BioBrick Transformations Plasmid Isolation PCRs Restriction digests Ligations Screening
The five BioBricks we are using were transformed into E. coli strain DH5α — chosen for its recA and endA mutations that allow for high-yield minipreps.
INSERT IMAGES OF PLATES
Plasmid Isolation
Minipreps were performed from three colonies on each plate to confirm the presence of the plasmid. One colony from each positive transformants was used to make a glycerol stock.
INSERT GEL IMAGES
PCRs: Adding Restriction Sites and Linkers
Our assembly begins with our PCRs: using carefully-designed primers with 5'-overhangs, we add restriction sites, linkers and other features to our parts of interest. Since most of our PCRs have such overhangs, our annealing temperature changes after the first few cycles — our PCR cycle parameters account for this variation. In addition, we used NEB's Q5 MasterMix and NEB's Phusion polymerase, both high-fidelity DNA polymerases which have a higher annealing temperature than usual.
PCRs for the T7 expression backbone
PCR 1 — T7 Expression Backbone (Piece 1)
Template
BBa_K525998 (T7 promoter+RBS)
Forward primer
gactaccacggcatgatgaacctgaatcgc
Splits the CmR gene, includes NcoI site
Reverse primer
gaattcAAGCTTtttctcctctttccctatagtgagtcg
Adds HindIII site downstream
Amplicon size
886 bp
PCR 2 — T7 Expression Backbone (Piece 2)
Template
BBa_K731721 (T7 terminator)
Forward primer
gtatcacgaggcagaatttcag
Keeps the NheI site
Reverse primer
gagaatatgtttttcgtctcagcc
Splits the CmR gene, includes NcoI site
Amplicon size
1533 bp
PCRs for sfGFP-SpyCatcher
PCR 3 — sfGFP
Template
BBa_K1321337 (sfGFP in Freiburg format)
Forward primer
gaattcAAGCTTatgACCGGTcgtaaaggcgaagagctgttc
Adds BBa_K2319001 (HindIII+ATG+AgeI scar) upstream
Reverse primer
gGAATTCggatccTGACCCTCCtttgtacagttcatccataccatg
Adds Gly-Gly-Ser and BamHI site downstream
Amplicon size
753 bp
PCR 4 — SpyCatcher
Template
BBa_K1650037 (SpyCatcher)
Forward primer
GAATTAggatccGGGAGTAGCtcttattatcatcatcaccatcacc
Adds BamHI and Gly-Ser-Ser upstream
Reverse primer
gacgtcGCTAGCTTAaatatgagcatcgcccttgg
Adds stop codon (TAA) and NheI site downstream
Amplicon size
450 bp
PCRs for 6xHis-mCherry
PCR 5 — mCherry
Template
BBa_J18932 (mCherry RFP)
Forward primer
CACCATCATCACCATGTGAGCAAAGGCGAGGAAG
Adds 5xHis upstream
Reverse primer
cgtatgGCTAGCTTATTTATACAGTTCATCCATGCCG
Adds stop codon (TAA) and NheI site downstream
Amplicon size
735 bp
PCR 6 — mCherry
Template
Product of PCR5
Forward primer
aattcgAAGCTTATGCACCACCATCATCACCATGTGAG
Adds HindIII, a start codon (ATG) and His upstream
Reverse primer
cgtatgGCTAGCTTATTTATACAG
Keeps stop codon (TAA) and NheI site downstream
Amplicon size
753 bp
PCRs for mCherry-SpyTag
PCR 7 — mCherry-SpyTag
Template
BBa_J18932 (mCherry RFP)
Forward primer
CACCATCATCACCATGTGAGCAAAGGCGAGGAAG
Adds 5xHis upstream
Reverse primer
accgatGGATCCtttatacagttcatccatgccg
Adds BamHI site downstream
Amplicon size
732 bp
Restriction Digests
Restriction digests for T7 expression backbone
Restriction enzymes used in our assembly
Restriction Enzyme
Sequence
Activity in NEBuffers (%)
Incubation temperature
Heat inactivation
1.1
2.1
3.1
CutSmart
AgeI
A\CCGGT
100
75
25
75
37°C
65°C
AgeI-HF
A\CCGGT
100
50
10
100
37°C
65°C
BamHI
G\GATCC
75*
100*
100
100*
37°C
—
BamHI-HF
G\GATCC
100
50
10
100
37°C
—
HindIII
A\AGCTT
25
100
50
50
37°C
80°C
HindIII-HF
A\AGCTT
10
100
10
100
37°C
80°C
HindIII
A\AGCTT
25
100
50
50
37°C
80°C
HindIII-HF
A\AGCTT
10
100
10
100
37°C
80°C
NcoI
C\CATGG
100
100
100
100
37°C
80°C
NcoI-HF
C\CATGG
50
100
10
100
37°C
80°C
NdeI
CA\TATG
75
100
100
100
37°C
65°C
NheI
G\CTAGC
100
100
10
100
37°C
65°C
NheI-HF
G\CTAGC
100
25
10
100
37°C
80°C
* denotes star activity| NOTE ADD NdeI TOO
Multi-Ligation: Coming Full Circle
Screening Transformants: Colony PCR with VF2/VR
