Team:IISc-Bangalore/Assembly

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1. Transformations
2. Plasmid Isolation
3. PCRs
4. Restriction digests
5. Ligations
6. Screening

BioBrick Transformations

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.

Transformations for T7 expression system

Figure 1: BBa_K525998 and BBa_K731721

Transformations for sfGFP-SpyCatcher

Figure 2: BBa_K1650037 and BBa_K1321337

Transformations for mCherry

Figure 3 : BBa_J18932

Plasmid Isolation

Minipreps were performed using three colonies on each transformation plate to confirm the presence of the plasmid. One positive transformant for each BioBrick was used to make a glycerol stock.

PCRs

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
	Amplifies the CmR gene, includes NcoI site
Reverse primer	gaattcAAGCTTtttctcctctttccctatagtgagtcg
	Adds HindIII site downstream
Amplicon size	886 bp

PCR 1

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

PCR 2

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 3

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

PCR 4

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 5

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

PCR 6

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

Double-digest PCR1 product with NcoI and HindIII (D1)

Double-digest PCR2 product with NheI and NcoI (D2)

Restriction digests for sfGFP-SpyCatcher

Double-digest PCR3 product with HindIII and BamHI (D3)

Double-digest PCR4 product with BamHI and NheI (D4)

Restriction digest for 6xHis-mCherry

Double-digest PCR6 product with HindIII and NheI (D6)

Restriction digests for mCherry-SpyTag

Double-digest Oligo 1 with HindIII and NdeI (DO1)

Double-digest PCR7 product with NdeI and BamHI (D7)

Double-digest Oligo 2 with BamHI and NheI (DO2)

Gel-purification of our digested products

Figure 4: Digested PCR products (D1, D2, D3, D4, D6, DO1, DO2)

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

Multi-Ligation

Ligating sfGFP-SpyCatcher

Plasmid map of BBa_K2319000 (sfGFP-SpyCatcher)

Ligating 6xHis-mCherry

Plasmid map of BBa_K2319009 (6xHis-mCherry)

Screening Transformants

We screened 30 transformants from each plate using colony PCR with primers VF2 and VR, which we standardized using Taq polymerase (annealing temperature of 56°C).

Figure 6 : Colony PCR of sfGFP-SpyCatcher colonies (B1-B21) and 6xHis-mCherry colonies (D1-D21)