

• Agarose
• 1xTAE
• Microwave
• EtBr
• Tray and comb

Methods

1. Measure 1 g of agarose
2. Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
3. Microwave for 1-3 min until the agarose is completely dissolved
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
6. Pour the agarose into a gel tray with the well comb in place.
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.

Gel Purification

Materials

• Finished Gel
• Microtube
• DNA Purification Kit

Methods

1. Once you have run your gel, move it to an open UV box.
2. Remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel.
3. Place the gel in a labeled microfuge tube.
4. Using a scale, weigh the tube with the gel fragment. The weight of the gel is directly proportional to its liquid volume and this is used to determine how much of each buffer to add during the DNA isolation step.
5. Isolate the DNA from the gel using any gel purification kit. Follow kit instructions.

Creating Constructs

Materials

• DNA samples
• Transformation efficiency DNA, specific plasmid (for us: pUC 19, 10 pg/ µL), Lucigen, Cat No. F92078-1
• LB Carbenicillin 50 agar plates, Cat No. Teknova, L1801
• E coli BL21 DE3, Life Technology, Cat No. 60106-1
• SOC (Recovery Medium), Lucigen, Cat No. F98226
• 15 mL culture tube
• Vortex
• Tooth pick
• Incubator shaker
• 42°C Water bath
• Ice and ice bucket
• Pipet

Methods

1. Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
2. Transform 1 µL of DNA of plasmid (for us: pUC19) as transformation efficiency control
3. Turn on incubator-shaker at 37°C.
4. Turn on incubator for plates at 37°C.
5. Set up water bathat 42°C.
6. Bring to room temperature S.O.C medium.
7. Bring LB plates supplemented with appropriate antibiotic at room temperature.
8. Thaw competent cells on ice.
9. Aliquots competent cells in as many tubes as needed.
10. Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix
11. Incubate on ice for 20 minutes
12. Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.
13. Add 200 µl of 18-25°C SOCmedium and transfer mixture to 15mL Falcon tube
14. Incubate in shaker at 37°C, 225 rpm for 30 min
15. Plate 2 volumes (50 µL and ~100µL) of the mixture onto 2 different plates of LB agar plates supplemented with Carbenicillin 100 µg/mL
16. Incubate plates at 37°C overnight
17. Count colonies and estimate transformation efficiency
18. Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Cloning with pUC Plasmid of Lac Operation (CCA_San_Diego Specific)

Materials

• Synthetic DNA
  • Synthetic flnB, dbfA1, dbfA2: 3219 bp
  • Synthetic flnE, flnD1, ORF16, flnC: 3545 bp
  • Synthetic phnF, phnE, phnC, phnD: 4227 bp
  • Synthetic phnAc, phnAd, phnB: 3174 bp
• HindIII restriction enzyme, Thermo Fisher, Cat No. ER0501
• Restriction digest Buffer R, Thermo Fisher, Cat No. 00267554
• Antarctic Phosphatase, New England Biolabs Cat No. M0289G
• Agarose, 100 g, Fisher, Cat No. BP-164-100
• 50XTAE Electrophoresis Buffer, 1L, (1X: 40 mMTris, 20mM Acetic Acid, 1 mM EDTA), Thermofisher, Cat No. B49
• Sybr Safe DNA Gel Stain, Invitrogen, Cat No. S33102
• 100 bp DNA marker, Invitrogen, Cat No. 15628-019
• 10X Blue Juice DNA loading buffer, Invitrogen, 10816-015
• Reagent grade water
• Gel DNA Recovery Kit, Zymo Research, Cat No. D4007
• 1.5 mL tubes
• Vortex
• Ice bucket and ice

Methods

Preparation of Recipient Vector (Insert 1)

1. This insert contains the vector (origin of replication of pUC and the first part of the catabolic pathway under the control of the T7 promoter + inducible lacI +RBS)
2. Digest clone containing synthetic gene containing insert 1 with the restriction enzyme HindIII that cuts only at one site.
3. Restriction Digest Set-up (20µl reaction) in a 1.5 mL tube
4. Add the reagents as described below.
5. Incubate at 37°C for 1 hours
6. Add 2 µl of phosphatase buffer
7. Add 1 µl of phosphatase
8. Incubate at 37°C for 30 minutes
9. Add 2.3 µl of loading buffer at the end of the reaction
10. Load the reaction on a 1% Agarose gel
11. After running the electrophoresis, cut the linearized band.
12. Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit
13. Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O

Component	Volume (μl)	Final condition
Reagent grade water	8.0 μl
10X Buffer R	2.0 μl	1x
Vector-Insert 1	1.0 μl	0.5 µg
HindIII (10U/µl)	1.0 μl	10U

Preparation of Insert (Insert 2)

1. This insert contains the second part of the catabolic pathway under the control of the T7 promoter + inducible lacI +RBS)
2. Digest plasmid containing synthetic gene designated as insert 2 with the restriction enzyme HindIII that cuts at 2 sites (HindIII sites are flanking the genes)
3. Set-up restriction digestion (20µl reaction) in a 1.5 mL tube
4. Turn on water bath at 37°C
5. Add the reagents in the order and with volume described in the table below.
6. Spin the tube briefly for 15 seconds at 10,000 rpm
7. Incubate the tube at 37°C for 1 hours
8. Add 2 µl of loading buffer in the tube at the end of the reaction
9. Load the digestion reaction on a 1% Agarose gel, TAE
10. After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size.
11. Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit.
12. Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O

Component	Volume (μl)	Final condition
Reagent grade water	12.5 μl
10X Buffer R	1.5 μl	1x
Vector-Insert 2	1.0 μl	~0.4 µg
HindIII (10U/µl)	1.0 μl	10U

Cloning with Constructive Promoter of Different Strengths (CCA_San_Diego Specific)

Materials

• Synthetic DNA [Fluorene F1, F2] and [Phenanthrene P1 and P2]
  • Synthetic F1 : flnB, dbfA1, dbfA2: 3219 bp
  • Synthetic F2: flnE, flnD1, ORF16, flnC: 3545 bp
  • Synthetic P1: phnF, phnE, phnC, phnD: 4227 bp
  • Synthetic P2: phnAc, phnAd, phnB: 3174 bp
• Synthetic DNA [Promoter areas 100, 101, 110]
  • Synthetic Promoter 100: 88 bp
  • Synthetic Promoter 101: 88 bp
  • Synthetic Promoter 110: 88 bp
• Agarose, 100 g, Fisher, Cat No. BP-164-100
• 50XTAE Electrophoresis Buffer, 1L, (1X: 40 mMTris, 20mM Acetic Acid, 1 mM EDTA), Thermofisher, Cat No. B49
• Sybr Safe DNA Gel Stain, Invitrogen, Cat No. S33102
• 1kb plus DNA ladder DNA marker, Thermo Scientific, Cat No. SM1334
• 10X Blue Juice DNA loading buffer, Invitrogen, 10816-015
• Reagent grade water
• Gel DNA Recovery Kit, Zymo Research, Cat No. D4007
• 1.5 mL tubes
• Vortex
• Ice bucket and ice
• Cryo box for restriction enzyme
• P1000 and P200 with corresponding tips

Methods

Preparation of Promoter Areas with RBS

1. The promoters/RBS regions were designed by the CCA_IGEM team and synthetized by IDT.
2. For the writing on plates, the designations for the promoters + RBS were as follows:
   1. BBa_J23100 + RBS =100
   2. BBa_J23101 + RBS =101
   3. BBa_J23110 + RBS =110
3. Double Digestion of promoter + RBS region with restriction enzymes EcoRI and SpeI
5. Restriction Digest Set-up (20µl reaction) in a 1.5 mL tube
6. Add the reagents as described below.
7. Incubate the tube at 37°C for 15 min
8. Note: there is no need to add a loading buffer because the digestion buffer already includes it.
9. Load the reaction on a 1.5 % Agarose gel, TAE
10. After running the electrophoresis for 1 hour at 80V, cut the linearized band.
11. Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit
12. Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O

Component	Volume (μl)	Final condition
Reagent grade water	12.0 μl
10X Buffer R	2.0 μl	1x
Promoter+RBS Region	4.0 μl	~1 µg
EcoRI (10U/µl)	1.0 μl
SpeI (10U/µl)	1.0 μl

Preparation of insert F1, F2, P1, and P2

1. The following polycistronic sequences are cloned behind the 3 different promoter regions:
   1. For fluorene catabolic pathway
      1. FLUO-Insert 1 or F1 [Synthetic flnB, dbfA1, dbfA2]
      2. FLUO-Insert 2 or F2 [Synthetic flnE, flnD1, ORF16, flnC]
   2. For phenanthrene catabolic pathway
      1. PHE-Insert 1 or P1 [Synthetic phnF, phnE, phnC, phnD]
      2. PHE-Insert 2 or P2 [Synthetic phnAc, phnAd, phnB]
   3. The fragments are digested with 2 enzymes: XbaI and Pst I
   4. Digest plasmid containing synthetic gene designated F1, F2, P1, and P2 with the restriction enzyme XbaI and PstI
   5. Set-up restriction digestion (15 µl reaction) in a 1.5 mL tube
   6. Turn on water bath at 37°C
   7. Add the reagents in the order and with volume described in the table below.
   8. Spin the tube briefly for 15 seconds at 10,000 rpm
   9. Incubate the tube at 37°C for 15 min
   10. Note: there is no need to add a loading buffer because the digestion buffer already has it.
   11. Load the digestion reaction on a 1% Agarose gel, TAE
   12. After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size.
   13. Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit.
   14. Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O

       Component	Volume (μl)	Final condition
       Reagent grade water	10.0 μl
       10X Buffer	1.5 μl	1X
       Preparation of Insert F1, F2, P1 and P2	1.5 μl	~0.4 µg
       Xbal	1.0 μl
       Pstl	1.0 μl

2. For fluorene catabolic pathway FLUO-Insert 1 or F1 [Synthetic flnB, dbfA1, dbfA2] FLUO-Insert 2 or F2 [Synthetic flnE, flnD1, ORF16, flnC] For phenanthrene catabolic pathway PHE-Insert 1 or P1 [Synthetic phnF, phnE, phnC, phnD] PHE-Insert 2 or P2 [Synthetic phnAc, phnAd, phnB] The fragments are digested with 2 enzymes: XbaI and Pst I Digest plasmid containing synthetic gene designated F1, F2, P1, and P2 with the restriction enzyme XbaI and PstI Set-up restriction digestion (15 µl reaction) in a 1.5 mL tube Turn on water bath at 37°C Add the reagents in the order and with volume described in the table below. Spin the tube briefly for 15 seconds at 10,000 rpm Incubate the tube at 37°C for 15 min Note: there is no need to add a loading buffer because the digestion buffer already has it. Load the digestion reaction on a 1% Agarose gel, TAE After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size. Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit. Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O

Cloning for depository (CCA_San_Diego Specific)

Materials

• DNA miniprrep
• LB agar plates, Cat No. Teknova, appropriate antibiotic
• DH5a competent cells, Invitrogen, Cat 18265-017
• SOC (Recovery Medium), Lucigen, Cat No. F98226
• 1.5 mL tube
• Vortex
• Pipet and tips
• Ice bucket and ice
• Water bath (42°C)
• Incubator (37°C) and shaker

Methods

1. Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
2. For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells
3. Turn on incubator-shaker at 37°C.
4. Turn on incubator for plates at 37°C.
5. Set up water bath at 42°C.
6. Bring to room temperature S.O.C medium.
7. Bring LB plates supplemented with appropriate antibiotic at room temperature.
8. Thaw competent cells on ice.
9. Aliquots competent cells in as many tubes as needed.
10. Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix
11. Incubate on ice for 20 minutes
12. Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.
13. Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube
14. Incubate in shaker at 37°C, 225 rpm for 30 min
15. Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)
16. Incubate plates at 37°C overnight
17. Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Clone verification

Materials

• DNA miniprrep
• LB agar plates, Cat No. Teknova, appropriate antibiotic
• DH5a competent cells, Invitrogen, Cat 18265-017
• SOC (Recovery Medium), Lucigen, Cat No. F98226
• 1.5 mL tube
• Vortex
• Pipet and tips
• Ice bucket and ice
• Water bath (42°C)
• Incubator (37°C) and shaker

Methods

1. Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
2. For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells
3. Turn on incubator-shaker at 37°C.
4. Turn on incubator for plates at 37°C.
5. Set up water bath at 42°C.
6. Bring to room temperature S.O.C medium.
7. Bring LB plates supplemented with appropriate antibiotic at room temperature.
8. Thaw competent cells on ice.
9. Aliquots competent cells in as many tubes as needed.
10. Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix
11. Incubate on ice for 20 minutes
12. Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.
13. Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube
14. Incubate in shaker at 37°C, 225 rpm for 30 min
15. Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)
16. Incubate plates at 37°C overnight
17. Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Glycerol Stock

Materials

• Glycerol
• LB liquid medium
• Antibiotic stock solution
• 15 mL culture tube
• Incubator-Shaker
• 10 mL pipette
• Pipet aid
• Vortex
• Rack
• Toothpick

Methods

Set-up Culture and Prepare Glycerol Stocks

1. Grow selected clones that have been checked (so they are correct) in 3 mL LB medium supplemented with appropriate antibiotics overnight at 37°C in the incubator/shaker at 220 rpm.
2. Prepare LB medium with 40% glycerol and add 0.5 mL to a cryogenic vial
3. Add 0.5 mL of culture sample to be stored
4. Gently vortex the cryogenic vial to ensure the culture and glycerol is well mixed
5. Label tube with date and identifier
6. Organize in a freezer box and label box
7. Prepare excel spreadsheet with all information
8. Store freezer box at -80°C

email igemcca@gmail.com Canyon Crest Academy iGEM 2017 CC;