Team:GO Paris-Saclay/NucleasesCharacterization/A1

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Nuclease_A1 (from bacteriophage T5)

Project: Nucleases characterizations
Authors: iGEM_GO_Paris_saclay
Monday, 1/7/2019
Aim: Insert the gene A1 in pBD24MoClo with golden gate method in order to express A1 nuclease under the control of the inducive promoter arabinose-dependent promoter Para.
Heat shock transformation of competent cells Escherichia coli KeioZ1 F’ with pBAD24GG and pBAD24A1 : Brahim _ Arnaud _ Laura
See the protocol "heat shock transformation".
The transformants were selected on LB agar plates plus Ampicilline (100µg/mL) and 0.2% glucose.
Tuesday, 2/7/2019
RESULTS of transformation of KeioZ1 F' cells
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pBAD24A1pBAD24GGWithout Plasmid
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Amp / Without AmpYesYesYesNoYesYesYesNoYesYesYesNo
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Volume spread (µL)50100100 concentrated10050100100 concentrated10050100100 concentrated100
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ResultsColoniesColoniesColoniesColoniesColoniesColoniesColoniesColoniesNo coloniesNo coloniesNo coloniesColonies
Table3
Controls are OK. Isolated colonies are available to continue our experiment.
Isolation of single colony : Laura_Arnaud_Brahim
Single colonies are picked and spread onto a new agar plate with LB+Amp+Glucose in an attempt to isolate single colonies.
Agar plates are then incubated at 37°C overnight.
Wednesday, 3/7/2019
Culture of isolated clones : Laura_Arnaud_Brahim
Isolated clones are put in liquid LB+Amp (100µg/µL) + 0.2% Glucose overnight at 37°C.
Friday, 5/7/2019
Culture of isolated clones (Results) : Laura_Arnaud_Brahim_Marie
Results :
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GG (futur Ara)GGA1 (futur Ara)A1
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DO0,580,620,630,60
Table1
Beginning of induction with Ara and first measurement at 30min then every hour.
Spread on petri dishes from transformed cells
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A1-30 min+30 min1h30 min2H 30Demain
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Amp GluAmpGluAmp GluAmpGluAmp GluAmpGluAmp GluAmpGluLB GluLB AmpLB Amp Glu
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araTapis4241288715686
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433111382126128
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237141212132584
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Non araTapis242T1310586000
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325T-6244000
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393T-656-000
9
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CFU in 10^8 (cell/mL)
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GG-30 min+30 min1h30 min2H 30Demain
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Amp GluAmpGluAmp GluAmpGluAmp GluAmpGluAmp GluAmpGluLB Amp GluLB AmpLB Glu
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araTapis581991715122003
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3420151612231131
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5420151117234110
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Non araTapis536203730100535
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443T34T011655
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4311537T000436
CFU in 10^8 ( cell/mL)
Second step: reading the OD using the plate spectrophotometer.
Measurement automatically of the DO every 20min during 12hours.
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1234
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AA1 AraA1GG AraGG
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BA1 AraA1GG AraGG
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CA1 AraA1GG AraGG
Plate Plan
Results :
image.png
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image.png
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Monday, 8/7/2019
1.
Characterization of the A1 nuclease : Marie - Laura
Transformation verification : colony PCR. For each sample : pBAD24A1.1, pBAD24A1.2, pBAD24GG.1, pBAD24GG.2 transformed in E.coli on 1st/2nd july :
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Primer namePrimer sequenceTm °C
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2019GO-5-Seq-pBAD-F5'- TGGCTATGCCATAGCATTTTTATCC55,6
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2019GO-6-Seq-pBAD-R5'- GGT CTG ATT TAA TCT GTA TCA GGC TG55,3
Primer sequences for checking insertion of the inserts
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2,5 μLGreen taq Buffer
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1 μLdNTP
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1 μLAmorce5
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1 μLAmorce6
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10 μLColony ADN + 10 μL of water
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0,15 μLTaq polymerase
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9,5 μLH2O
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Vtot = 25 μL
Table2
PCR program (named "IGM") :
94°C/5'
30 cycles : 94°C/30''
51°C/30''
72°C/2min
72°C/10'
Electrophoresis : 100V, 10 μL sample/well, 25 minutes.
Results :
Gel IGEm 09.07.19.001.jpeg
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Pre-culture : From glycerol stock on 1st/2nd july (pBAD24A1.1, pBAD24A1.2, pBAD24GG.1, pBAD24GG.2 transformed in E.coli), for each sample : 2 mL LB + 100μg/mL Ampicilline + 20% glucose, incubation with agitation overnight.
Tuesday, 9/7/2019
Culture of isolated clones (Results) : Laura_Arnaud_Marie
60mL culture of A1 and GG. Then beginning of induction with Ara in 30mL of A1 and GG, and first measurement of Optic Density at 30min each sample, then every hour twice.
Results :
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A1 (60mL)GG (60mL)A1+Ara (30mL)A1 (30mL)GG+Ara (30mL)GG (30mL)
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t = -45min0,120,13
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t = -15min0,220,23
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t = t0 induction0,330,38
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t = +30min0,650,740,800,81
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t = +1h300,661,391,311,48
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t = +2h300,631,891,681,97
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t = +18h301,93,83,13,8
Table4
Rq : We can notice that the population of A1+Ara bacteria decreases over time until 2h30. Then, during the night bacteria develop. We can assume that bacteria mutate during the night and grow.
Box spreading from transformers' solutions.
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Time-30min+30min+1h30+2h30+tomorrow
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GluGlu+AmpGluGlu+AmpGluGlu+AmpGluGlu+AmpGluGlu+Amp
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A1Ara10—210—310—210—410—610—610—2
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11102517
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108241921
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10152911116
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No Ara10—410—410—510—510—610—610—610—610—6
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22191214716511
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18241186921710
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21271517917714
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GGAra10—510—510—510—610—610—610—610—6
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16203591610138
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612271114696
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65348141016
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No Ara10—410—410—510—510—610—510—610—610—610—6
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15331516132510161012
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122612187341115813
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10172012331912
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Results
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Time-30min+30min+1h30+2h30+tomorrow
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GluGlu+AmpGluGlu+AmpGluGlu+AmpGluGlu+AmpGluGlu+Amp
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A1Ara1,03E+051,10E+062,00E+046,00E+061,00E+069,00E+081,23E+09
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5,77E+033,61E+050,00E+002,65E+060,00E+002,00E+081,10E+09
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No Ara2,03E+072,33E+071,27E+071,30E+077,33E+089,00E+081,80E+096,33E+081,17E+09
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2,08E+064,04E+062,08E+064,58E+061,53E+080,00E+002,65E+081,15E+082,08E+08
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GGAra9,33E+071,23E+083,20E+08