Team:NEFU China/Labnote

From 2014.igem.org

Labnote

Abbreviations
B	smtB	Trans-acting regulator
OP	smtO-P	Smt operator/promoter region, a bi-directional promoter
A	smtA	Encoding MT-like protein that can sequester metal ions
C	amilCP	Encoding a chromoprotein that has a blue/purple color visible to the naked eyes. A registered part from iGEM11_Uppsala-Sweden
R	RFP	Red Fluorescent Protein. A registered part from iGEM11_Uppsala-Sweden
Flo	Flocculation gene	It can improve the flocculent activity of our host cells (Rosetta pLysS)
CP25		A constitutive strong promoter
CDS7		Encoding a short peptide that can bind to CdS and induce the formation of CdS nanocrystals
BCP		According to priority: smtB, smtO-P(omit here), amilCP
BRP		According to priority: smtB, smtO-P(omit here), RFP
OPA		According to priority: smtO-P, smtA
FCDS7		According to priority: flocculation gene, CP25(omit here), CDS7

BCP or BRP(smtB, smtO-P and amilCP/RFP )

The smt locus was successfully cloned from Synechococcus elongates PCC7942. Show sequence

Fig.1 PCR product of the smt locus(640bp); Marker (DL2000)

BCP/BRP

Molecular biology techniques: SOE(Splicing by overlap extension) PCR

A. Primary PCR reaction

Segment1-smtBOP

primers	F		5' CGCGGATCCCTAGCGACACTCTTGTAAGTGA 3'
	R		5' TTTAGCGATCACACTCATGACAGCAACTCCTTTGA 3'
PCR system (50ul)			parameters
			procedure	temperature	time
pfu		0.5ul	PreDenature	94 ℃	2 min
primer F		2ul	Denature	94 ℃	30 sec
primer R		2ul	Annealing	53 ℃	30 sec
smt locus(PCR product) diluted 100×		3ul	Extension	72 ℃	30 sec
dNTPs		8ul	Final Elongation	72 ℃	5 min
buffer		10ul	Final Hold	16 ℃	∞
H₂O		24.5ul	Cycle	30 cycles

Segment2-amilCP(BBa_K592009) or RFP(BBa_E1010)

primers	amilCP		F	5' GGAGTTGCTGTCATGAGTGTGATCGCTAAACAAATG 3'
			R	5' CCGGAATTCTTATTAGGCGACCACAGGTT 3'
	RFP		F	5' GGAGTTGCTGTCATGGCTTCCTCCGAAGACG 3'
			R	5' CCGGAATTCGCGATCTACACTAGCACTATCAG 3'
PCR system (50ul)				parameters
				procedure	temperature	time
pfu		0.5ul		PreDenature	94 ℃	2 min
primer F		2ul		Denature	94 ℃	30 sec
primer R		2ul		Annealing	53 ℃	30 sec
registered parts diluted 100×		3ul		Extension	72 ℃	1 min
dNTPs		8ul		Final Elongation	72 ℃	5 min
buffer		10ul		Final Hold	16 ℃	∞
H₂O		24.5ul		Cycle	30 cycles

Fig.2 PCR product of primary reaction: 1. Marker (DL2000); 2, 3. amilCP(669bp); 6,7. RFP(708bp); 4, 5, 8 and 9. BOP(469bp)

Gel Extraction with TIANgel Midi Purification Kit(TIANGEN BIOTECH) see protocol

B. Overlapping and elongation

PCR system (50ul)		parameters
PCR system (50ul)		procedure	temperature	time
pfu	0.5ul	PreDenature	94 ℃	2 min
primer F&R	0ul	Denature	94 ℃	30 sec
segment 1	31.5ul in total (mole number of segment 1 and 2=1:1)	Annealing	55 ℃	30 sec
segment 2		Extension	72 ℃	1 min
H₂O		Final Elongation	72 ℃	5 min
dNTPs	8ul	Final Hold	16 ℃	∞
buffer	10ul	Cycle	10 cycles

Fig.3 separation gel of step B 1. Marker (DL2000); 2. mixture containing BCP; 3. mixture containing BRP; the left arrow points at BCP; the right arrow points at BRP

Gel Extraction with TIANgel Midi Purification Kit(TIANGEN BIOTECH)

C. Second PCR reaction

primers	F		5' CGCGGATCCCTAGCGACACTCTTGTAAGTGA 3'
	R	amilCP	5' CCGGAATTCTTATTAGGCGACCACAGGTT 3'
		RFP	5' CCGGAATTCGCGATCTACACTAGCACTATCAG 3'
PCR system (50ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)	25ul		PreDenature	94 ℃	5 min
primer F	2ul		Denature	94 ℃	30 sec
primer R	2ul		Annealing	53 ℃	30 sec
purified B’s product diluted 100×	1ul		Extension	72 ℃	1.5 min
dNTPs	included in premix		Final Elongation	72 ℃	10 min
buffer			Final Hold	16 ℃	∞
H₂O	20ul		Cycle	30 cycles

Fig.4 PCR product of second reaction: 1. Marker; 2.BCP(1138bp); 3.BRP(1177bp); Marker (DL2000)

Gel Extraction with TIANgel Midi Purification Kit(TIANGEN BIOTECH) Ligation and transformation with pEASY-T5 Zero Cloning Kit from TransGen Biotech. See protocol

The sequencing result is consistent with our designation.

OPA(smtO-P and smtA)

primers	F		5' TTGGCGCGCGAGCCAATCACGGTTTGTCC 3'
	R		5' CCAATGCATTTAGCCGTGGCAGTTACAGC 3'
PCR system (50ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)		25ul	PreDenature	94 ℃	2 min
primer F		2ul	Denature	94 ℃	30 sec
primer R		2ul	Annealing	59 ℃	30 sec
smt locus(PCR product) diluted 100×		1ul	Extension	72 ℃	30 sec
dNTPs		included in premix	Final Elongation	72 ℃	5 min
buffer			Final Hold	16 ℃	∞
H₂O		20ul	Cycle	30 cycles

Fig.5 PCR product of OPA(271bp); Marker (DL2000)

Gel Extraction with TIANgel Midi Purification Kit(TIANGEN BIOTECH) Ligation and transformation with pEASY-T5 Zero Cloning Kit from TransGen Biotech. The sequencing result is consistent with our designation.

FC(Flocculation gene, CP25 and CDS7)show sequence

The flocculation gene was successfully cloned from Bacillussp. F2.

While CP25 and CDS7 and the backbone sequence adjacent to them was synthesized by BGI Tech.And they are inserted in pMV.

We also used SOE PCR to splice flocculation gene and the rest ones.

A. Primary PCR reaction

Segment1-flocculation gene

primers	F		5' GGAATTCCATATGATGAGTCTACTTGCTGTTTTGTTTT 3'
	R		5'AAGGGGTTATGCTAGTTACGAATTCGAGCTC 3'
PCR system (50ul)			parameters
			procedure	temperature	time
pfu		0.5ul	PreDenature	94 ℃	2 min
primer F		2ul	Denature	94 ℃	30 sec
primer R		2ul	Annealing	58 ℃	30 sec
Flocculation gene(PCR product) diluted 100×		3ul	Extension	72 ℃	1.5 min
dNTPs		8ul	Final Elongation	72 ℃	10 min
buffer		10ul	Final Hold	16 ℃	∞
H₂O		24.5ul	Cycle	30 cycles

Segment2-including CP25 and CDS7

primers	F		5' GAGCTCGAATTCGTAACTAGCATAACCCCTT 3'
	R		5' CCCAAGCTTTTATTAAATATCCGCATGTTCCG 3'
PCR system (50ul)			parameters
			procedure	temperature	time
pfu		0.5ul	PreDenature	94 ℃	2 min
primer F		2ul	Denature	94 ℃	30 sec
primer R		2ul	Annealing	58 ℃	30 sec
synthesized fragment(plasmid) diluted 100×		3ul	Extension	72 ℃	30 sec
dNTPs		8ul	Final Elongation	72 ℃	5 min
buffer		10ul	Final Hold	16 ℃	∞
H₂O		24.5ul	Cycle	30 cycles

Fig.6 PCR product of the flocculation gene(1038bp) (arrows); Marker (DL2000)

Fig.7 PCR product of segment2(containing CP25 and CDS7, 217bp in total); Marker (DL2000)

Gel Extraction with TIANgel Midi Purification Kit(TIANGEN BIOTECH)

B. Overlapping and elongation

PCR system (50ul)		parameters
PCR system (50ul)		procedure	temperature	time
pfu	0.5ul	PreDenature	94 ℃	2 min
primer F&R	0ul	Denature	94 ℃	30 sec
segment 1	31.5ul in total (mole number of segment 1 and 2=1:1)	Annealing	60 ℃	30 sec
segment 2		Extension	72 ℃	1.5 min
H₂O		Final Elongation	72 ℃	10 min
dNTPs	8ul	Final Hold	16 ℃	∞
buffer	10ul	Cycle	10 cycles

Fig.8 seperation gel of step B arrow points at FC; Marker (DL2000)

Gel Extraction with TIANgel Midi Purification Kit(TIANGEN BIOTECH)

C. Second PCR reaction

primers	F		5' GGAATTCCATATGATGAGTCTACTTGCTGTTTTGTTTT 3'
	R		5' CCCAAGCTTTTATTAAATATCCGCATGTTCCG 3'
PCR system (50ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)		25ul	PreDenature	94 ℃	5 min
primer F		2ul	Denature	94 ℃	30 sec
primer R		2ul	Annealing	58 ℃	30 sec
purified B’s product diluted 100×		1ul	Extension	72 ℃	1.5 min
dNTPs		included in premix	Final Elongation	72 ℃	10 min
buffer			Final Hold	16 ℃	∞
H₂O		20ul	Cycle	30 cycles

Fig.9 PCR product of second reaction: FC(1267bp); Marker (DL2000)

Gel Extraction with TIANgel Midi Purification Kit(TIANGEN BIOTECH)

Ligation and transformation with pEASY-T5 Zero Cloning Kit from TransGen Biotech. The sequencing result is consistent with our designation.

All designed fragments would be replicated by PCR when needed in plasmid construction.

Plasmid construction

1. pHY300PLK-BCP-OPA

Insert BCP

Miniprep (pHY300PLK without BCP and OPA; pEASY-T5 cloning vector with BCP or OPA) with TIANprep Mini Plasmid Kit.see protocol

Double digestion (NEB)

substrate		BamH I-HF	EcoR I-HF	Cutsmart Buffer	H₂O	total	temperature	time
pHY300PLK	30ul	3ul	3ul	10ul	54ul	100ul	37℃	16 h
PCR product	30ul	3ul	3ul	10ul	54ul	100ul	37℃	16 h

Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)

Solution I	-plasmid- & -BCP-	total	temperature	time
5ul	5ul in total (see note)	10ul	16℃	30 min
Note:-plasmid-:-BCP-(mole number)=1:2~1:8

Transformation see protocol

Colony PCR

primers	F		5' CGCGGATCCCTAGCGACACTCTTGTAAGTGA 3'
	R		5' CCGGAATTCTTATTAGGCGACCACAGGTT 3'
PCR system (20ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)		10ul	PreDenature	94 ℃	5 min
primer F		0.8ul	Denature	94 ℃	30 sec
primer R		0.8ul	Annealing	53 ℃	30 sec
template: pick a single colony and dip in H₂O		8.4ul	Extension	72 ℃	1.5 min
			Final Elongation	72 ℃	10 min
dNTPs		included in premix	Final Hold	16 ℃	∞
buffer			Cycle	30 cycles
Note: The template was heat denatured at 100℃in metal bath, then freeze on ice for 2 min before running through parameters on the right.

Fig.10 1. Marker (DL2000); 2-5. each for one single colony(all positive); 6. positive control; 7. H₂O control

Miniprep (pHY300PLK with maybe BCP) with TIANprep Mini Plasmid Kit.as before Double digestion (NEB) for detection

Plasmids with H₂O	BamH I-HF	EcoR I-HF	Cutsmart Buffer	total	temperature	time
16.8ul	0.6ul	0.6ul	2ul	20ul	37℃	16 h

Fig.11 digestion detection: 1. digestion product of positive clone plasmid DNA; 2. linearized vector; 3. BCP; 4. Marker (DL15000)

The sequencing result is consistent with our designation.

Insert OPA

Miniprep (pHY300PLK with only BCP; pEASY-T5 cloning vector with OPA) with TIANprep Mini Plasmid Kit.

Two-step enzyme digestion(NEB)(total 100ul)

substrate		Nsi I	Buffer 3.1	H₂O	temperature	time	BssH II	temperature	time
pHY300PLK-BCP	30ul	3ul	10ul	54ul	37℃	3 h	3ul	50℃	3 h
PCR product	30ul	3ul	10ul	54ul	37℃	3 h	3ul	50℃	3 h

Ligation (TaKaRa DNA Ligation Kit Ver.2.1 as before)

Solution I	-plasmid- & -OPA-	total	temperature	time
5ul	5ul in total (see note)	10ul	16℃	30 min
Note:-plasmid-:-OPA-(mole number)=1:2~1:8

Transformation

Colony PCR

primers	F		5' TTGGCGCGCGAGCCAATCACGGTTTGTCC 3'
	R		5' CCAATGCATTTAGCCGTGGCAGTTACAGC 3'
PCR system (20ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)		10ul	PreDenature	94 ℃	5 min
primer F		0.8ul	Denature	94 ℃	30 sec
primer R		0.8ul	Annealing	59 ℃	30 sec
template: pick a single colony and dip in H₂O		8.4ul	Extension	72 ℃	30 sec
			Final Elongation	72 ℃	5 min
dNTPs		included in premix	Final Hold	16 ℃	∞
buffer			Cycle	30 cycles
Note: The template was heat denatured at 100℃ in metal bath, then freeze on ice for 2 min before running through parameters on the right.

Fig.12 1-4. each for one single colony(all positive); 5.positive control; 6. H₂O control; 7. Marker Miniprep (pHY300PLK with BCP and maybe OPA) with TIANprep Mini Plasmid Kit.

Two-step enzyme digestion(NEB) for detection

Plasmids with H₂O	Nsi I	Buffer 3.1	temperature	time	BssH II	temperature	time	total
16.8ul	0.6ul	2ul	37℃	3 h	0.6ul	50℃	3 h	20ul

Fig.13 digestion detection: 1. Marker (DL5000); 2. digestion product of positive clone plasmid DNA; 3. linearized vector; 4. OPA

The sequencing result is consistent with our designation.

2. pHY300PLK-BRP-OPA

Insert BRP

Miniprep (pHY300PLK without BRP and OPA; pEASY-T5 cloning vector with BRP) with TIANprep Mini Plasmid Kit.

Double digestion (NEB)

substrate		BamH I-HF	EcoR I-HF	Cutsmart Buffer	H₂O	total	temperature	time
pHY300PLK	30ul	3ul	3ul	10ul	54ul	100ul	37℃	16 h
PCR product	30ul	3ul	3ul	10ul	54ul	100ul	37℃	16 h

Ligation (TaKaRa DNA Ligation Kit Ver.2.1 as before)

Solution I	-plasmid- & -BRP-	total	temperature	time
5ul	5ul in total (see note)	10ul	16℃	30 min
Note:-plasmid-:-BRP-(mole number)=1:2~1:8

Transformation

Colony PCR

primers	F		5' CGCGGATCCCTAGCGACACTCTTGTAAGTGA 3'
	R		5' CCGGAATTCGCGATCTACACTAGCACTATCAG 3'
PCR system (20ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)		10ul	PreDenature	94 ℃	5 min
primer F		0.8ul	Denature	94 ℃	30 sec
primer R		0.8ul	Annealing	53 ℃	30 sec
template: pick a single colony and dip in H₂O		8.4ul	Extension	72 ℃	1.5 min
			Final Elongation	72 ℃	10 min
dNTPs		included in premix	Final Hold	16 ℃	∞
buffer			Cycle	30 cycles
Note: The template was heat denatured at 100℃ in metal bath, then freeze on ice for 2 min before running through parameters on the right.

Fig.14 1. Marker (DL2000); 2-5. each for one single colony(all positive); 6.H₂O control; 7. positive control

Miniprep (pHY300PLK with maybe BRP) with TIANprep Mini Plasmid Kit.

Double digestion (NEB) for detection

Plasmids with H2O	BamH I-HF	EcoR I-HF	Cutsmart Buffer	total	temperature	time
16.8ul	0.6ul	0.6ul	2ul	20ul	37℃	16 h

Fig.15 digestion detection: 1. Marker (DL15000); 3. digestion product of positive clone plasmid DNA

The sequencing result is consistent with our designation.

Insert OPA

Miniprep (pHY300PLK with only BRP; pEASY-T5 cloning vector with OPA) with TIANprep Mini Plasmid Kit.

Two-step enzyme digestion(NEB)(total 100ul)

substrate		Nsi I	Buffer 3.1	H2O	temperature	time	BssH II	temperature	time
pHY300PLK-BRP	30ul	3ul	10ul	54ul	37℃	3 h	3ul	50℃	3 h
PCR product	30ul	3ul	10ul	54ul	37℃	3 h	3ul	50℃	3 h

Ligation (TaKaRa DNA Ligation Kit Ver.2.1 as before)

Solution I	-plasmid- & -OPA-	total	temperature	time
5ul	5ul in total (see note)	10ul	16℃	30 min
Note:-plasmid-:-OPA-(mole number)=1:2~1:8

Transformation

Colony PCR

primers	F		5' TTGGCGCGCGAGCCAATCACGGTTTGTCC 3'
	R		5' CCAATGCATTTAGCCGTGGCAGTTACAGC 3'
PCR system (20ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)		10ul	PreDenature	94 ℃	5 min
primer F		0.8ul	Denature	94 ℃	30 sec
primer R		0.8ul	Annealing	59 ℃	30 sec
template: pick a single colony and dip in H2O		8.4ul	Extension	72 ℃	30 sec
			Final Elongation	72 ℃	5 min
dNTPs		included in premix	Final Hold	16 ℃	∞
buffer			Cycle	30 cycles
Note: The template was heat denatured at 100℃ in metal bath, then freeze on ice for 2 min before running through parameters on the right.

Fig.16 1. Marker (DL2000); 2-15. each for one single colony(8 positive); 16. H₂O control; 17. positive control

Miniprep (pHY300PLK with BRP and maybe OPA) with TIANprep Mini Plasmid Kit.

Two-step enzyme digestion(NEB) for detection

Plasmids with H₂O	Nsi I	Buffer 3.1	temperature	time	BssH II	temperature	time	total
16.8ul	0.6ul	2ul	37℃	3 h	0.6ul	50℃	3 h	20ul

Fig.17 digestion detection: 1. Marker (DL5000); 2. digestion product of positive clone plasmid DNA; 3. linearized vector; 4. OPA

The sequencing result is consistent with our designation.

3. PACYC184-BCP-OPA

Insert OPA

Miniprep (pACYC184 without BCP and OPA; pEASY-T5 cloning vector with OPA) with TIANprep Mini Plasmid Kit.

Single enzyme digestion (TaKaRa)

substrate		Xba I	0.1% BSA	10×M Buffer	H₂O	total	temperature	time
pACYC184	30ul	3ul	10ul	10ul	47ul	100ul	37℃	16 h
PCR product	30ul	3ul	10ul	10ul	47ul	100ul	37℃	16 h

Ligation (TaKaRa DNA Ligation Kit Ver.2.1 as before)

Solution I	-plasmid- & -OPA-	total	temperature	time
5ul	5ul in total (see note)	10ul	16℃	30 min
Note:-plasmid-:-OPA-(mole number)=1:2~1:8

Transformation

Colony PCR

primers	F		5' TGCTCTAGAGAGCCAATCACGGTTTGTCC 3'
	R		5' TGCTCTAGATTAGCCGTGGCAGTTACAGC 3'
PCR system (20ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)		10ul	PreDenature	94 ℃	5 min
primer F		0.8ul	Denature	94 ℃	30 sec
primer R		0.8ul	Annealing	59 ℃	30 sec
template: pick a single colony and dip in H₂O		8.4ul	Extension	72 ℃	30 sec
			Final Elongation	72 ℃	5 min
dNTPs		included in premix	Final Hold	16 ℃	∞
buffer			Cycle	30 cycles
Note: The template was heat denatured at 100℃in metal bath, then freeze on ice for 2 min before running through parameters on the right.

Fig.18 1-6. each for one single colony(1,4,6 negative; 2,3,5 positive); 7.positive control; 8. H₂O control; 9. Marker

Miniprep (pACYC184 with maybe OPA) with TIANprep Mini Plasmid Kit.

Single enzyme digestion (TaKaRa) for detection

Plasmids with H₂O	Xba II	0.1% BSA	10×M Buffer	total	temperature	time
15.75ul	0.25ul	2ul	2ul	20ul	37℃	16 h

Fig.19 digestion detection: 1. 15000bp marker 2-5. digestion product of positive clone plasmid DNA; 6. 2000bp marker; the upper arrow points at linearization vector; the lower arrow points at OPA

The sequencing result is consistent with our designation.

Insert BCP

Miniprep (pACYC184 with only OPA; pEASY-T5 cloning vector with BCP) with TIANprep Mini Plasmid Kit. as before

Single enzyme digestion (TaKaRa)

substrate		Sac II	0.1% BSA	10×T Buffer	H₂O	total	temperature	time
pACYC184-OPA	30ul	3ul	10ul	10ul	47ul	100ul	37℃	16 h
PCR product	30ul	3ul	10ul	10ul	47ul	100ul	37℃	16 h

Ligation (TaKaRa DNA Ligation Kit Ver.2.1 as before)

Solution I	-plasmid- & -BCP-	total	temperature	time
5ul	5ul in total (see note)	10ul	16℃	30 min
Note:-plasmid-:-BCP-(mole number)=1:2~1:8

Transformation

Colony PCR

primers	F		5' TCCCCGCGGCTAGCGACACTCTTGTAAGTGA 3'
	R		5' TCCCCGCGGTTATTAGGCGACCACAGGTT 3'
PCR system (20ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)		10ul	PreDenature	94 ℃	5 min
primer F		0.8ul	Denature	94 ℃	30 sec
primer R		0.8ul	Annealing	58 ℃	30 sec
template: pick a single colony and dip in H₂O		8.4ul	Extension	72 ℃	1.5 min
			Final Elongation	72 ℃	10 min
dNTPs		included in premix	Final Hold	16 ℃	∞
buffer			Cycle	30 cycles
Note: The template was heat denatured at 100℃in metal bath, then freeze on ice for 2 min before running through parameters on the right.

Fig.20 1. Marker (DL2000); 2. H₂O control; 3-13. each for one single colony (3-11 negative; 12, 13 positive); 14. positive control

Miniprep (pACYC184 with OPA and maybe BCP) with TIANprep Mini Plasmid Kit. as before

Single digestion(TaKaRa) for detection

Plasmids with H₂O	Sac II	0.1% BSA	10×T Buffer	total	temperature	time
15.75ul	0.25ul	2ul	2ul	20ul	37℃	16 h

Fig.21 digestion detection: 1. Marker (DL5000) 2. digestion product of positive clone plasmid DNA; 3. linearized vector; 4. BCP

The sequencing result is consistent with our designation.

4.PACYC184-BRP-OPA

Insert BRP

Miniprep (pACYC184 with only OPA; pEASY-T5 cloning vector with BRP) with TIANprep

Mini Plasmid Kit.

Single enzyme digestion (TaKaRa)

substrate		Sac II	0.1% BSA	10×T Buffer	H₂O	total	temperature	time
pACYC184-OPA	30ul	3ul	10ul	10ul	47ul	100ul	37℃	16 h
PCR product	30ul	3ul	10ul	10ul	47ul	100ul	37℃	16 h

Ligation (TaKaRa DNA Ligation Kit Ver.2.1 as before)

Solution I	-plasmid- & -BRP-	total	temperature	time
5ul	5ul in total (see note)	10ul	16℃	30 min
Note:-plasmid-:-BCP-(mole number)=1:2~1:8

Transformation

Colony PCR

primers	F		5' TCCCCGCGGCTAGCGACACTCTTGTAAGTGA 3'
	R		5' TCCCCGCGGGCGATCTACACTAGCACTATCAG 3'
PCR system (20ul)			parameters
			procedure	temperature	time
premix taq(TaKaRa)		10ul	PreDenature	94 ℃	5 min
primer F		0.8ul	Denature	94 ℃	30 sec
primer R		0.8ul	Annealing	53 ℃	30 sec
template: pick a single colony and dip in H₂O		8.4ul	Extension	72 ℃	1.5 min
			Final Elongation	72 ℃	10 min
dNTPs		included in premix	Final Hold	16 ℃	∞
buffer			Cycle	30 cycles
Note: The template was heat denatured at 100℃in metal bath, then freeze on ice for 2 min before running through parameters on the right.

Fig.22 1. Marker (DL2000); 2-4. each for one single colony (4 positive); 5. positve control; 6. H₂O control

Miniprep (pACYC184 with OPA and maybe BRP) with TIANprep Mini Plasmid Kit.

Single digestion(TaKaRa) for detection

Plasmids with H₂O	Sac II	0.1% BSA	10×T Buffer	total	temperature	time
15.75ul	0.25ul	2ul	2ul	20ul	37℃	16 h

Fig.23 digestion detection: the upper arrow points at linearization vector; the lower arrow points at BRP The sequencing result is consistent with our designation. Marker (DL2000)

5.pET-28b(+)-Flo-CDS7

Miniprep (pET-28b(+) without FC; pEASY-T5 cloning vector with FC) with TIANprep Mini Plasmid Kit.

Double digestion (NEB)

substrate		Hind III-HF	Nde I	Cutsmart Buffer	H₂O	total	temperature	time
pET-28b(+)	30ul	3ul	3ul	10ul	54ul	100ul	37℃	16 h
PCR product	30ul	3ul	3ul	10ul	54ul	100ul	37℃	16 h

Ligation (TaKaRa DNA Ligation Kit Ver.2.1 as before)

Solution I	-plasmid- & -FC-	total	temperature	time
5ul	5ul in total (see note)	10ul	16℃	30 min
Note:-plasmid-:-FC-(mole number)=1:2~1:8

Transformation

Colony PCR

primers	F	5' GGAATTCCATATGATGAGTCTACTTGCTGTTTTGTTTT 3'
primers	R	5' CCCAAGCTTTTATTAAATATCCGCATGTTCCG 3'
PCR system (20ul)		parameters
PCR system (20ul)		procedure	temperature	time
premix taq(TaKaRa)	10ul	PreDenature	94 ℃	5 min
primer F	0.8ul	Denature	94 ℃	30 sec
primer R	0.8ul	Annealing	58 ℃	30 sec
template: pick a single colony and dip in H₂O	8.4ul	Extension	72 ℃	1.5 min
template: pick a single colony and dip in H₂O	8.4ul	Final Elongation	72 ℃	10 min
dNTPs	included in premix	Final Hold	16 ℃	∞
buffer	included in premix	Cycle	30 cycles
Note: The template was heat denatured at 100℃in metal bath, then freeze on ice for 2 min before running through parameters on the right.

Fig.23 1-5. each for one single colony(all positive); 6.positive control; 7. H₂O control; 8. Marker (DL2000)

Miniprep (pET-28b(+) with maybe FC) with TIANprep Mini Plasmid Kit. Double digestion (NEB) for detection

Plasmids with H₂O	Hind III-HF	Nde I	Cutsmart Buffer	total	temperature	time
16.8ul	0.6ul	0.6ul	2ul	20ul	37℃	16 h

Fig.25 digestion detection: 1. 2000bp marker; 3. negative result for worse digestion; 4-6. pET-14b(++) vector; 7. 15000bp marker

The sequencing result is consistent with our designation.

Protein characterization

Protein characterization by SDS-PAGE. see protocol

Fig.26 result of SDS-PAGE 1,2,3. Analysis of amilCP; 5,6,7. Annalysis of RFP; 1&5. not induced; 2&6. Proteins from IPTG induced(0.8mM) E.coli.; 3&7. Control without reporter gene; 4. Protein marker