Team:Heidelberg/Template/Del week17 Gibson

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Contents

19-08-2013

Colony PCRs for testing ligation of pSB4K5 and DelL

Reaction mixture
Reagent E13w E14w E15w F25w F26w F27w
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 E13w liquid culture Colony E14w liquid culture Colony E15w liquid culture Colony F25w liquid culture Colony F26w liquid culture Colony F27w liquid culture
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

Result:

  • Colony F26w was screened positive, as it shows the expected band at 1.4kbp, the others were screened negative
  • 8 ml of colony D8w and colony D26w (pFSN-1) were midi-preped according to protocol 'Preparation of large plasmids'.


Colony PCRs for testing ligation of pSB4K5 and DelAF, as well as DelAF and DelFG

Sceening of colonies D8w and D26w for fragments pSB4K5, DelAF, and DelFG.
Reaction mixture
Reagent D8w D8w F26w F26w
Primer_fw (1/10) 2 µl FS_47 2 µl FS_51 2 µl FS_47 2 µl FS_51
Primer_rv (1/10) 2 µl FS_48 2 µl FS_52 2 µl FS_48 2 µl FS_52
Colonies Colony D8w liquid culture Colony D8w liquid culture Colony F26w liquid culture Colony F26w liquid culture
DreamTaq 10 µl 10 µl 10 µl 10 µl
dd H2O 5 µl 5 µl 5 µl 5 µl
Expected band 547 bp 724 bp 547 bp 724 bp

Results:

  • The screening was positive for clone D8w with both primer combinations.
  • Thus this clone contains the backbone pSB4K5, the genes DelA, DelB, DelC, DelD, DelE, DelF, DelL and at least a part of the gene DelG.
  • For colony F26w the screenings were negativ.

Restriction digest of pFSN (31.435 kbp)

Restriction digest of pFSN (19-08) with l2:EcoRI, l3:BamHI, l4:BglII (17-08); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 6 hours

Reagent µl
pFSN 1
EcoRI BamH1 BglII 2
CutSmart Buffer 0.5
dd H2O 1.5
Expected fragment lengths [bp] EcoRI 9798, 7856, 5276, 4624, 2529, 1212
Expected fragment lengths [bp] BamHI 23860, 7435
Expected fragment lengths [bp] BglII 14223, 11919, 3291, 1892

Results:

  • All digests displayed the expected bands and thus were positive.
  • Therefore the pFSN plasmid is very likely our desired construct.

20-08-2013

Sequencing

  • The construct pFSN was isolated from overnight culture using the 'Preparation of large plasmids' protocol.
  • Single read sequencing was carried out by GATC Biotech
  • obtained .abi files were analyzed with UniPro Ugene, part of sequence showing convenient chromatogram was chosen for alignment using ClustalOmega


Sequence-Files from GATC Biotech

Sequencing of pFSN-1 with Primer FS_13_short

Sequencing of pFSN-1 with Primer FS_22

Sequencing of pFSN-1 with Primer VFII

Sequencing of pFSN-1 with Primer VR

Alignments

Alignment for sequencing of pFSN with primer FS_13s_rv against the expected construct to validate sequence of DelOP

Alignement for sequencing of pFSN with primer FS_57_rv against the expected construct to validate the sequence for the overlap of DelOP to DelG

Alignement for sequencing of pFSN with primer FS_58_fw against the expected construct to validate the sequence for the overlap of DelOP to DelL

Alignement for sequencing of pFSN with primer FS_63_rv against the expected construct to validate the sequence for the overlap of DelL to mRFP

Alignement for sequencing of pFSN with primer VF2 against the expected construct to validate the sequence for the overlap of pSB4K5 to DelAF


Results:

  • Sequencing of the final construct pFSN for all internal Gibson sites validated the intended sequence
  • Sequence of mRFP shows mutations within the primer site, therefore it can be concluded that mRFP is not functional
  • Colonies of D8w will be further analyzed by FACs to investigate whether mRFP is functional or not
  • Additionaly, SDS-page of clone D8w will be conducted to check for expression of the inserted Del genes
  • Concluding the results of colony PCRs, restriction digests and sequencing it can be concluded, that we successfully amplified 28 kbp of genomic DNA from our donator organism Delftia acidovorans SPH-1 distributed from the DSMZ, ligated the thereby obtained 6 fragments including the standard biobrick backbone pSB4K5 sucessfully in the intended order and consequently transformed a plasmid of 32 kbp in total into E. Coli using electroporation

21-08-2013

  • Another 8 ml of colony D8w (pFSN-1) were midi-preped according to protocol 'Preparation of large plasmids'.
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