Team:NEFU China/Parts

From 2014.igem.org

General Description

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 (E. coli DH5α)

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

For Detecting

BBa_K1509000

This is a new coding region part. It encodes smtB protein that functions as a trans-acting regulator and binds at two inverted repeats in smt operator/promoter sites, a bi-directional promoter. In the original intact smt locus, this interaction leads to the repression of the gene smtA, orientated in the opposite direction of the gene smtB. Previous cases selected smtA whose product functions by encoding MT-like protein that can sequester metal ions to deplete heavy metal ions from environment, while we take advantage of the interaction between SmtB protein and smt operator/promoter, the bi-directional promoter. This part is essential for our designed function of detection. With BBa_K1509001(smt operator/promoter), it’s the core member of the cadmium sensor.

BBa_K1509001

This is a bi-directional promoter, a new non-coding region part. It contains two inverted repeats that are the binding sites of SmtB protein. And the activity of one direction of this promoter can be repressed after the interaction with SmtB protein. With BBa_K1509000(the gene smtB), it’s the core member of the cadmium sensor.

For Recycling

BBa_K1509003

This is a constitutive strong promoter, a new non-coding region part. The promoter was designated as CP25. CP25 is a stronge promoter in Lactococcus lactis.(Jensen and Hammer, 1998) In this article, they measured the activities of the CP promoters in E. coli and assayed the expression of a reporter gene(lacLM) to rank the activities of those CP promoters. The result suggests CP25 is the strongest promoter. We picked it and insert it upstream of the gene CDS7(BBa_K643000), a part registered by Columbia-Cooper in 2011 iGEM competition, to make sure the gene CDS7 can be transcript constantly.

For Flocculating

BBa_K1509002

This is a new coding region part. It is responsible for the flocculent ability of Bacillus sp. F2 and has been converted to E. coli JM109. It has been proved that strain of E. coli positive clone FC2 could express flocculent activity, with the flocculent efficiency of 90%, thus can be a good choice in environmental applications.(Guangyu Chang et al., 2007) The 1071bp gene consists of 29% T, 18% C, 25% G and 29% A. Considering the subsequent treatment of our reconstructed bacteria, we use this gene to enhance the flocculent activity of our bacteria. This is to make it easier to collect nanocrystals formed by our bacteria and also a second thought of the problem of secondary pollution. It’s very promising in future application.

Construction and Standardize

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 (E. coli DH5α)

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

Miniprep (default pSB1C3 with RFPBBa_E1010, pEASY-T5 cloning vector with BCP, OPA and FC) with TIANprep Mini Plasmid Kit.see protocol Part preparation by PCR (smtB, smtO-P and CP25)

primers

smtB

F

5’ CGGAATTCCTAGCGACACTCTTGTAAGTGATCG 3’

R

5' GGACTAGTATGACAAAACCAGTGCTGCAG 3'

smtO-P

F

5’ CGGAATTCGAGCCAATCACGGTTTGTCCACCCA 3’

R

5’ AACTGCAGGACAGCAACTCCTTTGAATATCTGA 3’

CP25

F

5’ CGGAATTCCTTTGGCAGTTTATTCTTGACA 3’

R

5’ AACTGCAGAACAGTACTATGTGATTATACCAGC 3’

PCR system (50ul)

parameters

procedure

temperature

time

premix taq(TaKaRa)

25ul

PreDenature

94

10 min

primer F

2ul

Denature

94

30 sec

primer R

2ul

Annealing

55

30 sec

plasmids

diluted 100×

1ul

Extension

72

30 sec

dNTPs

included in premix

Final Elongation

72

10 min

buffer

Final Hold

16

H2O

20ul

Cycle

30 cycles

Part preparation by PCR (F, short for Flocculation)

primers

F

5’ GCTCTAGAATGAGTCTACTTGCTGTTTTGTTTT 3’

R

5' AACTGCAGTTACGAATTCGAGCTCGGTAC 3'

PCR system (50ul)

parameters

procedure

temperature

time

premix taq(TaKaRa)

25ul

PreDenature

94

10 min

primer F

2ul

Denature

94

30 sec

primer R

2ul

Annealing

55

30 sec

plasmids

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

P1 parts’ PCR product 1,2. smtO-P; 3-6. smtB; 8-11. F; Marker (DL2000)

P2 PCR product of CP25; Marker (DL2000)

Double digestion (NEB)

SmtB

substrate

EcoR I

Spe I

Cutsmart

Buffer

H2O

total

temperature

time

BBa_E1010

30ul

3ul

3ul

10ul

54ul

100ul

37

16 h

PCR product

30ul

3ul

3ul

10ul

54ul

100ul

37

16 h

SmtO-P, CP25

substrate

EcoR I

Pst I

1×H

Buffer

H2O

total

temperature

time

BBa_E1010

30ul

3ul

3ul

10ul

54ul

100ul

37

16 h

PCR product

30ul

3ul

3ul

10ul

54ul

100ul

37

16 h

F

substrate

Xba I

Pst I

1×M

Buffer

H2O

total

temperature

time

BBa_E1010

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-   & -Part-

total

temperature

time

5ul

5ul in total (see note)

10ul

16

30 min

Note:-plasmid-:-BCP-(mole number)=1:2~1:8

Transformationsee protocol

Colony PCR

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

55

30 sec

template:   pick a single colony and dip in H2O

8.4ul

Extension

72

30 sec/1.5   min

Final   Elongation

72

10 min

dNTPs

included   in premix

Final Hold

16

buffer

Cycle

30 cycles

Note:1.  The   template was heat denatured at 100 in metal bath, then freeze on ice for 2   min

before   running through parameters on the right.

2. Extension time: 30 sec for smtB, smtO-P and CP25; 1.5   min for F

3. Use corresponding primers

Miniprep (pSB1C3 with maybe smtB or smtO-P or F or CP25) with TIANprep Mini Plasmid Kit.see protocol

Double digestion (NEB) for detection (refer to the double digestion tables above for details)

plasmids with H2O

enzyme1

enzyme2

buffer

total

temperature

time

16.8ul

0.6ul

0.6ul

2ul

20ul

37

16 h

The sequencing results are consistent with our designation.