Team:Heidelberg/Template/Del week16 Gibson

From 2013.igem.org

Contents

15-08-2013

Gibson Assembly and electroporation

Assembly of our fragments

Those gel-purified fragments were added for the Gibson assembly of our construct pFSN.

Fragment Primer Date Concentration (ng/µl) Volume (µl)
pSB4K5 FS_01 - FS_16 14-08-13 13 1.35
AF FS_02 - FS_05 13-08-13 49.75 2.65
FG FS_21 - FS_26 07-08-13 24.5 2.69
G FS_35_SR_01 - FS_23 08-08-13 34.9 2.1
OP FS_22 - FS_13 13-08-13 36.3 0.89
L FS_14 - FS_15 07-08-13 51 0.31
Gibson MasterMix NEB 2x 10
  • Gibson Assembly according to protocols of NEB, 1h at 50°C

Gibson backbone (pSB4K5)

The backbone pSB4K5 was also incubated with the Gibson mastermix. We will use this as one negative control in the electroporation to test the efficiency.

Fragment Date Concentration (ng/µl) Volume (µl)
pSB4K5 14-08-13 13 1.35
H2O 8.65
Gibson MasterMix NEB 2x 10
  • Gibson Assembly according to protocols of NEB, 1h at 50°C

Electroporation

The following samples were electroporated

mixture volume used cells
5 µl Gibson Assembly of pFSN construct 10 µl H2O 1 µl 50 µl DH10β old
5 µl Gibson Assembly of pFSN construct 10 µl H2O 14 µl 50 µl DH10β old
5 µl Gibson Assembly of Backbone (control) 10 µl H2O 1 µl 50 µl DH10β old
pSB4K5 14-08-13 1.35 µl 50 µl DH10β old
pSB6A1 Hanna [69.57 ng/µl] 1:10 1 µl 50 µl DH10β Fanny new


  • Cells were transdered into 400 µl SOC-medium (NEB) after electroporation
  • incubation and growth of cells for 1 hour at 37°C
  • cells were centrifuged for 3 min at 6000rpm, supernatant discarded and cells resuspended in remaining SOC-medium
  • cells were plated on agar plates containing kanamycine, 10µl on one plate, remaining µl on another plate for electroporation of the construct pFSN and on agar plates containing ampicillin for electroporation of the control backbone pSB6A1


16-08-2013

Colony PCRs

As a lot of colonies grew we screened if DelL and pSB4K5 are present.

File:20130816 2log screeningsophieDelRESTbesch.png
Colony PCRs of red colonies after gibson assembly and electroporation (16-08); run at 100 V, 0.8 % gel (TAE)

Different probes:

  • A: 1 µl of diluted Gibson electroporated; 10 µl of E.coli plated
  • B: 1 µl of diluted Gibson electroporated; Rest of E.coli plated
  • C: 14 µl of diluted Gibson electroporated; 10 µl of E.coli plated
  • D: 14 µl of diluted Gibson electroporated; Rest of E.coli plated


  • Only red colonies were chosen for screening
  • The expected amplicon size is 1.5 kb


Reaction mixture
Reagent A1, A2 B1-B5 C1-C3 D1-D40
VR-Primer: (1/10) 2 µl 2 µl 2 µl 2 µl
FS_14: (1/10) 2 µl 2 µl 2 µl 2 µl
Colonies 2 red colonies A (A1, A2) 5 red colonies B (B1-B5) 3 red colonies C (C1-C3) 40 red colonies D (D1-D40)
DreamTaq 10 µl 10 µl 10 µl 10 µl
dd H2O 5 µl 5 µl 5 µl 5 µl


PCR Conditions

--> T100

Cycles-PCR temperature [°C] Time [s]
1 95 2:00
12 95 1:00
66 ↓ 0.5 5
72 1:30 min
18 95 1:00
63 5
72 1:30 min
1 12 inf

Result:

  • None of the screened colonies led to the correct amplicon, therefore the correct assembly has not worked in any of the white colonies
  • screening will be repeated, with white colonies as template, as bacteria including the correct 32 kbp backbone might not be capable of expressing mRFP anymore due to the large insert between lac promotor and mRFP

17-08-2013

Colony PCRs

File:20130817 2log DelRestwhiteColonyPCRsbesch.png
Colony PCRs of white colonies after gibson assembly and electroporation (17-08); run at 100 V, 0.8 % gel (TAE)

Different probes:

  • A 1 µl of diluted Gibson Assembly electroporated; 10 µl of E.coli plated
  • B 1 µl of diluted Gibson Assembly electroporated; remaining E.coli plated
  • C 14 µl of diluted Gibson Assembly electroporated; 10 µl of E.coli plated
  • D 14 µl of diluted Gibson Assembly electroporated; remaining E.coli plated


  • Only white colonies where chosen for screening
  • The expected amplicon size is 1.5 kb


Reaction mixture
Reagent B1w C1w D1w-D10w
VR-Primer: (1/10) 2 µl 2 µl 2 µl
FS_14: (1/10) 2 µl 2 µl 2 µl
Colonies 1 white colony B (B1w) 1 white colony C (C1w) 10 white colonies D (D1w-D10w)
DreamTaq 10 µl 10 µl 10 µl
dd H2O 5 µl 5 µl 5 µl


PCR Conditions

--> T100

Cycles-PCR temperature [°C] Time [s]
1 95 2:00
12 95 1:00
66 ↓ 0.5 5
72 1:30 min
18 95 1:00
63 5
72 1:30 min
1 12 inf

Result:

  • Colony D8w shows expected amplicon of 1.5kbp
  • The other colonies were screened negative.
  • D8w will be transfered to liquid culture to repeat screening PCR as well as for further validation beeing screening for the other insert fragments as well as test restriction digest
  • Furthermore another electorporation will be carried out to yield more clones positive for screening with the Primers VR and FS_14, therefore the remaining 10µl of the Gibson assembly will be purified using isopropanol precipitation


Electroporation

We electroporated the Gibson assembly (15-08) again to increase the chance of a positive clone. We purified it by isopropanol purification.

mixture volume used Cells
Assembled fragments (15-08) isopropanol precipitated 15 µl 50 µl DH10β


  • Cells were transdered into 400 µl SOC-medium (NEB) after electroporation
  • incubation and growth of cells for 1 hour at 37°C
  • cells were centrifuged for 3 min at 6000rpm, supernatant discarded and cells resuspended in remaining SOC-medium
  • cells were plated on agar plates containing kanamycine, 10µl on one plate, remaining µl on another plate for electroporation of the construct pFSN

18-08-2013

Colony PCRs

File:20130818 log2 D8w D11w-D23w E1w-E19w E1r-E10r F1w-F28w F1r-F133 F19r F16r.png
Colony PCRs to test if pSB4K5 and DelL are ligated; run at 100 V, 0.8 % gel (TAE)(14.08)

Different probes:

  • D: 14 µl of diluted Gibson Assembly electroporated; remaining E.coli plated
  • E: 1 µl isopropanol precipitated Gibson Assembly (15-08) ; 10 µl of E.coli plated
  • F: 1 µl isopropanol precipitated Gibson Assembly (15-08); remaining E.coli plated
  • The expected amplicon size is 1.5 kb
  • 3 colonies were sceened in one PCR
Reaction mixture
Reagent D8w D11w-D24w E1w-E19w F1w-F20w E1r-E10 F1r-F20r
VR-Primer: (1/10) 2 µl 2 µl 2 µl 2 µl 2 µl 2 µl
FS_14: (1/10) 2 µl 2 µl 2 µl 2 µl 2 µl 2 µl
Colonies Colony D8w liquid culture 24 white colonies D (D1w-D24w) 19 white colonies E (E1w-E19w) 30 white colonies F (F1w-F20w) 10 red colonies E (E1r-E10r) 20 red colonies F (F1r-F20r)
DreamTaq 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
dd H2O 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl


PCR Conditions

--> T100

Cycles-PCR temperature [°C] Time [s]
1 95 2:00
12 95 1:00
66 ↓ 0.5 5
72 1:30 min
18 95 1:00
63 5
72 1:30 min
1 12 inf

Result:

  • colony D8w again showed the expected amplicon in the colony PCR and will therefore be further analyzed for the other fragments as well as restriction digested and partially sequenced as soon as screening primers arive
  • Furthermore colonies E16w and F25w are screened positive for the fragment DelL