|
|
| Line 1: |
Line 1: |
| − | == Advancement on ''E. coli'' ==
| |
| | | | |
| − | The whole brainbow system will be integrated chromosomally at the GalK site. It will be synthesized as three gBlocks.
| |
| − |
| |
| − | It should allow expression of mCherry first, then upon expression of the CRE recombinase, differentiation in two states (YFP and CFP).
| |
| − |
| |
| − | It also contains a landing pad that allow insertion of a third state, which would subsequently lower the probability of states 1 and 2 so the three outcomes are possible.
| |
| − |
| |
| − | === Writing the artificial gene ===
| |
| − |
| |
| − | Promoter J23199 from the Biobrick collection
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| − |
| |
| − | 4 LoxP sites used together in mammalian brainbow plasmids
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| − |
| |
| − | RBS from Ihab
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| − |
| |
| − | ORF for mCerulean and mVenus from Ihab
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| − |
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| − | ORF for mCherry from Antoine
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| − |
| |
| − | rrnBT1 terminator used on the pOSIP plasmids
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| − |
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| − | Landing Pad (PhiC31 attB TT site)
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| − |
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| − |
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| − |
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| − | Check for secondary structure in the RBS Done
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| − |
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| − | Check for RBS in the LoxP array Done
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| − |
| |
| − | Split to have two ~1500 bp gblocks Done
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| − |
| |
| − | Design overlaps for Gibson in R6K vector Done
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| − |
| |
| − | Check for Biobrick restriction sites Done
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| − |
| |
| − | Fix gBlocks problems
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| − |
| |
| − | * Palindroms between LoxP sites Done
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| − | * Repeats in terminator: replaced with Lambda T0 terminator Done
| |
| − | * Repeat in RBS Done
| |
| − | * More GC in the LoxP array Done
| |
| − | * More GC at the end of fragment 1Done
| |
| − | * Repeat at the end of mVenus and mCerulean Done
| |
| − | * More GC at the beginning of fragment 2 Done
| |
| − |
| |
| − | It is very difficult to solve these -> make 3 fragments Done
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| − |
| |
| − | Fragment A: 0 - 1143
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| − |
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| − | Fragment B: 1105 - 2006
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| − |
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| − | Fragment C: 1981 - 2946
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| − |
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| − | Change the RBS for Fragment A -> “New RBS” Done
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| − |
| |
| − | Order gBlocks Done 07/13
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| − |
| |
| − | === Design + order oligos for gBlocks amplification Done ===
| |
| − |
| |
| − | Overlaps melting points are 62, 67, 54, 74.
| |
| − |
| |
| − | The overlap between fragments B and C should be increased to >62.
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| − |
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| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="33%"|R6K LinR
| |
| − | |width="33%"|tagcattatacctaggactgagctagctgtcaaggcaaatttgcggccgcaag
| |
| − | |width="33%"|o15.80
| |
| − | |-
| |
| − | |R6K LinF
| |
| − | |CGGGCGCGTACTCCAgaagggcatcgatggc
| |
| − | |o15.81
| |
| − | |-
| |
| − | |Colibow A F
| |
| − | |ttgacagctagctcagtcctag
| |
| − | |o15.82
| |
| − | |-
| |
| − | |Colibow A R
| |
| − | |GGCCATTCACATCACCATC
| |
| − | |o15.83
| |
| − | |-
| |
| − | |Colibow B F
| |
| − | |GCCGATTCTTGTTGAACTTG
| |
| − | |o15.84
| |
| − | |-
| |
| − | |Colibow B R
| |
| − | |CCATGGTACCTCCTCCTTACTTCTATAACTTC
| |
| − | |o15.85
| |
| − | |-
| |
| − | |Colibow C F
| |
| − | |GATACTTTATACGAAGTTATAGAAGTAAGGAGGAG
| |
| − | |o15.86
| |
| − | |-
| |
| − | |Colibow C R
| |
| − | |gccatcgatgcccttcTGGAGTACGCGCCCG
| |
| − | |o15.87
| |
| − | |}
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| − |
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| − |
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| − |
| |
| − | Overlap melting points: 62, 67, 62, 72.
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| − |
| |
| − | === Design + order oligos for cassette sequencing Done ===
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| − |
| |
| − | >50 bp before the interest region
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| − |
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| − | 800 bp max contigs
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| − |
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| − |
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| − |
| |
| − | Obtain Pir+ strain Done
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| − |
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| − | Overnight of Pir+ strain Done
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| − |
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| − | Glycerol of Pir+ strain Done
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| − |
| |
| − | == Cloning inside the replication vector ==
| |
| − |
| |
| − | Order oligos linR and linF Done
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| − |
| |
| − | Obtain R6K vector from Ihab Done
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| − |
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| − | Overnight culture of the R6K vector propagation strain
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| − |
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| − | Failed New attempt with less harsh growth conditions. Done
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| − |
| |
| − | * Only 20 ug/ml of Kanamycine
| |
| − | * 6 ul of Thyamine in 3 ul of LB
| |
| − | * Culture at 30°C
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| − |
| |
| − | Miniprep of the R6K vector Done
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| − |
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| − | Glycerol of R6K strain Done
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| − |
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| − | Linearization of the R6K vector by PCR Done
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| − |
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| − |
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| − |
| |
| − | Primer linF:
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| − |
| |
| − | CCCTTGGGCTCCCCGGGCGCGTACTCCAgaagggcatcgatggc
| |
| − |
| |
| − | (28 bases: longest possible overlap without having a hairpin at 50°)
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| − |
| |
| − | Tm = 55°
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| − |
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| − |
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| − |
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| − | Primer linR:
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| − |
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| − | tagcattatacctaggactgagctagctgtcaaggcaaatttgcggccgcaag
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| − |
| |
| − | (33 bases overlap)
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| − |
| |
| − | Tm = 62°
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| − |
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| − |
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| − |
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| − | Product length: 2276 bp
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| − |
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| − | Synthetize, reconstitute and dilute primers Done
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| − |
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| − | Run gel for size checking Done
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| − |
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| − | PCR purification Done
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| − |
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| − |
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| − |
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| − | Obtain gBlocks Done
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| − |
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| − | PCR of colibow fragments A, B and C Done
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| − |
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| − | PCR purification of amplicon Done
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| − |
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| − |
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| − |
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| − | Obtain Gibson mix Done
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| − |
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| − | Make overnight culture of Pir+ strain Done
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| − |
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| − | Make electrocompetent cells out of the Pir+ strain Done
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| − |
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| − | Gibson assembly of 3 gblocks with the R6K backbone. Done
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| − |
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| − | The vector can only replicate in Pir+ strain. Transform into Pir+ strain Got colonies
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| − |
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| − | Check transformant: colony PCR before culture Failed
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| − |
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| − | Liquid culture + miniprep
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| − |
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| − | Analytical digestion or sequencing (find enzymes)
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| − |
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| − |
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| − |
| |
| − | {|
| |
| − | |width="33%"|SeqF1
| |
| − | |width="33%"|o15.88
| |
| − | |width="33%"|cttagtacgttagccatgagg
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| − | |-
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| − | |SeqF2
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| − | |o15.89
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| − | |CTAATTTTCCATCTGATGGCC
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| − | |-
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| − | |SeqF3
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| − | |o15.90
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| − | |CAAGCTCACGCTCAAATTC
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| − | |-
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| − | |SeqF4
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| − | |o15.91
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| − | |ACAACCATTACCTGTCGACG
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| − | |-
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| − | |SeqF5
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| − | |o15.92
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| − | |CGACATTAGGGTATGGGCTG
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| − | |-
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| − | |SeqR1
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| − | |o15.93
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| − | |TATAAACATTATGGCTATTATAG
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| − | |-
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| − | |SeqR2
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| − | |o15.94
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| − | |TTTAGAGAGTTTTGACTGCG
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| − | |-
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| − | |SeqR3
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| − | |o15.95
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| − | |TTTGCCAGTCGTACAGATGAA
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| − | |-
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| − | |SeqR4
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| − | |o15.96
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| − | |TCTTCTTCTGCATCACCGGGC
| |
| − | |}
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| − |
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| − |
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| − |
| |
| − | == Chromosomal integration with GalK ==
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| − |
| |
| − | PCR of the purified R6K plasmid. Done
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| − |
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| − |
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| − |
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| − | PCR-linerization of the R6K backbone
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| − |
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| − |
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| − |
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| − | * Miniprep du backbone R6K (20 ng/ul) -> 5 ul in PCR
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| − | * Primers: 15.80 (LinR) et 15.81(LinF) -> 1 ul each
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| − | * Eau qsp 25 ul
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| − | * Master mix 25 ul
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| − |
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| − |
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| − |
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| − | 1’30’’ extension, 50°C annealing.
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| − |
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| − |
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| − |
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| − | Find oligos (already there) Done
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| − |
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| − |
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| − |
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| − | IntR:
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| − |
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| − | GTTTGCGCGCAGTCAGCGATATCCATTTTCGCGAATCCGGAGTGTAAGAACCATATGAATATCCTCCTTAGTTCCTATTCCG
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| − |
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| − |
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| − |
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| − | Alternatively, with only one binding site:
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| − |
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| − | GTTTGCGCGCAGTCAGCGATATCCATTTTCGCGAATCCGGAGTGTAAGAAttagccatggtccatatgaatatcctccttag
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| − |
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| − |
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| − |
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| − | IntF:
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| − |
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| − | TTCATATTGTTCAGCGACAGCTTGCTGTACGGCAGGCACCAGCTCTTCCGGGTTGAACTGCGGATCTTGCGGC
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| − |
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| − |
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| − |
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| − | Italique: Homology region with E. coli GalK site.
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| − |
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| − | Length: 4908 bp
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| − |
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| − | Attention, autre site potentiel d’amorçage, produit de 3515 bp
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| − |
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| − |
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| − |
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| − | Purification
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| − |
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| − | Overnight culture de E. coli Lambda Red
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| − |
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| − | Competent cells out of Lambda Red strain
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| − |
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| − | Transformation of E. coli with pKD46 that carries Lambda Red recombinase
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| − |
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| − | Transformation of the Colibow PCR product
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| − |
| |
| − | == Function testing ==
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| − |
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| − | Tecan + Flux cytometry
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| − |
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| − | == CRE recombinase expression ==
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| − |
| |
| − | pFHC2938 should work.
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| − |
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| − | It expresses CRE and has a temperature sensitive ORI (30°C)
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| − |
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| − |
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| − |
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| − | === Monday 07/06 ===
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| − |
| |
| − | ==== Research about the synthetic integron ====
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| − |
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| − | It might make the landing pads better than with brainbow because the recombination site is always the same.
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| − |
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| − | ==== Plate culture of E. coli pIT5-KL and pIT5-KH ====
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| − |
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| − | Very important: grow @30
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| − |
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| − | -> Make a liquid culture of each and freeze at -80
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| − |
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| − | -> Miniprep from pIT5-KL for first construct
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| − |
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| − |
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| − |
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| − | These two strains produce the vector plasmids for clonetegration. They have to be linearized with EcoR1 and Pst1, and then Gibson assembled to get the self-integrating plasmids.
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| − |
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| − | The integrase they carry is expressed only at 37 degrees.
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| − |
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| − | They are resistant to Kanamycine.
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| − |
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| − | KH integrates into the HK022 phage’s site, while KL integrates into the Lambda phage site. We will probably only need one of them but I took both just in case. If needed there is a whole collection of such vectors.
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| − |
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| − | ==== Plate culture of E. coli pE-FLP ====
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| − |
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| − | Grow @ 30
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| − |
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| − | This strain produces the pE-FLP plasmid, which expresses the flippase. It will be useful to remove the backbone after the clonetegration. It’s resistant to ampicillin.
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| − |
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| − | It has a temperature-sensitive ORI and will disappear if grown at 37.
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| − |
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| − | ==== New primers ====
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| − |
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| − | LC81, LC85, LC82, LC86: used in the PCR to check integration with pIT5 KH.
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| − |
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| − | LC81, LC87, LC83, LC84: used in the PCR to check integration with pIT5 KL.
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| − |
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| − |
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| − |
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| − | == Thursday 07/09 ==
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| − |
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| − | * Prepared 10 tubes of 1 ml Kanamycine, 50 mg/ml
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| − | * Inoculated pIT5-KL and pIT5-KH E. coli in 2 ml LB Kan+50
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| − | * Inoculated pE-FLP E. coli in 2ml LB Amp+100
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| − | * Cultured these three tubes @ 30
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| − |
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| − | == Monday 07/20 ==
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| − |
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| − | === Reception of two plates from Ihab ===
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| − |
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| − | * Shortened R6K vector -> Kan+ and thyamine auxotrophy. Thyamine 1000x is available in an eppendorf tube.
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| − | * Pir 116 strain for propagation of the vector.
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| − |
| |
| − | -> Overnight culture for miniprep for PCR and gleezing
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| − |
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| − | * R6K in LB Kan Thyamine (in antibiotics box)
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| − | * Pir116 in LB with a control tube
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| − |
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| − | === Started cultures for Colibow ===
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| − |
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| − | pThy 1.0 aka pBrainbow 1.0 in 3 ml LB-Amp -> Miniprepcr
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| − |
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| − | pR6K in LB-Kan-Thyamine -> Miniprepcr and Glycerol freezing
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| − |
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| − | Pir116 for replication of pR6K-vectors -> LB
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| − |
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| − | Control without innoculation -> LB
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| − |
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| − |
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| − |
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| − | All of them, culture @ 37° overnight.
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| − |
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| − |
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| − |
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| − | No need to culture pKT174 because we already have the purified version. It may be a good idea to transform DH5a with it in case we run out of plasmid.
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| − |
| |
| − | === Reception of oligos o15.76 to o15.96 ===
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| − |
| |
| − | * Yeastbow SOE
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| − | * R6K linearization
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| − | * Colibow gBlock Amp
| |
| − | * Colibow Sequencing
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| − |
| |
| − | The ultramers (o15.78, o15.79) for Leu2 flanking are not ready yet.
| |
| − |
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| − |
| |
| − |
| |
| − | -> Oligo reconstitution and dilution
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| − |
| |
| − | -> Miniprep of R6K vector
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| − |
| |
| − | -> PCR of R6K vector
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| − |
| |
| − | -> Miniprep of pBrainbow 1.0
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| − |
| |
| − | -> PCR of pBrainbow 1.0
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| − |
| |
| − | -> PCR of pThy Ura3
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| − |
| |
| − |
| |
| − |
| |
| − | Reconstituted all primers to 100 ug/ml.
| |
| − |
| |
| − | === Miniprep of pR6K and pBrainbow 1.0 ===
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| − |
| |
| − | For PCR, using Promega kit w/ double wash. Elution in 30 ul
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| − |
| |
| − | Final concentration: 93 ng/ul
| |
| − |
| |
| − | === Re-start of the R6K culture ===
| |
| − |
| |
| − | The first culture failed: let’s try again with less harsh conditions.
| |
| − |
| |
| − | * Only 20 ug/ml of Kanamycine
| |
| − | * 6 ul of Thyamine in 3 ul of LB
| |
| − | * Culture at 30°C
| |
| − |
| |
| − | == Wednesday 07/22 ==
| |
| − |
| |
| − | === PCR-linerization of the R6K backbone ===
| |
| − |
| |
| − |
| |
| − |
| |
| − | * Miniprep du backbone R6K (20 ng/ul) -> 5 ul in PCR
| |
| − | * Primers: 15.80 (LinR) et 15.81(LinF) -> 1 ul each
| |
| − | * Eau qsp 25 ul
| |
| − | * Master mix 25 ul
| |
| − |
| |
| − |
| |
| − |
| |
| − | 1’30’’ extension, 50°C annealing -> 2276 bp
| |
| − |
| |
| − |
| |
| − |
| |
| − | === Bad news from IDT about the gene synthesis ===
| |
| − |
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| − |
| |
| − |
| |
| − | « I would like to notify you about a best effort for gBlock ‘Colibow C’. Even after multiple attempts at trying to correct this issue, the Final QC data for the best prep is not meeting our quality standards. The final prep (mfg# 188635671) has desired peak. A secondary peak is also present. The target mass was found in the chromatogram, but not as the most abundant product in any of the chromatographic peaks for mass spectrometry, and the sequence has been verified.
| |
| − |
| |
| − | If you are using this sequence for cloning and/or qPCR you should not have any problems with this sequence. It may require you to screen additional colonies. However, if your intentions with using this gBlock was for other purposes, we might need to cancel the gBlock and redesign. I have provided the trace below. »
| |
| − |
| |
| − | == Thursday, 07/23 ==
| |
| − |
| |
| − | === Glycerol stock for pR6K ===
| |
| − |
| |
| − | 1 ml of overnight culture of E. coli pR6K.
| |
| − |
| |
| − | 1 ml of glycerol.
| |
| − |
| |
| − | -> -20°C
| |
| − |
| |
| − | === Gel for linearized R6K pcr ===
| |
| − |
| |
| − | 1% agar TAE, with 1 kb+ generuler.
| |
| − |
| |
| − | 5 ul PCR product + 1 ul LB.
| |
| − |
| |
| − | -> band at the right size (2276 bp) , the PCR worked.
| |
| − |
| |
| − |
| |
| − |
| |
| − | === linearized R6K pcr cleanup ===
| |
| − |
| |
| − | Using the Qiagen kit, taken back in 50 ul water
| |
| − |
| |
| − | Titration: about 80 ng/ul, but very bad 260/230 ratio (presence of organic molecules).
| |
| − |
| |
| − |
| |
| − |
| |
| − | === Pir116 electrocompetent for bowcoli transformation ===
| |
| − |
| |
| − |
| |
| − |
| |
| − | 2 ml overnight culture in 100 ml LB, at 37°C w/ shaking.
| |
| − |
| |
| − |
| |
| − |
| |
| − | When OD reaches 0.6, they were put in ice for half an hour.
| |
| − |
| |
| − | Electrocompetent cells were made by Mukit along with DH5a competent cells.
| |
| − |
| |
| − | == Tuesday, 07/28 ==
| |
| − |
| |
| − | === Reception of gBlocks Colibow A, B, C ===
| |
| − |
| |
| − | Reconstitution to the concentration of 10 ng/ul (from spec sheet):
| |
| − |
| |
| − | * Centrifuge @ 11kG
| |
| − |
| |
| − | * Add 100 ul of RNase-free water
| |
| − | * Vortex
| |
| − | * Incubate at 50°C for 20 minutes
| |
| − | * Vortex/Centrifuge
| |
| − |
| |
| − | === PCR of Colibow gBlocks ===
| |
| − |
| |
| − |
| |
| − |
| |
| − | Colibow A
| |
| − |
| |
| − | Primers: 82 (56°) + 83 (54°) -> 1172 bp
| |
| − |
| |
| − | Colibow B
| |
| − |
| |
| − | Primers: 84 (53°) + 85 (54°) -> 909 bp
| |
| − |
| |
| − | Colibow C
| |
| − |
| |
| − | Primers: 86 (54°) + 87 (57°) -> 992 bp
| |
| − |
| |
| − |
| |
| − |
| |
| − | Reaction in 50 ul:
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="50%"|Compound
| |
| − | |width="50%"|Volume (ul)
| |
| − | |-
| |
| − | |Water
| |
| − | |19
| |
| − | |-
| |
| − | |Phusion 2x
| |
| − | |25
| |
| − | |-
| |
| − | |Primer 1
| |
| − | |2.5
| |
| − | |-
| |
| − | |Primer 2
| |
| − | |2.5
| |
| − | |-
| |
| − | |gBlock diluted 10 times
| |
| − | |1 ul
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | Program:
| |
| − |
| |
| − | 98 (30)
| |
| − |
| |
| − | 98 (10) 58 (25) 72 (45) x35
| |
| − |
| |
| − | 72 (600)
| |
| − |
| |
| − | 12 (hold)
| |
| − |
| |
| − |
| |
| − |
| |
| − | In parallel from running the gel, the PCR products were purified with the QIAGEN kit and eluted in 40 ul of water.
| |
| − |
| |
| − |
| |
| − |
| |
| − | Titration
| |
| − |
| |
| − | Fragment A: 90 ng/ul
| |
| − |
| |
| − | Fragment B: 88 ng/ul
| |
| − |
| |
| − | Fragment C: 85 ng/ul
| |
| − |
| |
| − |
| |
| − |
| |
| − | Gel plan
| |
| − |
| |
| − | 1% agar, SYBRsafe, 5 ul of PCR product + 1 ul of LD.
| |
| − |
| |
| − | The 100 bp+ marker was used.
| |
| − |
| |
| − | The apparatus didn’t work. It stopped two times, the gel stayed to diffuse in the tank for 30 minutes. At the end it was imaged even though it was far from finished.
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="25%"|Ladder 100 bp+
| |
| − | |width="25%"|Colibow A
| |
| − | |width="25%"|Colibow B
| |
| − | |width="25%"|Colibow C
| |
| − | |-
| |
| − | |Expected size
| |
| − | |1172
| |
| − | |909
| |
| − | |992
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | [[File:images/image04.jpg|Colibow Amp Results.jpg]][[File:images/image02.jpg|1438103355.jpg]]
| |
| − |
| |
| − |
| |
| − |
| |
| − | Conclusion
| |
| − |
| |
| − | There are a lot of non-specific binding.
| |
| − |
| |
| − | Due to the awful quality of the gel, it’s not possible to determine their size or which bands are the good ones. The top ones seem more consistent regarding their relative sizes.
| |
| − |
| |
| − |
| |
| − |
| |
| − | To do:
| |
| − |
| |
| − | # Run the whole purified PCR product in a gel and perform a gel extraction. Hopefully the right band can be determined this time.
| |
| − | # Try the PCR again with a higher annealing temperature.
| |
| − | # Gibson assemble directly the gBlocks fragments.
| |
| − |
| |
| − | == Wednesday, 07/29 ==
| |
| − |
| |
| − | === Gel for Colibow A, B, C and extraction ===
| |
| − |
| |
| − | Agar 1%, SYBRsafe, TAE
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="25%"|Ladder 100 bp+
| |
| − | |width="25%"|Colibow A
| |
| − | |width="25%"|Colibow B
| |
| − | |width="25%"|Colibow C
| |
| − | |-
| |
| − | |Expected size
| |
| − | |1172
| |
| − | |909
| |
| − | |992
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | [[File:images/image01.jpg|Colibow Amp Extracted.jpg]]
| |
| − |
| |
| − | For each sample, the top band was extracted. The Colibow B sample was extracted twice: Bg (grande, top band) and Bp (petite, bottom band).
| |
| − |
| |
| − | The bottom band is the right one.
| |
| − |
| |
| − | -> add DMSO 3% next time
| |
| − |
| |
| − |
| |
| − |
| |
| − | Titration (in 30 ul EB)
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="14%"|Name
| |
| − | |width="14%"|1
| |
| − | |width="14%"|U
| |
| − | |width="14%"|A
| |
| − | |width="14%"|Bp
| |
| − | |width="14%"|Bg
| |
| − | |width="14%"|C
| |
| − | |-
| |
| − | |C (ng/ul)
| |
| − | |13
| |
| − | |41
| |
| − | |6
| |
| − | |29
| |
| − | |11
| |
| − | |12.1
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | The concentrations of Colibow A and C are critically low. It is necessary to fix the PCR and reduce the non-specific priming before performing the Gibson.
| |
| − |
| |
| − | === New attempt at Colibow A and C PCRs ===
| |
| − |
| |
| − |
| |
| − |
| |
| − | Mix
| |
| − |
| |
| − | Phusion 25
| |
| − |
| |
| − | Colibow A 1
| |
| − |
| |
| − | o15.82 2.5
| |
| − |
| |
| − | o15.83 2.5
| |
| − |
| |
| − | Water 19
| |
| − |
| |
| − | DMSO 1.5 (3%)
| |
| − |
| |
| − | = Two tubes of 50 ul each -> 1172 bp
| |
| − |
| |
| − |
| |
| − |
| |
| − | Mix
| |
| − |
| |
| − | Phusion 25
| |
| − |
| |
| − | Colibow C 1
| |
| − |
| |
| − | o15.86 2.5
| |
| − |
| |
| − | o15.87 2.5
| |
| − |
| |
| − | Water 19
| |
| − |
| |
| − | DMSO 1.5 (3%)
| |
| − |
| |
| − | = Two tubes of 50 ul each -> 992 bp
| |
| − |
| |
| − |
| |
| − |
| |
| − | This is stored in 4° for now (29/07)
| |
| − |
| |
| − | === Received Gibson assembly mixes from Ihab ===
| |
| − |
| |
| − | I need better quality products before doing it.
| |
| − |
| |
| − |
| |
| − |
| |
| − | == Thursday, 07/30 ==
| |
| − |
| |
| − |
| |
| − |
| |
| − | === Gel for A, C and 1 ===
| |
| − |
| |
| − |
| |
| − |
| |
| − | *Sophie’s sample
| |
| − |
| |
| − | {|
| |
| − | |width="8%"|*
| |
| − | |width="8%"|A
| |
| − | |width="8%"|A
| |
| − | |width="8%"|A
| |
| − | |width="8%"|C
| |
| − | |width="8%"|C
| |
| − | |width="8%"|C
| |
| − | |width="8%"|100+
| |
| − | |width="8%"|1
| |
| − | |width="8%"|1
| |
| − | |width="8%"|1
| |
| − | |width="8%"|1kb
| |
| − | |-
| |
| − | |*
| |
| − | |1172
| |
| − | |1172
| |
| − | |1172
| |
| − | |992
| |
| − | |992
| |
| − | |992
| |
| − | |
| |
| − | |3965
| |
| − | |3965
| |
| − | |3965
| |
| − | |
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | 50 ul sample + 10 ul LD -> 40 ul in each well (3 wells)
| |
| − |
| |
| − | Attention C sample accidentally added to the well containing 100 bp + ladder !!!
| |
| − |
| |
| − |
| |
| − |
| |
| − | [[File:images/image00.jpg|Colibow A et C amp 29.07.jpg]]
| |
| − |
| |
| − | Gel extraction
| |
| − |
| |
| − | With Qiagen kit, product recovered in 30 ul of water.
| |
| − |
| |
| − | Name: “product” X+ 30/07
| |
| − |
| |
| − | The C product mixed with ladder was labeled Cl.
| |
| − |
| |
| − |
| |
| − |
| |
| − | Titration
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="20%"|Sample
| |
| − | |width="20%"|1
| |
| − | |width="20%"|A
| |
| − | |width="20%"|C
| |
| − | |width="20%"|Cl
| |
| − | |-
| |
| − | |c (ng/ul)
| |
| − | |5.2
| |
| − | |15.4
| |
| − | |12.9
| |
| − | |4.4
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | === Gibson assemblies of Colibow ===
| |
| − |
| |
| − | General mix:
| |
| − |
| |
| − | 15 ul of Gibson supermix (MMII)
| |
| − |
| |
| − | 10-100 ng of backbone for a 6 kb fragment
| |
| − |
| |
| − | Equimolar amount of DNA fragments
| |
| − |
| |
| − | Total: 20 ul
| |
| − |
| |
| − |
| |
| − |
| |
| − | Colibow PCR products
| |
| − |
| |
| − | Using the most concentrated PCR products known to date.
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="20%"|Nom
| |
| − | |width="20%"|Taille (bp)
| |
| − | |width="20%"|Concentration
| |
| − | |width="20%"|Volume (ul)
| |
| − | |width="20%"|Quantité (ng)
| |
| − | |-
| |
| − | |R6K pcr
| |
| − | |2276
| |
| − | |80
| |
| − | |0.6
| |
| − | |50
| |
| − | |-
| |
| − | |Colibow A+ e (in water)
| |
| − | |1172
| |
| − | |15
| |
| − | |1.7
| |
| − | |25
| |
| − | |-
| |
| − | |Colibow Bp
| |
| − |
| |
| − | (in EB)
| |
| − | |909
| |
| − | |29
| |
| − | |0.9
| |
| − | |25
| |
| − | |-
| |
| − | |Colibow C+ e
| |
| − |
| |
| − | (in water)
| |
| − | |992
| |
| − | |13
| |
| − | |1.6
| |
| − | |25
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | Colibow gBlocks
| |
| − |
| |
| − | Using directly the gBlocks from IDT dna synthesis
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="20%"|Nom
| |
| − | |width="20%"|Taille (bp)
| |
| − | |width="20%"|Concentration
| |
| − | |width="20%"|Volume (ul)
| |
| − | |width="20%"|Quantité (ng)
| |
| − | |-
| |
| − | |R6K pcr
| |
| − | |2276
| |
| − | |80
| |
| − | |0.5
| |
| − | |30
| |
| − | |-
| |
| − | |Colibow A
| |
| − | |1172
| |
| − | |10
| |
| − | |1.5
| |
| − | |15
| |
| − | |-
| |
| − | |Colibow B
| |
| − | |909
| |
| − | |10
| |
| − | |1.5
| |
| − | |15
| |
| − | |-
| |
| − | |Colibow C
| |
| − | |992
| |
| − | |10
| |
| − | |1.5
| |
| − | |15
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | Incubation during one hour at 50°C.
| |
| − |
| |
| − | === 3 LB ampicillin plates ===
| |
| − |
| |
| − |
| |
| − |
| |
| − | “30/07 ANTOINE”
| |
| − |
| |
| − | 50 ml of hot LB-agar.
| |
| − |
| |
| − | === Transformation of Colibow in Pir116 by Electroporation ===
| |
| − |
| |
| − | pColibow gBlock
| |
| − |
| |
| − | pColibow PCR
| |
| − |
| |
| − | Biobrick plasmid with RFP (3 ul of 10 pg tube, not dialysed)
| |
| − |
| |
| − |
| |
| − |
| |
| − | Protocol from OWW:
| |
| − |
| |
| − | * Thaw cells from -80°C to ice for >20 min
| |
| − | * Chill cuvettes
| |
| − | * Dialyse 6 ul of Gibson products on dWater during >20 min
| |
| − | * Mix 6 ul of dialysed DNA with 50 ul Pir116 electrocompetent cells
| |
| − | * Mix with tip
| |
| − | * Pulse (1.5 kV, 2 mm cuvette)
| |
| − | * Add 1 ml LB (at 18h43)
| |
| − |
| |
| − | Incubate for 1 hour at 37°C.
| |
| − |
| |
| − |
| |
| − |
| |
| − | 2 Kanamycine 25 plates for each colibow plasmid: 100 ul and 900 ul
| |
| − |
| |
| − | 1 Chloramphenicol plate for RFP control
| |
| − |
| |
| − | Incubation overnight @37.
| |
| − |
| |
| − | === Transformation of pKT174 in DH5a by Heat shock ===
| |
| − |
| |
| − | Protocol from addgene:
| |
| − |
| |
| − | * Thaw cells on ice (20 min)
| |
| − | * 3 ul DNA + 50 ul chemically competent cells, mix gently
| |
| − | * 20-30 min on ice
| |
| − | * 45s at 42°C
| |
| − | * 2 min on ice
| |
| − | * Add 1 ml LB (at 18h50)
| |
| − | * Incubate 1 hour @ 37°C
| |
| − |
| |
| − |
| |
| − |
| |
| − | 2 ampicillin plates: 100 ul and 900 ul.
| |
| − |
| |
| − | Incubation overnight @37.
| |
| − |
| |
| − | === Gel for SOE 29/07 ===
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="16%"|1 kb ladder
| |
| − | |width="16%"|SOE
| |
| − | |width="16%"|SOE
| |
| − | |width="16%"|SOE
| |
| − | |width="16%"|1 kb ladder
| |
| − | |width="16%"|*
| |
| − | |}
| |
| − |
| |
| − | Expected size: 5600 for all of them.
| |
| − |
| |
| − | [[File:images/image03.jpg|SOE+29.07.jpg]]
| |
| − |
| |
| − | It didn’t work at all. Try to Gibson-assemble them.
| |
| − |
| |
| − | === New electroporation of Pir116 ===
| |
| − |
| |
| − | Using the old electroporator (with the square cuvette holder).
| |
| − |
| |
| − | Dialysis of 6 ul and sampling of 5 ul of Colibow gBlock assembly
| |
| − |
| |
| − | Dialysis of 5 ul and sampling of 5 ul of 5 pg/ul
| |
| − |
| |
| − | {|
| |
| − | |width="33%"|Compound
| |
| − | |width="33%"|x1
| |
| − | |width="33%"|x8
| |
| − | |-
| |
| − | |10x Taq Buffer
| |
| − | |2.5
| |
| − | |20
| |
| − | |-
| |
| − | |10 nM dNTPs
| |
| − | |0.5
| |
| − | |4
| |
| − | |-
| |
| − | |10 uM primer 90
| |
| − | |0.5
| |
| − | |4
| |
| − | |-
| |
| − | |10 uM primer 94
| |
| − | |0.5
| |
| − | |4
| |
| − | |-
| |
| − | |taq polymerase
| |
| − | |0.125
| |
| − | |1
| |
| − | |-
| |
| − | |water
| |
| − | |17.875
| |
| − | |167
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | Then 22 ul of mix is added to 3 ul of template (2 ul water + 1 ul sample for positive control). Template is obtained by soaking part of a colony in 20 ul water.
| |
| − |
| |
| − | ==== Program (saved as Colony): ====
| |
| − |
| |
| − | 95 (6’00)
| |
| − |
| |
| − | 95 (15) 49 (25) 68 (45) x30
| |
| − |
| |
| − | 68 (5’00)
| |
| − |
| |
| − | ==== Gel: ====
| |
| − |
| |
| − | P900 B1 B2 B3 ColibowB 100bp+
| |
| − |
| |
| − | ==== Results: ====
| |
| − |
| |
| − | * One band at 508 bp on the positive control.
| |
| − | * No band at all on the colonies
| |
| − |
| |
| − | -> They are contaminations, the transformation did not work, probably because the Gibson itself did not work due to bad DNA concentration.
| |
| − |
| |
| − | == Wednesday, 8/5 ==
| |
| − |
| |
| − | ==== Pir116 electrocompetent cells ====
| |
| − |
| |
| − | From the overnight culture, 1 ml of cells were diluted in 50 ml of LB and incubated at 37°C (10h30).
| |
| − |
| |
| − | Centrifuge steps were done 10 min at 8500 rpm.
| |
| − |
| |
| − | * 50 ml culture -> centrifuge and remove well the supernatant
| |
| − | * 50 ml glycerol 10% -> centrifuge
| |
| − | * 50 ml glycerol 10% -> centrifuge
| |
| − |
| |
| − | Cells in residual glycerol were aliquoted in eppendorf tubes (6 in the end) and frozen @-80.
| |
| − |
| |
| − | ==== Testing of these EC Pir116 cells function ====
| |
| − |
| |
| − | 4 ul of pSB1C3 20 pg/ul were dialysed, 3 ul were picked after 20 min and mixed with 100 ul of cells.
| |
| − |
| |
| − | Pulse duration: 5.7 ms
| |
| − |
| |
| − | After 1 h of preculture @37, it was plated on a chloramphenicol 0.5 plate and grown @37.
| |
| − |
| |
| − | ==== New colibow PCR ====
| |
| − |
| |
| − | Performed by Chloé and Émilie.
| |
| − |
| |
| − |
| |
| − |
| |
| − | [[File:images/image06.png|1438872081.png]]
| |
| − |
| |
| − | Program:
| |
| − |
| |
| − | 98 (30)
| |
| − |
| |
| − | 35x 98 (10), Gradient (30), 72 (2’20)
| |
| − |
| |
| − | 72 (5)
| |
| − |
| |
| − | 10 (hold)
| |
| − |
| |
| − |
| |
| − |
| |
| − | Gradient:
| |
| − |
| |
| − | 59, 57.8, 55.3, 53.4
| |
| − |
| |
| − | The very long extension time is used to avoid promoting small non-specific fragments.
| |
| − |
| |
| − |
| |
| − |
| |
| − | Gel:
| |
| − |
| |
| − | 1% agarose, SYBRsafe, 2.5 ul sample + 0.5 ul LD
| |
| − |
| |
| − | {|
| |
| − | |width="6%"|L
| |
| − | |width="6%"|A
| |
| − | |width="6%"|A
| |
| − | |width="6%"|A
| |
| − | |width="6%"|A
| |
| − | |width="6%"|L
| |
| − | |width="6%"|B
| |
| − | |width="6%"|B
| |
| − | |width="6%"|B
| |
| − | |width="6%"|B
| |
| − | |width="6%"|L
| |
| − | |width="6%"|C
| |
| − | |width="6%"|C
| |
| − | |width="6%"|C
| |
| − | |width="6%"|C
| |
| − | |width="6%"|L
| |
| − | |-
| |
| − | |1 kb
| |
| − | |1172
| |
| − | |
| |
| − | |
| |
| − | |
| |
| − | |1 kb
| |
| − | |909
| |
| − | |
| |
| − | |
| |
| − | |
| |
| − | |1 kb
| |
| − | |992
| |
| − | |
| |
| − | |
| |
| − | |
| |
| − | |
| |
| − | |}
| |
| − |
| |
| − | [[File:images/image07.jpg|08.05 Colibow Amp gradient.jpg]]
| |
| − |
| |
| − | === Thursday, 8/6 ===
| |
| − |
| |
| − | ==== PCR purification of Colibow gBlocks ====
| |
| − |
| |
| − | The homologous tubes were mixed together, except for C2 that didn’t work.
| |
| − |
| |
| − | Elution in 50 ul of water.
| |
| − |
| |
| − | A second gel was ran in order to know whether the light band at the bottom is still present.
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="25%"|1 kb ladder
| |
| − | |width="25%"|A
| |
| − | |width="25%"|B
| |
| − | |width="25%"|C
| |
| − | |}
| |
| − |
| |
| − |
| |
| − |
| |
| − | Is it. This has to be taken into account when calculating the concentrations for the Gibson assembly.
| |
| − |
| |
| − |
| |
| − |
| |
| − | Titration (in 50 ul of water):
| |
| − |
| |
| − | A: 137 ng/ul
| |
| − |
| |
| − | B: 167 ng/ul
| |
| − |
| |
| − | C: 99 ng/ul
| |
| − |
| |
| − | [[File:images/image05.jpg|08.06 Colibow gradient after purification.jpg]]
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| − |
| |
| − | === New R6K PCR ===
| |
| − |
| |
| − |
| |
| − |
| |
| − | {|
| |
| − | |width="33%"|Compound
| |
| − | |width="33%"|1x
| |
| − | |width="33%"|2x
| |
| − | |-
| |
| − | |water
| |
| − | |71
| |
| − | |142
| |
| − | |-
| |
| − | |Phu buffer
| |
| − | |20
| |
| − | |40
| |
| − | |-
| |
| − | |dNTP
| |
| − | |2
| |
| − | |4
| |
| − | |-
| |
| − | |80 primer
| |
| − | |1
| |
| − | |2
| |
| − | |-
| |
| − | |81 primer
| |
| − | |1
| |
| − | |2
| |
| − | |-
| |
| − | |pR6K shortened (template)
| |
| − | |1
| |
| − | |2
| |
| − | |-
| |
| − | |DMSO
| |
| − | |3
| |
| − | |6
| |
| − | |-
| |
| − | |Phusion polymerase
| |
| − | |1
| |
| − | |2
| |
| − | |}
| |
| − |
| |
| − | Program:
| |
| − |
| |
| − | 98 (30)
| |
| − |
| |
| − | 98 (10) 52 (30) 72 (1’30) x 35
| |
| − |
| |
| − | 72 (5’)
| |
| − |
| |
| − | Problem: There was only <1 ul of phusion left in the tube. The PCR did not work at all.
| |
| − |
| |
| − | ==== New attempt ====
| |
| − |
| |
| − | Mix (made twice)
| |
| − |
| |
| − | Phusion master mix 50 ul
| |
| − |
| |
| − | 80 primer 2 ul
| |
| − |
| |
| − | 81 primer 2 ul
| |
| − |
| |
| − | pR6K 3 ul
| |
| − |
| |
| − | DMSO 3 ul
| |
| − |
| |
| − | Water 40 ul
| |
| − |
| |
| − |
| |
| − |
| |
| − | Program:
| |
| − |
| |
| − | 98 (30)
| |
| − |
| |
| − | 98 (10) 50 (30) 72 (1’30) x 35
| |
| − |
| |
| − | 72 (5’)
| |
| − |
| |
| − |
| |
| − |
| |
| − | After this PCR, the product was:
| |
| − |
| |
| − | * ran on a gel: Ladder 1kb, tube 1, tube 2 (3 ul sample, 2 ul water, 1 ul LD)
| |
| − | * digested by DPN1:
| |
| − |
| |
| − | 1 ul of DPN1 added directly to the product, then incubated for 15 minutes at 37° in the thermocycler and inactivated (5 min at 80°).
| |
| − |
| |
| − |
| |
| − |
| |
| − | Gel results: To be uploaded (on my pendrive now).
| |
| − |
| |
| − | == Monday, 08/10 ==
| |
| − |
| |
| − | === PCR purification of R6K amp ===
| |
| − |
| |
| − | With QIAGEN kit, performed by Chloé & Émilie. At the end, the product was recovered in 50 ul of water and also 30 ul EB, for 80 ul total.
| |
| − |
| |
| − | The titration was done using a 5 ul + 3 ul mix as a blank.
| |
| − |
| |
| − | ==== Products summary: ====
| |
| − |
| |
| − | {|
| |
| − | |width="33%"|R6K pcr
| |
| − | |width="33%"|67.4 ng/ul
| |
| − | |width="33%"|2276 bp
| |
| − | |-
| |
| − | |Colibow A
| |
| − | |137
| |
| − | |1172 bp
| |
| − | |-
| |
| − | |Colibow B
| |
| − | |167
| |
| − | |909 bp
| |
| − | |-
| |
| − | |Colibow C
| |
| − | |99
| |
| − | |992 bp
| |
| − | |}
| |
| − |
| |
| − | A and C have a small band at ~200 bp, so the actual concentration of the product is half of what indicated by the Nanodrop.
| |
| − |
| |
| − | === Gibson assembly of Colibow ===
| |
| − |
| |
| − | Assuming half the DNA in A and C is the right one.
| |
| − |
| |
| − | {|
| |
| − | |width="33%"|Name
| |
| − | |width="33%"|Volume (ul)
| |
| − | |width="33%"|Final amount (pmol)
| |
| − | |-
| |
| − | |A
| |
| − | |1.11
| |
| − | |0.10
| |
| − | |-
| |
| − | |B
| |
| − | |0.4
| |
| − | |0.11
| |
| − | |-
| |
| − | |C
| |
| − | |1.30
| |
| − | |0.10
| |
| − | |-
| |
| − | |R6K
| |
| − | |2.19
| |
| − | |0.10
| |
| − | |}
| |
| − |
| |
| − | Added to a MMII Gibson assembly master mix and incubated during 1h at 50°C.
| |
| − |
| |
| − | ==== Gel for Colibow’s Gibson ====
| |
| − |
| |
| − | 6 ul SYBRsafe in 20 ml agar 1%, with 1 kb ladder.
| |
| − |
| |
| − | The sample consisted in 10 ul of Gibson product and 2 ul of LD.
| |
| − |
| |
| − | Unfortunately the comb for making the wells went through the gel, resulting in loss of the sample.
| |
| − |
| |
| − | === Electroporation of Pir116 E. coli with newly assembly pColibow ===
| |
| − |
| |
| − | * Dialyse of the assembly product during 20 min. 5 ul were deposed and 5 ul were taken back.
| |
| − | * Mixed with already tested Pir116 Electrocompetent cells
| |
| − | * Program Ec2, bacterial, in 2 mm cuvette. Pulse time: 4.7 ms.
| |
| − | * Incubation 1h @37
| |
| − | * Plating on “pColibow Gibson” plates (K50 but K20 written on the box). 100 ul on the first box, centrifuged 900 ul on the second one.
| |
| − | * Incubation overnight @37.
| |