Team:Lethbridge/Notebook August

iGEM

3

Digestion

Digest of Device 1 Plasmids and Subsequent Agarose Gel Electrophoresis

Component Volume (μL)
Plasmid DNA 5
10x FD green buffer 1
FD EcoRI 0.5
dH2O 3.5

Total volume: 10μL

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4

Miniprep & PCR

Miniprep Colonies 1 to 5

Part is TP ribozyme test #1 currently in Pjet1.2 blunt cloning vector. Will bio drop minipreps and PCR amplify out the TP ribozyme test #1 using BB prefix/suffix primers. Spin down 4 mL LB at 13,300 rpm for 2 minutes. Cultures were taken at 3:30 am to be miniprepped.

  • 100μL solution I pipette up and down
  • 200μL solution II mix by inverting
  • 300μL solution III mix by inverting

Spin at 13,300 for 5 minutes

Pour supernatant onto column

Rinse 2x with wash solution

2x elute in 50μL using the eluta in elution #2

Bio Drop

Colony Concentration (ng/μL)
1 204.2
2 237.2
3 51.24
4 66.6
5 150.9

PCR

Component Volume (μL) Master Mix (x5 reactions)
MilliQ H2O 39 195
10x Pfu buffer 5 25
10 nM dNTPs 2 10
BB prefix/suffix primer (10μM) 1 2
Template DNA 1 -
Pfu polymerase (Thermo Sci) 1 5

Total volume: 50μL

Thermo Cycler

Temperature (°C) Time (seconds)
98 180
98 30
53 30
72 30
72 600

Note: All 30 seconds runs were repeated 35 times.

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6

Miniprep

Miniprep of Device 2

12% native PAGE

  • 1.8 mL polyacrylamide (40-1)
  • 1.2 mL TBE (5x)
  • Fill to 6 mL with dH2O
  • Put in over to help dissolve. Vortex.
  • Add 10μL APS. Invert
  • Add 12μL TEMED. Vortex.

12% 8M Urea PAGE

  • 2.88g urea
  • 1.8μL polyacrylamide
  • 1.2μL TBE (x5)
  • Fill to 6 mL with dH2O
  • Add 40μL APS. Invert
  • Add 6μL TEMED. Vortex
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10

Miniprep

Sending device 1 for sequencing

  1. Rep Device 1 col 2 - BB prefix
  2. Rep Device 1 col 2 - BB suffix
  3. Device 1 - BB prefix
  4. Device 1 - BB suffix
  1. I3504 in pSBIA2 – gel extracted (1% agarose gel)
    • 5μL 10x cutsmart buffer
    • 10μL I3504 minipreps
    • 28μL MilliQ H2O
    • 1μL XbaI (20U/μL)
    • 1μL PstI (20U/μL)
    • J23117 in pSBIC3
      • 5μL 10x cutsmart buffer
      • 15μL miniprep DNA
      • 28μL MilliQ H2O
      • 1μL SpeI
      • 1μL PstI
      • J23106 in pSBIC3
        • 5μL 10x cutsmart buffer
        • 15μL miniprep DNA
        • 28μL MilliQ H2O
        • 1μL SpeI
        • 1μL PstI

Elute in 35μL water after clean-up

Ligation Overnight

Device I → J23101 + I13504

Component Volume (μL)
T4 DNA ligase buffer 1
T4 DNA ligase 0.5
I13504 (gel extracted) 2.5
J23101 backbone 1
dH2O 5

Total volume: 10μL

Device II → J23106 + I13504

Component Volume (μL)
T4 DNA ligase buffer 1
T4 DNA ligase 0.5
I13504 2.5
J23106 backbone 1
dH2O 5

Total volume: 10μL

Device III → J23117 + I13504

Component Volume (μL)
T4 DNA ligase buffer 1
T4 DNA ligase 0.5
I13504 2.5
J23117 backbone 1
dH2O 5

Total volume: 10μL

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11

In Vitro Transcription

Clone Tt ribozyme test 1 into pJET again. Test with PCR and subsequent in vitro transcription

Component Volume (μL)
2x reaction buffer 10
Construct (ribozyme) 1
MilliQ H2O 6
DNA blunting enzyme 1
pJET vector 1
T4 DNA ligase 1

Total volume: 18μL

  • Incubate the mix of the first 4 components at 20°C for 5 minutes
  • After adding the pJET vector and T4 DNA ligase incubate at room temperature for 5 minutes

Cleaving pSBIC3

Component Volume (μL)
pSBIC3 2
EcoRI 1
PstI 1
Cutsmart buffer 10x 2
MilliQ H2O 14

Incubate for 1 hour at 47°C

Transformations of 1μL construct in pJET1.2 (Devices I, II, III) into 25μL of E. coli DH5α Competent Cells

Protocol:

  • Add 1μL of DNA to 25μL of competent cells
  • Incubate for 30 minutes on ice
  • Heat shock at 47°C for 45 seconds
  • Incubate on ice for 5 minutes
  • Add 400μL of LB
  • Incubate in RNA common room at 37°C for an hour
  • Plate 400μL on LB + Amp
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12

PCR

Add overnight cultures to LB media with 5μL of Cu antibiotic. Device I colony I (DICI), DIC2, DIIC1, DIICII, DIIICI, DIIICII.

Cultivate overnight cultures to LB media. Test construct in pJET, colonies 1 and 4. Add 5μL of Amp.

Ligate High and Low Affinity Ribozyme into pJET

Component Volume (μL)
2x reaction buffer 10
Construct 1
DNA blunting enzyme 1
MilliQ H2O 6
  • Ran at 70°C for 5 minutes on a heat block
  • Add 1μL of pJET blunt vector
  • Add 1μL of T4 ligase
  1. 1μL of high and low affinity ribozyme added to DH5α cells
  2. Sit on ice for 30 minutes
  3. Heat shock for 45 seconds at 42°C
  4. Put on ice for 5 minutes
  5. Add 400μL of outgrowth media
  6. Incubate for 1 hour in shaker
  7. Plate 200μL on LB + Amp plates

Colony PCR of theophylline ribozyme test I using Pfu polymerase

Component Volume (μL) # of reactions Master Mix
MilliQ H2O 39 4 156
10X Pfu buffer 5 4 20
10 nM dNTPs 2 4 8
Pfu polymerase 1 4 4
BB prefix primer 1 4 4
BB suffix primer 1 4 4

1μL of colony in 20μL of dH2O added to each reaction. 1% agarose gel ran using the PCR products

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13

Miniprep

Overnight Cultures Placed in LB Media

  1. Add 5&L of Amp
  2. Low affinity culture 1 (low 1), low 2, low 3, high 1, high 2, high 3
  3. Incubate in the shaker

Overnight media cultures test construct 1 and 4, DICI, DICII, DIICI, DIICII, DIIICI, DIIICII were taken off shaker at 10:30am.

Miniprep of Ttr 1 and 4, DICI, DICII, DIICI, DIICII, DIIICI and DIIICII.

  1. Add xx mL of culture and centrifuge at xxx
  2. Add 100μL of solution I
  3. Add 200μL of solution II
  4. Add 300μL of solution III
  5. Centrifuge
  6. Transfer to EE columns, centrifuge at 10,000 rpm for 2 minutes. Discard flow through
  7. Add 700μL and centrifuge at 10,000 rpm. Discard flow through was. Repeat once
  8. Centrifuge at 10,000 rpm for 1 minute. Discard flow through.
  9. Transfer column to 1.5mL tube, add 50μL of MilliQ H2O, centrifuge for 2 min at 10,000 rpm
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17

In Vitro Transcription

In vitro transcription of PCR products of TT ribozyme. As on page 20 using PCRs 1 and 4 as template

  • Digest of RAP constructs and insertion into pSBIC3 → E+P
  • Digest of TtR test 1 and 4 for insertion into pSBIC3 as well → E+P
Component Volume (μL) pJET plasmid 2 EcoRI 1 PstI 1 Custmart buffer 10x 2 MilliQ H2O 14

Total volume: 20μL

Labels

  1. Tt Test col 1
  2. Tt test col 4
  3. RAP high 1
  4. RAP high 2
  5. RAP high 3
  6. RAP low 1
  7. RAP low 2
  8. RAP low 3

In vitro transcriptions in 300μL left overnight at 37°C

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19

PCR

Streak device I, II and III on CM plates

Component Volume (μL)
Digest 6
pSBIC3 digest 1
Ligase 0.5
Ligase buffer 1
MilliQ H2O 1.5

Total volume: 10μL

Put on heat block at 16°C

R0040 - Kit plate 2, well 6F

I20270 - kit plate 3, well 8P

  • Resuspend in 10μL of dH2O.
  • Transformed 1μL of DNA to 25μL of DH5α E. coli competent cells.

Protocol

  • Add 1μL of R0040 to 25μL of competent cells, repeat with I20270
  • Incubate them for 30 minutes on ice
  • Heat shock them at 47°C for 45 seconds
  • Incubate on ice for 5 minutes
  • Add 400μL of LB
  • Incubate in RNA common room at 37°C for an hour
  • Plate 400μL on LB + CM

Cultivate overnight cultures in LB media, 3 colonies for I20270 and R0040 each

  • Add 5μL of CM
  • Incubate in shaker

Cultivate overnight streak devices in LB media

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20

PCR & Thermo Cycler

PCR

Volume (μL)395210.51.0
Component
MilliQ dH2O
10X Pfu buffer
10 nM dNTPs
BB prefix/suffix primers
Template DNA
Pfy pol (Thermo Sci)

Thermo Cycler

Temperature (°C) Time (seconds)
98 180
98 30
53 30
72 30
72 600

Note: All 30 seconds runs were repeated 35 times.

Red → TT test 1

Green → TT test 2

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21

Overexpression

Overexpression of Devices 1, 2 and 3; and Positive and Negative Controls all with n=6

Device Sample OD reading for a 1/2 dilution Volume(μL)
1 1 0.34 0.89
2 0.40 0.75
3 0.38 0.78
4 0.39 0.77
5 0.41 0.73
6 0.38 0.78
2 1 0.38 0.78
2 0.39 0.77
3 0.43 0.70
4 0.40 0.75
5 0.33 0.91
6 0.38 0.78
3 1 0.47 0.64
2 0.41 0.73
3 0.29 1.03
4 0.44 0.68
5 0.42 0.71
6 0.48 0.63
Positive control 1 0.45 0.66
2 0.46 0.65
3 0.48 0.63
4 0.28 1.07
5 0.41 0.73
6 0.46 0.65
Negative control 1 0.41 0.73
2 0.46 0.65
3 0.44 0.68
4 0.41 0.73
5 0.44 0.68
6 0.42 0.71
  1. 3.4/5.6 = 0.6 OD (5μL/5.6) add 0.89μL
  2. 4/0.6 = 6.66/J add 0.75
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24

E. coli Cell Extract

Preparation of E. coli Cell Extract by Lysozyme

Buffers and solutions: binding/opening buffer DNAse lysozyme sodium deoxycholat

Method

  1. Resuspend the cell pellet in 3.5mL of binding/opening buffer by stirring the mixture slowly on ice.
  2. Add 350μL of lysozyme and incubate the cell suspension on ice for 30 minutes
  3. Add sodium deoxycholat (12.5mg/g)
  4. Stir the mixture slowly on ice
  5. Add few crystals of DNAse while stirring
  6. Remove debris by centrifugation at 3000g for 30 minutes at 4°C
  7. Collect the supernatant (cell lysate) in fresh tube
  8. Centrifugation for 45 minutes at 30,000g will result in the S-30 extract
  • 0.5g of cells per tube
  • Lysozyme stock solution
  • 20mg/mL lysozyme
  • 0.05mL lysozyme solution per mL cell suspension
  • 900μL binding buffer
  • 12.5mg/g with 0.5g x 18 = 112.5mg
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25

PCR Amplification

PCR amplification of Theophylline aptazyme template for in vitro transcription

  • 50μL 2x DreamTaq Mastermix
  • 1μL BB Prefix (fwd) primer (10μM)
  • 1μL BB Suffix (rev) primer (10μM)
  • 0.5 Theophylline aptazyme in pJET 1.2
  • 47.5μL MilliQ ddH2O

Thermocycler Conditions

  1. 95 - 2 min
  2. 95 - 20 sec
  3. 58 - 20 sec
  4. 68 - 30 sec (return to step 2 x25)
  5. 68 - 2 min
  6. 4 - hold
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26

Miniprep & In Vitro Transcription

Miniprep of Tt test 2-1, Tt test 2-2, Tt test 1-1 RAP low 5-3, RAP low 5-1, RAP high 4-1, RAP low 6-1, RAP low 8-1

  1. Add 100μL of solution I
  2. Add 200μL of solution II
  3. Add 350μL of solution III
  4. Centrifuge at 12,000 rpm for 5 minutes
  5. Discard flow through, add 750μL wash solution
  6. Centrifuge at 10,000 rpm for 2 minutes. Discard flow through
  7. Repeat previous 2 steps
  8. Centrifuge at 10,000 rpm for 2 minutes, discard flow through
  9. Centrifuge at 10,000 rpm for 1 minute. Discard flow through.
  10. Add 50μL of elution buffer (MilliQ H2O), centrifuge at 10,000 rpm for 2 minutes
  11. Store DNA at -20°C

Pfu PCR of RAP and Tt test constructs

Component Volume (μL)
10x Pfu buffer 5
pSBIC3 plasmid + insert p
10mM dNTPs 2
Forward primer → BB prefix 1
Reverse primer → BB suffix 1
MilliQ dH2O 35
Pfu DNA polymerase 1

Total volume: 50μL

Temperature (°C) Time (seconds)
98 180
98 30
53 40
72 40
72 600

Note: All 30 seconds runs were repeated 30 times.

Agarose Gel

Lane Sample Volume (μL)
1 1 kb ladder 3
2 TT test 2-1 5μL sample + 1μL 6x dye
3 RAP low RBS 5-3
4 TT test 1-1
5 RAP low RBS 5-1
6 RAP high RBS 4-1
7 TT test 2-2
8 RAP low RBS 8-2
9 RAP low RBS 8-1
10 1 kb ladder 3

In vitro transcriptions in 300μL left overnight at 37°C

Volume (μL) Part
20 5x Transcription Buffer
10 100mM DTT
12 25mM NTPs
5 100mM GMP
2 0.5 units/μL iPPase
0.5 40 units/μL RNase inhibitor
10 previous PCR product (~30ng/μL)
30.5 MilliQ ddH2O
10 T7-RNA Polymerase (9.4μM)

Incubate at 37°C for 4 hours

Digest using DNaseI (1μL) for one hour, sample purified using BioBasic spin column

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31

Digest

Triple Digest of RAP Contructs in PSB1C3:

Volume (μL) Part
10 Plasmid
1 Kpu1 FD
1 EcoR1
1 BmaH1
2 FD Buffer
5 dH20

Total Volume: 20μL

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