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Foundation
Detecting
Recycling
Future application

Lab note

Foundation

Obtaining the target genes

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 eye. 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

10ul

54ul

100ul

37℃

16 h

PCR product

30ul

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.

P10 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

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-:-BCP-(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

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

47ul

100ul

37℃

16 h

PCR product

30ul

3ul

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

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

47ul

100ul

37℃

16 h

PCR product

30ul

3ul

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

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

47ul

100ul

37℃

16 h

PCR product

30ul

3ul

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

10ul

54ul

100ul

37℃

16 h

PCR product

30ul

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-:-BCP-(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

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

@@ Line 2,477: / Line 2,477: @@
 <p class="text-center"><img src="https://static.igem.org/mediawiki/2014/d/de/NEFU_China_labnote_fig36.png" class="img-thumbnail"></p>
 <p>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) <em>E.coli</em>.;  3&;7. Control without reporter gene; 4. Protein marker</p>
-</div>
-<h2 class="subtitle"><a name="Detecting">Detecting</a></h2>
-<h3>Introductions, Results and Discussions. </h3>
-<p>As we know, some metal ions are toxic to  bacterial cells at all concentrations, therefore detoxification and resistance  systems that employ a variety of mechanisms to rid the cell of these  potentially lethal toxins have evolved employ. In most cases, the expression of  such resistance systems is controlled at the level of transcription by metal  sensor proteins that sense specific metal ions via their direct coordination  [1]. The most famous resistance system is smtB-OP-smtA device which defend  metal ions including Zn<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup>, Pb<sup>2+</sup>  and Cd<sup>2+</sup> [1-3]. Briefly, the protein SmtB generally function as a  repressor in the absence of metal ions and become activators upon metal  binding, by driving a metal-induced DNA conformational switch that converts a  sub-optimal promoter (OP-promoter) into a potent one, than activate the  expression of SmtA. In previous reports, the smtB-OP-smtA element which located  in <em>Staphylococcus </em>may alsofunctions as a metal ions (Zn<sup>2+</sup>,  Co<sup>2+</sup> and Cd<sup>2+</sup>) responsive repressor in <em>E.coli </em>[4-6]. In this case, we focused  on detecting the content of Cd<sup>2+</sup>, whether this well-known element  could be employed in our project still remained in uncertain. Therefore, we  took steps of experiments and yielded several interesting data below.</p>
-<p>Firstly, we obtained the smtB-OP-smtA device  from <em>Staphylococcus </em>Genome and tested  the growth of bacterium containing this element in Cd<sup>2+</sup> present  condition (Figure 1). The result displayed a significant growth advantage with  smtB-OP-smtA positive <em>E.coli</em>,  indicating the device worked in <em>E.coli.</em> Thus, we wanted to use pigments as reporters in our designed genetic construct  which can be recognizable by the naked eye. According to the previous work, we  have chosen an identified pigment in the Registry: the biobrick of RFP (<a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>) was  used as reporter gene in our metal detection device. After exchanging the  biobrick part with SmtA in smtB-OP-smtA device, the pigment gene was under  control of metal-induced promoter (smtB-OP). </p>
-<p>It was important to us that the similar  device which contained <em>Lac-Z</em> as  reporter gene had ability to sense Zn<sup>2+</sup> and Cd<sup>2+</sup> in <em>E. coli </em>[7, 8]. Thus, there are four  potential metal binding sites on protein SmtB as known as α3, α3N, α5 and α5C  [9], different from Zn<sup>2+</sup> resistance mechanisms, Cd<sup>2+</sup> binds  SmtB at the site α3N [10]. To gain deep insight regarding the detailed  comparison of smtB-OP-reporter affected by these two metal ions in <em>E. coli</em>, we adopted <em>Rosetta-plysS </em>and detected the expression of reporter gene by  measuring OD value (OD450 for RFP). As shown in Figure.2 A-B, pigment gene can  be both activated by Zn<sup>2+</sup> and Cd<sup>2+</sup>, the up-regulation  from Cd<sup>2+</sup> was with more sensitive with shorter period and lower consistency  than Zn<sup>2+</sup>. With higher density of bacteria(1x107 cells ml-1), the pigment expression  induced by Cd<sup>2+</sup> in 2 hours could be easily distinguished by our  naked eye (Fig.3C). Taken together, these data provided basic but necessary  information that we could take this recombinant device to report the content of  Cd<sup>2+</sup> with certain degree (2-20μM in 2 hours) not affected by Zn<sup>2+</sup>.</p>
-<p>Based on the conclusions above, we focused  on the details of our detecting device resulting from Cd<sup>2+</sup> only with  certain degree. For quality control, we detected the standard substances by Optima 8300  ICP-OES Spectrometer the credible interval of this method  displayed to land at 50-500μM (Table.1). For the pigment expression differences  with Cd<sup>2+</sup> at 10-50μM in 2 hours are not obvious, our team has  coupled a more impressible biobrick part amilCP (<a href="http://parts.igem.org/Part:BBa_K592009:Experience?title=Part:BBa_K592009:Experience">BBa_K592009</a>)  with the smtB-OP element. Results from Fig 3 A-B indicated that, the diversity  of amilCP expression (measured by OD600) was able to represent the continuous concentrations of Cd<sup>2+</sup> precisely within 0-50μM in one hour. The  visualization is another potential advantage of this system, we raised the dose  of the engineering bacteria and extended the sense time. Finally, blue color  produced by our device was strong enough to be observed by the human naked eye,  however, similar lightness among 100-500μM Cd<sup>2+</sup>-treatment samples  were shown which matched the our previous data (Fig.3 C). </p>
-<p>In summary, the <em>Rosetta-plysS</em> strain made our system convenient to be applied, the  classical smtB-OP-smtA device from <em>Staphylococcus</em> supported our system a responsive Cd<sup>2+</sup> inducible-promoter, and the  viewable pigment gene provided our system a reliable and macroscopic  observation. After theoretical prediction, genetic engineering, experimental  optimization and reasonable model analysis (deeply discussed in Modeling&nbsp;), our detecting system residing in the engineering  bacteria was able to sensitively represent the content of Cd<sup>2+</sup>  (1-100μM) in 1-2 hours. </p>
-<p>Although the inducible operator in our case  might also response to other metal ions including Zn<sup>2+</sup>, our date at least did point out that Cd<sup>2+</sup> has acuter stimulus to the  pigment gene than Zn<sup>2+</sup> which was confirmed both from  experimental data and model analysis. We achieved to our goal at a certain  degree. Finally, our system is easier to utilize and exhibits improved  flexibility as a tool to detect Cd<sup>2+</sup> which belongs to the toxical  heavy metal ions.</p>
-<h3>Protocols</h3>
-<h3>Plasmid design and construction</h3>
-<p>The vector pHY300 PLK and vector PACYC184  were constructed based on the smtBCP/smtBRP backbone (see obtaining  the target genes&nbsp;) using standard cloning techniques. The sensor vector  included an improved metal ions inducible promoter (smtO-P), a pigment gene  (RFP or amilCP) marker. Detailed vector maps, sequence information and cloning  protocols has been described above in Foundation&nbsp;.</p>
-<h3>Establishment of the detecting clonal cell lines</h3>
-<p>DNA transfection was performed as standard molecular  cloning techniques, the <em>Rosetta-plysS</em> strain (kindly supported by our instructors) was cultured with LB medium. All  the clonal cells were validated by PCR (see Foundation&nbsp;). </p>
-<h3>OD value measurement and analysis</h3>
-<p>All measurement procedures were performed  using an Eppendorf BioSpectrometer basic instrument. Briefly, the cells were  centrifuged at 3,000g for 5 min. Then, resulting pellet was twice-washed with ddH<sub>2</sub>O  and resuspended at a density of 105-6 cells ml-1.  After that, the cells were treated with metal ions using a shaker culture box  with 200rpm at 37。C. All the data were analyzed using Sigmaplot software.  For each experiment, triplicate cultures were measured.</p>
-<h3>Quality control of the Cd<sup>2+</sup> standard samples by emission spectrometric detection</h3>
-<p>Briefly, the CdCl<sup>2</sup> (Sigma-Aldrich) was taken for preparing the standard samples.  183.32g (1M) was weighed by analytical balance and diluted at a  concentration of 1M with ddddH<sub>2</sub>O. After 10-times-dilution step by  step, we got several standard samples of Cd<sup>2+</sup> at  different concentrations (1μM, 10μM, 50μM, 100μM and 500μM). Then, these  samples were measured via Optima 8300 ICP-OES  Spectrometer (this section was performed  in HIT). Detailed condition for the detection was shown in table.2. For  each experiment, 8 separate repeats were measured. </p>
-<h3>Figure and Table</h3>
-<h4>Figure 1</h4>
-<p class="text-center"><img src="https://static.igem.org/mediawiki/2014/0/0d/NEFU_CHINA_detecting_fig1.png" class="img-thumbnail"></p>
-<p>Fig.1. Growth of cells containing  smtB-OP-smtA element in LB medium supplemented with 2μM Cd<sup>2+</sup>.  Cells were inoculated at a density of 1x106 cells ml-1,  and growth was monitored by measuring the OD540 value. Data points  represent the mean values from three separate cultures with SD.</p>
-<p class="text-center"><img src="https://static.igem.org/mediawiki/2014/f/f4/NEFU_CHINA_detecting_fig2.png" class="img-thumbnail"></p>
-<p>Fig.2. A. Metal-induced expression of the  pigments (RFP), <em>Rosetta-plysS</em> (1x10<sup>6</sup>  cells ml<sup>-1</sup>) carrying the smtB-OP-RFP element were grown with  Cd<sup>2+</sup> and Zn<sup>2+</sup> (1-50μM) supplement for 2h immediately  before assay and expression was monitored by measuring the OD<sub>450</sub> value;  B. Metal-induced expression of the pigments (RFP). <em>Rosetta-plysS</em> carrying the smtB-OP-RFP element were grown with Cd<sup>2+</sup>  and Zn<sup>2+</sup> (2μM) supplement for 1-12h immediately before measurement;  C. Cadmium-induced expression of the pigment (RFP) at different concentrations. <em>Rosetta-plysS</em> (1x107 cells  ml-1) carrying the smtB-OP-RFP element were grown with Cd<sup>2+</sup> (1-20μM)  supplement for 2h. The data points shown in A and B represent the means of  three separate assays with SD.</p>
-<h4>Table 1</h4>
-<table align="center" class="MsoNormalTable" border="0" cellspacing="0" cellpadding="0" style="background:#D9D9D9;border-collapse:collapse;">
-  <tr style="height:27.65pt;">
-    <td width="140" style="width:104.65pt;border-top:none;border-left:none;border-bottom:dashed windowtext 1.0pt;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:27.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">Expected concentration</span><span style="font-size:7.5pt; "> (μM)</span></p></td>
-    <td width="76" style="width:2.0cm;border-top:none;border-left:none;border-bottom:dashed windowtext 1.0pt;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:27.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">1</span></p></td>
-    <td width="76" style="width:2.0cm;border-top:none;border-left:none;border-bottom:dashed windowtext 1.0pt;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:27.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">10</span></p></td>
-    <td width="76" style="width:2.0cm;border-top:none;border-left:none;border-bottom:dashed windowtext 1.0pt;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:27.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">50</span></p></td>
-    <td width="85" style="width:63.8pt;border-top:none;border-left:none;border-bottom:dashed windowtext 1.0pt;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:27.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">100</span></p></td>
-    <td width="85" style="width:63.75pt;border:none;border-bottom:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:27.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">500</span></p></td>
-  </tr>
-  <tr style="height:20.4pt;">
-    <td width="140" style="width:104.65pt;border:none;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">Reported measured</span><span style="font-size:7.5pt; ">     (μM)</span></p></td>
-    <td width="76" style="width:2.0cm;border:none;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">0.85<u>+</u>0.19</span></p></td>
-    <td width="76" style="width:2.0cm;border:none;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">9.68<u>+</u>1.28</span></p></td>
-    <td width="76" style="width:2.0cm;border:none;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">55.57<u>+</u>5.66</span></p></td>
-    <td width="85" style="width:63.8pt;border:none;border-right:dashed windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">107.52<u>+</u>11.98</span></p></td>
-    <td width="85" style="width:63.75pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-size:7.5pt; ">528.07<u>+</u>33.15</span></p></td>
-  </tr>
-</table>
-<h4>Figure 3</h4>
-<p class="text-center"><img src="https://static.igem.org/mediawiki/2014/0/0b/NEFU_CHINA_detecting_fig3.png" class="img-thumbnail"></p>
-<p>Fig.3. A. Cadmium-induced expression of the  pigment (amilCP) at constant concentration. <em>Rosetta-plysS</em> (1x106 cells ml-1) carrying the smtB-OP-amilCP element  were grown with Cd<sup>2+</sup> (1μM) supplement for 1-2h immediately before  assay and the expression was monitored by measuring the OD600  value; B. Cadmium-induced expression of the pigment (amilCP) with  different concentrations. <em>Rosetta-plysS</em> carrying the smtB-OP-amilCP device were grown with Cd<sup>2+</sup> (1-100μM)  supplement for 1h immediately before assay；C. Cadmium-induced expression of the pigment (amilCP) with different  concentrations. <em>Rosetta-plysS</em> (1x107  cells ml-1) carrying the smtB-OP-amilCP element were grown  with Cd<sup>2+</sup> (10, 20, 50, 100, 200 and 500 μM) supplement for 2h. The  data points shown in A and B represent the means of three separate values with  SD.</p>
-<h4>Table 2</h4>
-<p class="text-center">
-<table align="center" border="1" cellspacing="0" cellpadding="0">
-  <tr>
-    <td width="284" valign="top"><p>RF    power</p></td>
-    <td width="148" valign="top"><p>1.3kw</p></td>
-  </tr>
-  <tr>
-    <td width="284" valign="top"><p>flow rate of plasma </p></td>
-    <td width="148" valign="top"><p>14L/min</p></td>
-  </tr>
-  <tr>
-    <td width="284" valign="top"><p>flow rate of assistant gas </p></td>
-    <td width="148" valign="top"><p>0.2    L/min</p></td>
-  </tr>
-  <tr>
-    <td width="284" valign="top"><p>flow rate of atomization </p></td>
-    <td width="148" valign="top"><p>0.55    L/min</p></td>
-  </tr>
-  <tr>
-    <td width="284" valign="top"><p>the flow velocity of peristaltic pump </p></td>
-    <td width="148" valign="top"><p>1.5    L/min</p></td>
-  </tr>
-  <tr>
-    <td width="284" valign="top"><p>the time of washing the sample</p></td>
-    <td width="148" valign="top"><p>40 sec</p></td>
-  </tr>
-  <tr>
-    <td width="284" valign="top"><p>integration time</p></td>
-    <td width="148" valign="top"><p>5 sec</p></td>
-  </tr>
-  <tr>
-    <td width="284" valign="top"><p>method</p></td>
-    <td width="148" valign="top"><p>axial observation </p></td>
-  </tr>
-  <tr>
-    <td width="284" valign="top"><p>the wavelength of Cd<sup>2+</sup></p></td>
-    <td width="148" valign="top"><p>228.802    nm</p></td>
-  </tr>
-</table>
-</p>
-<h4>Reference</h4>
-<ol class="refrence">
-<li>Busenlehner,  L.S., M.A. Pennella, and D.P. Giedroc, <em>The  SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural  insights into prokaryotic metal resistance.</em> FEMS Microbiol Rev, 2003. <strong>27</strong>(2-3): p. 131-43.</li>
-<li>Robinson,  N.J., S.K. Whitehall, and J.S. Cavet, <em>Microbial  metallothioneins.</em> Adv Microb Physiol, 2001. <strong>44</strong>: p. 183-213.</li>
-<li>Erbe,  J.L., K.B. Taylor, and L.M. Hall, <em>Metalloregulation  of the cyanobacterial smt locus: identification of SmtB binding sites and  direct interaction with metals.</em> Nucleic Acids Res, 1995. <strong>23</strong>(13): p. 2472-8.</li>
-<li>VanZile,  M.L., X. Chen, and D.P. Giedroc, <em>Allosteric  negative regulation of smt O/P binding of the zinc sensor, SmtB, by metal ions:  a coupled equilibrium analysis.</em> Biochemistry, 2002. <strong>41</strong>(31): p. 9776-86.</li>
-<li>Morby,  A.P., et al., <em>SmtB is a metal-dependent  repressor of the cyanobacterial metallothionein gene smtA: identification of a  Zn inhibited DNA-protein complex.</em> Nucleic Acids Res, 1993. <strong>21</strong>(4): p. 921-5.</li>
-<li>Huckle,  J.W., et al., <em>Isolation of a prokaryotic  metallothionein locus and analysis of transcriptional control by trace metal  ions.</em> Mol Microbiol, 1993. <strong>7</strong>(2):  p. 177-87.</li>
-<li>VanZile,  M.L., X. Chen, and D.P. Giedroc, <em>Structural  characterization of distinct alpha3N and alpha5 metal sites in the  cyanobacterial zinc sensor SmtB.</em> Biochemistry, 2002. <strong>41</strong>(31): p. 9765-75.</li>
-<li>Kar,  S.R., et al., <em>The cyanobacterial  repressor SmtB is predominantly a dimer and binds two Zn<sup>2+</sup> ions per subunit.</em> Biochemistry, 1997. <strong>36</strong>(49): p.  15343-8.</li>
-<li>Cook,  W.J., et al., <em>Crystal structure of the  cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory  proteins.</em> J Mol Biol, 1998. <strong>275</strong>(2):  p. 337-46.</li>
-<li>Busenlehner,  L.S., et al., <em>Spectroscopic properties of  the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC.</em> Biochemistry, 2001. <strong>40</strong>(14): p.  4426-36.</li>
-</ol>
-<h2 class="subtitle"><a name="Recycling">Recycling</a></h2>
-<h3>TEM Protocol</h3>
-<p>In order to verify the synthesis of the CdS nanocrystal in E.coli (Rosetta plysS), the bacteria was harvested by centrifuged in 3000*g for 3 min then suspended in resin and hardened at 60℃ for 16 hours.</p>
-<p>Then the hard pellets were cut into 60nm thin slices and the slices were floated on water and deposited on a carbon-coated copper TEM grid. Microscopy was performed with a JEM-1400 microscope at 120-keV electron energy.</p>
-<h3>Result</h3>
-<p>Shown as bellow</p>
-<p class="text-center"><img src="https://static.igem.org/mediawiki/2014/9/9c/NEFU_China_labnote_fig42.png" class="img-thumbnail"></p>
-<h3>Reference</h3>
-<p>Damage of the Bacterial Cell Envelope by Antimicrobial Peptides Gramicidin S and PGLa as Revealed by Transmission and Scanning Electron Microscopy Antimicrob Agents Chemother. Aug 2010; 54(8): 3132–3142.</p>
-<p>Biosynthesis and characterization of CdS quantum dots in genetically engineered Escherichia coli.
-Journal of biotechnology 153(2011) 125-132</p>
-<p>Silver  nanoparticles as antimicrobial agent: a case study on <em>E. coli</em> as a model  for Gram-negative bacteria. Journal of Colloid and Interface Science 275 (2004)  177–182</p>
-<h2 class="subtitle"><a name="Future_application">Future Application</a></h2>
-<h3>Purpose</h3>
-<p>Via successful application of module 1 and module 2 above, we achieved the goal that our engineering bacteria displayed the presence of Cd<sup>2+</sup> and synthetize nanocrystals. However, there is another problem coming, the 2nd pollution committed by bacterium themselves spreading limits its future application and conceals the function of module 2. In our opinion, the rapid flocculation of our engineering bacteria is viable in avoiding this bottleneck and helpful in collecting the solid contaminant. As reported, flocculating system are capable to increase the flocculent activity of the bacteria, a flocculation gene was tentatively adopted in our next work. In this section, our host bacteria containing a flocculation gene cloned from Bacillus sp. F2 was presented and detected.</p>
-<h3>Strains, media and plasmids</h3>
-<p>The<em> Bacillus</em> sp. F2 [1] that was  used in this study was stored at -40°C in 20% glycerol. The bacteria from  the stock cultures were pre-cultured in Luria-Bertani culture medium (LB) prior to  use.<em> DH5</em><em>α</em> was taken as the host for recombinant plasmids. The pET-28b (+) was prepared  as an overexpression vector to produce the target protein.<em> Rosetta pLysS</em> was used as the host for expression of the flocculation  gene under the control of the T7 promoter. <em>E.  coli</em> transformants were grown at 37°C in LB medium.</p>
-<h3>Cloning and overexpression of the flocculation gene in <em>E. coli.</em></h3>
-<p>The  extraction of total DNA from the strain of <em>Bacillus</em> sp. F2 was carried out according to standard  techniques. The putative flocculation activity gene was amplified from the  total DNA by using the primers introduced HindIII and NdeI restriction sites  for cloning to the pET-28b (+). The following primers were used: </p>
-<p>F,5'GGAATTCCATATGATGAGTCTACTTGCTGTTTTGTTTT3'.  R,5'AAGGGGTTATGCTAGTTACGAATTCGAGCTC3'  [2]. </p>
-<p>After  sequencing, the positive PCR product was digested with NdeI /HindIII and then  ligated into NdeI/HindIII-treated expression vector pET-28b (+) and transformed  into <em>Rosetta pLysS</em>. The <em>E. coli </em>cells transformed with this  plasmid were plated on LB agar containing 100 μg/ml Kanamycin. The transformant  was grown in a 100-ml flask containing 10 ml LB medium supplemented with 100  μg/ml Kanamycin at 37°C until the optical density at 600 nm reached to 0.6–1.0,  and then 0.8 mM IPTG were added to induce target protein expression. After  incubation at 37°C for more than 8 h with shaking at 200 rpm, cells were  harvested by centrifugation (6000g for 5 min at 4°C) and washed twice with cold  50 mM Tris-HCl buffer (pH7.0), and the cell pellet was stored at −20°C for  further use.</p>
-<h3>Preparation of biomass</h3>
-<p>Cells were incubated at 37°C on an orbital  shaker at 150 rpm for 24 h. After growth, cells were harvested by centrifugation  (6000g, 5 min), washed  two times with 30 mM ethylenediaminetetraacetic acid (EDTA) solution.  Subsequently, cells were washed twice with deionised water. Then the cells were  resuspended in phosphate buffer (10 mM, pH 7.0). </p>
-<h3>Measurement of sedimentation ability</h3>
-<p>The sedimentation ability was evaluated under standard  conditions. Briefly, cells suspensions were placed in a 25 ml cylinder, at 5 g  dry weight L-1 in phosphate buffer (10 mM, pH 7.0), containing 4 mM  Ca<sup>2+</sup>. Then, the sediment ability of the <em>E.coil </em>strain was tested in phosphate buffer (10 mM, pH 7.0). At  defined periods of time, samples were taken from a fixed position of the  cylinder (the level corresponding to 20 ml) and dispersed in 30 mM EDTA  solution. Cell concentration after a t time (Ct) was determined by measuring the absorbance of the  suspension at 600 nm. Calibration curves (absorbance versus either number of  cells or dry weight) were previously constructed.</p>
-<p>In order to determine the initial cell concentration, 5 g dry weight L-1 of cell suspensions were placed in 30 mM EDTA solution, in a 25 ml cylinder. The suspensions were agitated. Samples were taken and diluted in 30 mM EDTA solution, before absorbance was determined at 600 nm (Ci).</p>
-<p>The % of settled cells (%SC) was calculated by the following equation: %SC=100–(Ct / Ci) ×100, where Ci is the initial cell concentration and Ct is the cell concentration in suspension after t time [3].</p>
-<h3>Result</h3>
-<p>For comparative purposes, two <em>E.coli</em> strains with or without flocculation gene were used. The results were shown in Fig. 1. It was showed that the  control strain could not settle efficiently within a short time. After 20 min static  placement, only 55% of cells were flocculated. In contrast, the recombinant  strain was strongly flocculent, 70% of cells were flocculated within only 10  min. At last, about 80% of the cells were settled after 20 min. The results  indicated that the flocculation  gene was expressed successfully in the <em>E.coli</em> strain. The flocculation ability of the recombinant strain could give us a  new sight to remove cells from liquid environment. However, we only observed  the flocculation characters of the strain in phosphate buffer containing 4 mM Ca2+, and we did not test  the flocculation ability of the strain in the waste water which contains  various metal ions and organic pollutant, and that is what we will focus on in our further  study. </p>
-<p class="text-center"><img src="https://static.igem.org/mediawiki/2014/f/fe/NEFU_CHINA_fa_fig2.png" class="img-thumbnail"></p>
-<p class="text-center">Fig. 1. Settling profiles of E.coil with or without flocculation gene.</p>
-<h3>Conclusion</h3>
-<p>In total,  this work supported our project with convenience in enriching the nanocrystals  and reliability against secondary pollution. Eventually, we can realize our  double-win goal, safeguarding our environment by removing heavy metal ions and  yielding available nanocrystals. However, this work was limited in the  laboratory. </p>
-<h3>Reference</h3>
-<ol class="refrence">
-  <li>Fang Ma, Junliang Liu, Shugeng Li, Jixian  Yang, Liqiu Zhang, Bo Wu, Yanbin Zhu.<em> Development of complex microbial flocculant</em>.  China water and wastewater, 2003, 19(4):1-5.</li>
-  <li>Yuguang Chang, Fang Ma, Jingbo Guo, Nanqi  Ren. <em>Flocculent genomic clone and  flocculating mechanism analysis</em>. Environmental Science, 2007,  28(12):2849-2855.</li>
-  <li>Soares, E.V., <em>Flocculation in Saccharomyces cerevisiae: a review.</em> Journal of  Applied Microbiology,  2011. <strong>110</strong>(1): p. 1-18.</li>
-</ol>
          </div>
        </div>

Team:NEFU China/Labnote

From 2014.igem.org

Revision as of 06:16, 17 October 2014

Lab note

Foundation

Obtaining the target genes

Plasmid construction

1. pHY300PLK-BCP-OPA

2. pHY300PLK-BRP-OPA

3. PACYC184-BCP-OPA

4.PACYC184-BRP-OPA

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