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. |
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 |
|
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.