Difference between revisions of "Team:NKU China/Notebook"

Revision as of 02:15, 17 October 2016

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50μL PCR system ×2
2× Taq Master Mix	25μL
C2-F	2μL
C2-R	2μL
p-C2	2μL
ddH2O	19μL
Total	50μL

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	15 sec	30 cycles
72^oC	30 sec
72^oC	10 min
16^oC	∞

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
pMD19 T-Simple Vector	1μL
C2-luxS	4μL
ddH2O	12μL
Total	20μL
Reaction condition: 16^oC overnight

20μL digestion system
10× FastDigest Buffer	2μL
BamH Ⅰ	1μL
T-lsrACDB	1μL
ddH2O	7μL
Total	20μL
Reaction condition: 37^oC for 40 min

100μL methylation system
10× BamH Ⅰ methyltransferase Buffer	10μL
BamH Ⅰ methyltransferase	1μL
S-adenosylmethionine	0.5μL
pWH-C2-luxS	80μL
ddH2O	8.5μL
Total	100μL
Reaction condition: 37^oC for 1 hour

Groups divided in this experiment
GR286	wild strain as control group
GR286ΔluxS	GR286 without luxS gene
pWH-luxS	luxS overexpression plasmid in GR286; without induced by xylose
pWH-luxS + xyl	luxS overexpression plasmid in GR286; induced by xylose
pWH1520	empty plasmid in GR286 as control group
pHT-lsrACDB	lsrACDB overexpression plasmid in GR286ΔluxS
pHT-01	empty plasmid in GR286ΔluxS as control group

Selection of positive clones by colony PCR
(No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-*C2-luxS* into GR286)

Laboratory Notes

☞☟ Week1 (May 16–May 22)

begin
➀
➁
➂

In order to make sure the efficiency of our "consumer", we should first knock out the luxS gene in our engineering bacteria GR286(a simplified strain of Bacillus amyloliquefaciens LL3). We used a markerless gene replacement method to knock out the luxS gene.

Construction of targeting vector : the upstream and downstream of luxS gene were combined by over-lapping PCR and ligated into plasmid pKSU.

pKSU-ΔluxS was transformed into GR286, and positive clones were selected.

20μL PCR system
2× Taq Master Mix	10μL
pKSU-F	1μL
pKSU-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30 sec	30 cycles
72^oC	1 min 30 sec
72^oC	10 min
16^oC	∞

The transformants were cultured at 42^oC with chloramphenicol to select single-crossover clones.

20μL PCR system
2× Taq Master Mix	10μL
luxS-up-F	1μL
luxS-dn-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
56^oC	30 sec	30 cycles
72^oC	2 min
72^oC	10 min
16^oC	∞

Selection of single-crossover clones by PCR
(No.1-4 are single-crossover strains,
No.5 is positive control.)

☞☟ Week2 (May 23–May 29)

➀
➁

The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations.

The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. Regretfully, we didn't get the double-crossover clones.

20μL PCR system
2× Taq Master Mix	10μL
luxS-up-F	1μL
luxS-dn-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
56^oC	30 sec	30 cycles
72^oC	2 min
72^oC	10 min
16^oC	∞

Selection of double-crossover clones by PCR
(No.1-5 are experimental groups, No.6 is wild GR286. The result showed that we failed to get the double-crossover clones.)

☞☟ Week3 (May 30–Jun 05)

➀
➁
➂

Transformants were cultured at 42^oC with chloramphenicol again and the single-crossover clones were selected successfully.

20μL PCR system
2× Taq Master Mix	10μL
luxS-up-F	1μL
luxS-dn-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
56^oC	30 sec	30 cycles
72^oC	2 min
72^oC	10 min
16^oC	∞

Selection of single-crossover clones by PCR
(No.1&2&4 are single-crossover strains,No.5 is positive control. )

The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations.

The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. We finally obtained our aimed strain—GR286ΔluxS.

20μL PCR system
2× Taq Master Mix	10μL
luxS-up-F	1μL
luxS-dn-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
56^oC	30 sec	30 cycles
72^oC	2 min
72^oC	10 min
16^oC	∞

Selection of Δ*luxS* clones by PCR
(The No.4 is the aimed strain&horbar;GR286Δ*luxS*)

☞☟ Week4 (Jun 06–Jun 12)

➀
➁
➂
➃
➄
➅

The GR286ΔluxS strain was cultured and made competent for future use.

The lsrACDB gene from Bacillus thuringiensis was cloned and ligated to T-vector.

50μL PCR system ×2
2× Taq Master Mix	25μL
lsrACDB-F	2μL
lsrACDB-R	2μL
Bacterium solution	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
57^oC	30 sec	30 cycles
72^oC	4 min 30 sec
72^oC	10 min
16^oC	∞

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
pMD19 T-Simple Vector	1μL
lsrACDB	3μL
ddH2O	13μL
Total	20μL
Reaction condition: 16^oC overnight

The T-lsrACDB was transformed into DH5α and plate was coated, and then positive clones were selected.

20μL PCR system
2× Taq Master Mix	10μL
M13F	1μL
M13R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
59^oC	30 sec	30 cycles
72^oC	4 min 30 sec
72^oC	10 min
16^oC	∞

Selection of positive clones by PCR
(No. 3&4 are positive results)

After restriction enzyme digestion verification, the positive clones were then sequenced. Unfortunately, the sequencing result showed some mutations inside the target gene.

20μL digestion system
10× FastDigest Buffer	2μL
BamH Ⅰ	1μL
T-lsrACDB	1μL
ddH2O	7μL
Total	20μL
Reaction condition: 37^oC for 40 min

Restriction enzyme digestion verification
(No.1 are *lsrACDB* fragement, No.2 are linearized T-vector.)

The gene cloning process was repeated but there were still some mutations.

We finally decided to request the gene company to synthesize the lsrACDB gene.

☞☟ Week5 (Jun 13–Jun 19)

begin
➀
➁
➂
➃

This week, we started to construct another controller―supplier.

A strong promoter C2 was cloned from former kit and luxS gene was cloned from GR286.

50μL PCR system ×2
2× Taq Master Mix	25μL
C2-F	2μL
C2-R	2μL
p-C2	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	15 sec	30 cycles
72^oC	30 sec
72^oC	10 min
16^oC	∞

50μL PCR system ×2
2× Taq Master Mix	25μL
luxS-F	2μL
luxS-R	2μL
Bacterium solution	1μL
GR286	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
59^oC	30 sec	30 cycles
72^oC	30 sec
72^oC	10 min
16^oC	∞

PCR cloning product of promoter C2 and gene *luxS*
(NO.1&2 are C2, No.3&4 are *luxS*)

Two segments were fused together by fusion PCR and ligated into T-vector. After that, the vector was transformed into DH5α.

50μL PCR system ×2
2× Taq Master Mix	25μL
C2-F	2μL
luxS-R	2μL
C2	2μL
luxS	2μL
ddH2O	17μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
59^oC	30 sec	30 cycles
72^oC	40 sec
72^oC	10 min
16^oC	∞

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
pMD19 T-Simple Vector	1μL
C2-luxS	4μL
ddH2O	12μL
Total	20μL
Reaction condition: 16^oC overnight

Positive clones were selected by colony PCR.

20μL PCR system
2× Taq Master Mix	10μL
M13-F	1μL
M13-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
59^oC	30 sec	30 cycles
72^oC	40 sec
72^oC	10 min
16^oC	∞

PCR cloning product of promoter C2 and gene *luxS*

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
BamH Ⅰ	1μL
T-lsrACDB	1μL
ddH2O	7μL
Total	20μL
Reaction condition: 37^oC for 40 min

Restriction enzyme digestion verification
(No.1 are linearized T-vector, No.2 are *C2-luxS* fragment.)

☞☟ Week6 (Jun 20–Jun 26)

➀
➁
➂
➃
➄

The sequencing result showed that there was a correct strain and thus it could be used for the following experiments. We obtained the correct plasmid T-C2-luxS from DH5α. Then the fragment C2-luxS was obtained by digestion and gel extraction.

40μL digestion system
10× FastDigest Buffer	4μL
BamH Ⅰ	2μL
T-C2-luxS	25μL
ddH2O	9μL
Total	20μL
Reaction condition: 37^oC for 40 min

The C2-luxS fragment was ligated to linearized plasmid pWH1520, and then the ligation product was transformed into DH5α.

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
pMD19 T-Simple Vector	1μL
C2-luxS	5μL
ddH2O	11μL
Total	20μL
Reaction condition: 16^oC overnight

The plasmid pWH-C2-luxS was extracted from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.

The plasmid pWH-C2-luxS was extracted from JM110,and then it was treated with BamH Ⅰ methylase.

100μL methylation system
10× BamH Ⅰ methyltransferase Buffer	10μL
BamH Ⅰ methyltransferase	1μL
S-adenosylmethionine	0.5μL
pWH-C2-luxS	80μL
ddH2O	8.5μL
Total	100μL
Reaction condition: 37^oC for 1 hour

The plasmid was transformed into GR286 by electroporation.[Failed]

☞☟ Week7 (Jun 27–Jul 03)

➀
➁
➂

This week, we tried to use different voltages to transform the plasmid. Sadly, all of these attempts rendered negative results.

We considered whether the luxS gene is toxic for GR286, and the bacteria tends to reject the gene when a strong promoter is inserted upstream of it. So, we planned to use inducible promoter to reconstruct our expression vector.

The plasmid pWH1520 contains a strong xylA promoter originating from Bacillus megaterium, and transcription initiated by this promoter is xylose-inducible. Also, the gene of interest carries its own ribosome binding sequence (RBS) and translation initiation codon. Based on these points, we redesigned primers.

☞☟ Week8 (Jul 04–Jul 10)

➀
➁
➂
➃

luxS gene was cloned from GR286 using our new primers.

50μL PCR system ×2
2× Taq Master Mix	25μL
YD-luxS-F	2μL
YD-luxS-R	2μL
Bacterium solution	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30 sec	30 cycles
72^oC	40 sec
72^oC	10 min
16^oC	∞

The luxS fragment was purified by gel extraction, and ligated into linearized pWH1520. Then the vector was transformed into DH5α.

40μL digestion system ×2
10× FastDigest Buffer	4μL
BamH Ⅰ	2μL
pWH1520	25μL
ddH2O	9μL
Total	40μL
Reaction condition: 37^oC for 40 min

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
linearized pWH1520	1μL
luxS	3μL
ddH2O	13μL
Total	20μL
Reaction condition: 16^oC overnight

Positive clones were selected by colony PCR.

20μL PCR system
2× Taq Master Mix	10μL
pWH-F	1μL
pWH-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30 sec	30 cycles
72^oC	40 sec
72^oC	10 min
16^oC	∞

Selection of positive clones by colony PCR

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
BamH Ⅰ	1μL
pWH-luxs	10μL
ddH2O	7μL
Total	20μL
Reaction condition: 37^oC for 40 min

Restriction enzyme digestion verification
(No.1 are linearized pWH1520, No.2 are *luxS* fragments.)

☞☟ Week9 (Jul 11–Jul 17)

➀
➁
➂
end

The sequencing result showed there's three positive strains. So one positive strain was chosen to be used for the following experiments. The pWH-luxS plasmid was extracted from the chosen strain. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.

the plasmid pWH-luxS was extracted from JM110,and it was treated withBamH Ⅰ methylase.

100μL methylation system
10× BamH Ⅰ methyltransferase Buffer	10μL
BamH Ⅰ methyltransferase	1μL
S-adenosylmethionine	0.5μL
pWH-C2-luxS	80μL
ddH2O	8.5μL
Total	100μL
Reaction condition: 37^oC for 1 hour

The plasmid was transformed into GR286 by electroporation, and positive clones were selected.

The construction of supplier was accomplished!

☞☟ Week10 (Jul 18–Jul 24)

➀
➁

We have received the product of synthetic lsrACDB gene. We first used restriction-ligation method to ligate lsrACDB to plasmid pWH1520, but we failed to select positive after several tries.

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
linearized pWH1520	1μL
lsrACDB	3μL
ddH2O	13μL
Total	20μL
Reaction condition: 16^oC overnight

Considering that the lsrACDB gene is a large fragment (4500bp), we used ClonExpress technique to clone the gene again to improve the efficiency of ligation. The lsrACDB sequence was divided into two parts and they were cloned separately. Then the two segments were ligated to the plasmid pWH1520 and the recombinant vector was transformed into DH5α. After that, verification PCR was used to select the positive clones. However, we didn't get a good result.

Selection of positive clones by colony PCR
(No.6 is positive control, No.1-5 are experimental groups)

☞☟ Week11 (Jul 25–Jul 31)

➀

We learnt a new method called circular polymerase extension cloning (CPEC) for high-throughput cloning of complex and combinatorial DNA libraries, and we decided to use this method to try to ligate our lsrACDB gene. It's encouraging that we succeeded to ligate the lsrACDB gene to the plasmid pHT-01.

Selection of positive clones by colony PCR
(No.3-6 are positive clones)

Restriction enzyme digestion verification
(No.2&3 are positive results.)

☞☟ Week12 (Aug 1–Aug 7)

➀
➁

Since we have already successfully constructed "supplier" and part of "consumer", we decided to measure the growth curve to explore the function of our "controller".

Groups divided in this experiment
GR286	wild strain as control group
GR286ΔluxS	GR286 without luxS gene
pWH-luxS	luxS overexpression plasmid in GR286; without induced by xylose
pWH-luxS + xyl	luxS overexpression plasmid in GR286; induced by xylose
pWH1520	empty plasmid in GR286 as control group
pHT-lsrACDB	lsrACDB overexpression plasmid in GR286ΔluxS
pHT-01	empty plasmid in GR286ΔluxS as control group

OD₆₀₀ of different groups at specific times

Growth curve of pWH-*luxS*, pWH-*luxS* + xyl and pWH1520

Growth curve of pHT-*lsrACDB* and pHT-01

Cultured media of our supplier was tested for the presence of AI-2 by inducing luminescence of Vibrio harveyi reporter strain BB170.

Fluorescence intensity of different groups at specific times

Relative fluorescence intensity of different groups

☞☟ Week13 (Aug 8–Aug 14)

➀
➁

For our consumer, we should also overexpress the lsrK and lsrFG gene for phosphorylating and degrading phosphorylated AI-2. We used ClonExpress technique to clone the two genes and ligate them to plasmid pHT-01 successfully.

Restriction enzyme digestion verification
(No.1&3&4 are positive results. )

We co-cultured the supplier with BB170 and tested the fluorescence intensity to explore the function of supplier. (negative result)

Fluorescence intensity of BB170 and co-culture medium

☞☟ Week14 (Aug 15–Aug 21)

begin
outline
1.1
1.2
1.3

Since the controller we constructed with GR286 did not demonstrate positive results, we decided to substitute GR286 with Escherichia coli whose AI-2 metabolic pathway had been elucidated as the chassis of our controller.

The strains to be constructed are shown in the tables below.

AI-2 supplier
	LuxS	Mtn
pTrcHisB	Native	Native
pLuxS	Induced	Native
pLuxS-Mtn	Induced	Induced

AI-2 consumer
	LsrACDB	LsrK	LsrFG
pTrcHisB	Native	Native	Native
pLsrACDB	Induced	Native	Native
pLsrFG	Native	Native	Induced
pLsrK	Native	Induced	Native
pLsrACDBFG	Induced	Native	Induced
pLsrACDBK	Induced	Constitutive	Native
plsrACDBFGK	Induced	Constitutive	Induced

The sequences of lsrACDB, lsrFG, lsrK, luxS, mtn gene in E.coli K12 were searched in NCBI, and primers were designed according to the gene sequences. The primers used in the construction of AI-2 controller are shown in the table below.

Primers used in the construction of AI-2 controller
Primer name	Sequence
lsrACDBFG1-F	CGACGATGACGATAAGGATCCATGCAAACGAGTGATACCCGC
lsrACDBFG1-R	TGTTTGGCGTTTCCGGCAGCGGTGCGGAGAGC
lsrACDBFG2-F	TGTTTGGCGTTTCCGCGATTG
lsrACDBFG2-R	GCACTCTCACACCACGTTGCATGGCGCGTTTC
lsrACDBFG3-F	GTGGTGTGAGAGTGCTGAC
lsrACDBFG3-R	ACCAGCTGCAGATCTCGAGCTCGTCACGGCATCAACCCATTGAAC
lsrACDB1-F	CGACGATGACGATAAGGATCCATGCAAACGAGTGATACCCGC
lsrACDB1-R	ACCGGAACCGCCGAGCAAACTAATGCCGCCCAGCAC
lsrACDB2-F	CTCGGCGGTTCCGGTGCGAT
lsrACDB2-R	ACCAGCTGCAGATCTCGAGCTCGTCAGAAATCGTATTTGCCG
luxS-F	ATGACGATAAGGATCCGAGCTCGATGCCGTTGTTAGATAGCTTC
luxS-R	GGACTCCCCCGGGGGACTAAATGTGCAGTTCCTGCAACTTC
mtn-F	TCCCCCGGGGGAGTCCTCTCCCGCGTGAGAAATAC
mtn-R	TATGGTACCAGCTGCAGATCTCTTAGCCATGTGCAAGTTTCTG
luxS-Fd	GGAATTCCCTAAATGTGCAGTTCCTGCAACTTC
luxS-Rv	GGGGTACCCCATGCCGTTGTTAGATAGCTTCACAG
lsrK-Fd	GGGGTACCCCATGGCTCGACTCTTTACC
lsrK-Rv	GGAATTCCCTATAACCCAGGCGCTTTCC
lsrFG-Fd	CGGGATCCCGATGGCAGATTTAGACGATATTAAAGATGG
lsrFG-Rv	GAAGATCTTCTCACGGCATCAACCCATTGAAC
lsrK-F	AACTGCAGAACCAATGCATTGGTTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCAAAGAGGAGAAAATGGCTCGACTCTTTACC
lsrK-R	TTCGAACTATAACCCAGGCGCTTTCC
BK-JC-F	TTTCAGTGTATGTCGCGGATGC
GK-JC-F	TTGCGCTTCGATGTCTTACAGG
pTrc-JC-F	TGGGCACTCGACCGGAATTATC
pTrc-JC-R	GCTACTGCCGCCAGGCAAATTC

Construction of pLuxS-Mtn

luxS gene was cloned from E.coli K12.

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
luxS-F	2μL
luxS-R	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
63^oC	30 sec	30 cycles
72^oC	30 sec
72^oC	10 min
16^oC	∞

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
mtn-F	2μL
mtn-R	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
63^oC	30sec	30 cycles
72^oC	45 sec
72^oC	10 min
16^oC	∞

ClonExpress technique was used to ligate luxS and mtn genes to plasmid pTrcHisB. The recombinant vector pTrc-luxS-mtn was transformed into E.coli K12. After that, verification PCR was performed to select the positive clones.

20μL PCR system
PrimeSTAR Max Premix(2×)	10μL
pTrc-JC-F	1μL
pTrc-JC-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30sec	30 cycles
72^oC	1 min 30 sec
72^oC	10 min
16^oC	∞

Selection of positive clones by PCR
(NO.3&5&8 are positive results)

3 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
Sac Ⅰ	1μL
Bgl Ⅱ	1μL
pTrc-luxS-mtn	6μL
ddH2O	10μL
Total	20μL
Reaction condition: 37^oC for 40 min

Restriction enzyme digestion verification
(No.1 are linearized pTrcHisB, No.2 are *luxS*-*mtn* fragment. )

☞☟ Week15 (Aug 22–Aug 28)

outline
1.1
1.2
1.3
1.4
2.1
2.2
2.3
3.1
3.2
3.3

Construction of pLuxS
Construction of pLsrACDB
Construction of pLsrACDBFG

luxS gene was cloned from E.coli K12.

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
luxS-Fd	2μL
luxS-Rv	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
60^oC	30 sec	30 cycles
72^oC	30 sec
72^oC	10 min
16^oC	∞

The luxS gene was purified by gel extraction. Then, both the luxS segment and pTrcHisB vector were treated with restriction enzyme. After that, luxS gene was ligated to linearized pTrcHisB, and the ligation product was transformed into E.coli K12.

40μL digestion system ×2
10× FastDigest Buffer	4μL
Kpn Ⅰ	2μL
EcoR Ⅰ	2μL
luxS	25μL
ddH2O	7μL
Total	40μL
Reaction condition: 37^oC for 40 min

40μL digestion system ×2
10× FastDigest Buffer	4μL
Kpn Ⅰ	2μL
EcoR Ⅰ	2μL
pTrcHisB	20μL
ddH2O	12μL
Total	40μL
Reaction condition: 37^oC for 40 min

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
linearized pTrcHisB	1μL
luxS	3μL
ddH2O	13μL
Total	20μL
Reaction condition: 16^oC overnight

Verification PCR was performed to select the positive clones.

20μL PCR system ×2
2× Taq Master Mix	10μL
pTrc-JC-F	1μL
pTrc-JC-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30sec	30 cycles
72^oC	50 sec
72^oC	10 min
16^oC	∞

One positive strain was chosen to be cultured overnight and plasmid was extracted. After restriction enzyme digestion verification, the positive clone was sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
Kpn Ⅰ	1μL
EcoR Ⅰ	1μL
pTrc-luxS	6μL
ddH2O	10μL
Total	20μL
Reaction condition: 37^oC for 40 min

Restriction enzyme digestion verification
(No.1 is linearized pTrcHisB + *luxS*, No.2 is plasmid pTrc-*luxS*. )

The lsrACDBFG sequence was divided into three parts and they were cloned separately. Then the three segments were ligated to the linearized pTrcHisB by ClonExpress technique, and the recombinant vector was transformed into DH5α.

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
lsrACDB1-F	2μL
lsrACDB1-R	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
lsrACDB2-F	2μL
lsrACDB2-R	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
60^oC	30 sec	30 cycles
72^oC	2 min 20 sec
72^oC	10 min
16^oC	∞

PCR cloning product of segment *lsrACDB1*,*lsrACDB1*2

Verification PCR was performed to select the positive clones.

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
BamH Ⅰ	1μL
Sac Ⅰ	1μL
pTrc-lsrACDB	6μL
ddH2O	10μL
Total	20μL
Reaction condition: 37^oC for 40 min

The lsrACDBFG sequence was divided into three parts and they were cloned separately. Then the three segments were ligated to the linearized pTrcHisB by ClonExpress technique, and the recombinant vector was transformed into DH5α.

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
lsrACDBFG1-F	2μL
lsrACDBFG1-R	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
lsrACDBFG2-F	2μL
lsrACDBFG2-R	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
lsrACDBFG3-F	2μL
lsrACDBFG3-R	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30 sec	30 cycles
72^oC	2 min
72^oC	10 min
16^oC	∞

PCR cloning product of segment *lsrACDBFG*1,*lsrACDBFG*2, *lsrACDBFG*3

Verification PCR was performed to select the positive clones.

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
BamH Ⅰ	1μL
Sac Ⅰ	1μL
pTrc-lsrACDB	6μL
ddH2O	10μL
Total	20μL
Reaction condition: 37^oC for 40 min

Restriction enzyme digestion verification
(No.1-4 are linearized pTrcHisB + *lsrACDBFG*, No.5 is plasmid pTrc-*lsrACDBFG*.)

☞☟ Week16 (Aug 29–Sep 04)

outline
1.1
1.2
1.3
1.4
2.1
2.2
2.3
2.4
3.1
3.2
3.3
3.4

Construction of pLsrFG
Construction of pLsrACDBK
Construction of pLsrACDBFGK

lsrFG gene was cloned from E.coli K12.

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
lsrFG-Fd	2μL
lsrFG-Rv	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30 sec	30 cycles
72^oC	1 min 15 sec
72^oC	10 min
16^oC	∞

The lsrFG gene was purified by gel extraction. Then, both the lsrFG segment and pTrcHisB vector were treated with restriction enzyme. After that, lsrFG gene was ligated to linearized pTrcHisB, and the ligation product was transformed into E.coli K12.

40μL digestion system ×2
10× FastDigest Buffer	4μL
BamH Ⅰ	2μL
Bgl Ⅱ	2μL
lsrFG	25μL
ddH2O	7μL
Total	40μL
Reaction condition: 37^oC for 40 min

40μL digestion system ×2
10× FastDigest Buffer	4μL
BamH Ⅰ	2μL
Bgl Ⅱ	2μL
pTrcHisB	20μL
ddH2O	12μL
Total	40μL
Reaction condition: 37^oC for 40 min

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
pMD19 T-Simple Vector	1μL
luxSFG	3μL
ddH2O	13μL
Total	20μL
Reaction condition: 16^oC overnight

Verification PCR was performed to select the positive clones.

20μL PCR system ×2
2× Taq Master Mix	10μL
pTrc-JC-F	1μL
pTrc-JC-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30 sec	30 cycles
72^oC	1 min 30 sec
72^oC	10 min
16^oC	∞

Selection of positive clones by PCR
(NO. 1&3 are positive results)

Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
BamH Ⅰ	1μL
Bgl Ⅱ	1μL
pTrc-lsrFG	6μL
ddH2O	10μL
Total	20μL
Reaction condition: 37^oC for 40 min

lsrK gene was cloned from E.coli K12.

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
lsrK-F	2μL
lsrK-R	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
55^oC	30 sec	30 cycles
72^oC	1 min 40 sec
72^oC	10 min
16^oC	∞

The lsrK gene was purified by gel extraction. Then, both the lsrK segment and pTrc-lsrACDB vector were treated with restriction enzyme. After that, lsrK gene was ligated to linearized pTrc-lsrACDB, and the ligation product was transformed into E.coli K12.

40μL digestion system ×2
10× FastDigest Buffer	4μL
Pst Ⅰ	2μL
BstB Ⅰ	2μL
lsrK	25μL
ddH2O	7μL
Total	40μL
Reaction condition: 37^oC for 40 min

40μL digestion system ×2
10× FastDigest Buffer	4μL
Pst Ⅰ	2μL
BstB Ⅰ	2μL
pTrc-lsrACDB	25μL
ddH2O	7μL
Total	40μL
Reaction condition: 37^oC for 40 min

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
linearized pTrc-lsrACDB	1μL
lsrK	3μL
ddH2O	13μL
Total	20μL
Reaction condition: 16^oC overnight

Verification PCR was performed to select the positive clones.

20μL PCR system ×2
2× Taq Master Mix	10μL
BK-JC-F	1μL
pTrc-JC-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30 sec	30 cycles
72^oC	2 min
72^oC	10 min
16^oC	∞

Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
Pst Ⅰ	1μL
BstB Ⅰ	1μL
pTrc-lsrACDBK	6μL
ddH2O	10μL
Total	20μL
Reaction condition: 37^oC for 40 min

lsrK gene was cloned from E.coli K12.

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
lsrK-F	2μL
lsrK-R	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
55^oC	30 sec	30 cycles
72^oC	1 min 40 sec
72^oC	10 min
16^oC	∞

The lsrK gene was purified by gel extraction. Then, both the lsrK segment and pTrc-lsrACDBFG vector were treated with restriction enzyme. After that, lsrK gene was ligated to linearized pTrc-lsrACDBFG, and the ligation product was transformed into E.coli K12.

40μL digestion system ×2
10× FastDigest Buffer	4μL
Pst Ⅰ	2μL
BstB Ⅰ	2μL
lsrK	25μL
ddH2O	7μL
Total	40μL
Reaction condition: 37^oC for 40 min

40μL digestion system ×2
10× FastDigest Buffer	4μL
Pst Ⅰ	2μL
BstB Ⅰ	2μL
pTrc-lsrACDBFG	20μL
ddH2O	12μL
Total	40μL
Reaction condition: 37^oC for 40 min

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
linearized pTrc-lsrACDFGB	1μL
lsrK	3μL
ddH2O	13μL
Total	20μL
Reaction condition: 16^oC overnight

Verification PCR was performed to select the positive clones.

20μL PCR system ×2
2× Taq Master Mix	10μL
GK-JC-F	1μL
pTrc-JC-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30 sec	30 cycles
72^oC	2 min
72^oC	10 min
16^oC	∞

Selection of positive clones by PCR
(NO.3 is positive result)

Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
Pst Ⅰ	1μL
BstB Ⅰ	1μL
pTrc-lsrACDBKFGK	6μL
ddH2O	10μL
Total	20μL
Reaction condition: 37^oC for 40 min

☞☟ Week17 (Sep 05–Sep 11)

outline
1.1
1.2
1.3
1.4

Construction of pLsrK

lsrK gene was cloned from E.coli K12.

50μL PCR system ×2
PrimeSTAR Max Premix(2×)	25μL
lsrK-Fd	2μL
lsrK-Rv	2μL
E.coli K12	2μL
ddH2O	19μL
Total	50μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
55^oC	30 sec	30 cycles
72^oC	1 min 40 sec
72^oC	10 min
16^oC	∞

The lsrK gene was purified by gel extraction. Then, both the lsrK segment and pTrc-lsrACDBFG vector were treated with restriction enzyme. After that, lsrK gene was ligated to linearized pTrc-lsrACDBFG, and the ligation product was transformed into E.coli K12.

40μL digestion system ×2 ×2
10× FastDigest Buffer	4μL
Kpn Ⅰ	2μL
EcoR Ⅰ	2μL
lsrK	25μL
ddH2O	7μL
Total	40μL
Reaction condition: 37^oC for 40 min

40μL digestion system ×2 ×2
10× FastDigest Buffer	4μL
Pst Ⅰ	2μL
BstB Ⅰ	2μL
pTrc-HisB	20μL
ddH2O	12μL
Total	40μL
Reaction condition: 37^oC for 40 min

20μL ligation system
10× DNA Ligase Buffer	2μL
T4 DNA Ligase	1μL
linearized pTrcHisB	1μL
lsrK	3μL
ddH2O	13μL
Total	20μL
Reaction condition: 16^oC overnight

Verification PCR was performed to select the positive clones.

20μL PCR system ×2
2× Taq Master Mix	10μL
pTrc-JC-F	1μL
pTrc-JC-R	1μL
Bacterium solution	1μL
ddH2O	7μL
Total	20μL

}

PCR reaction condition
94^oC	10 min
94^oC	30 sec
58^oC	30 sec	30 cycles
72^oC	2 min
72^oC	10 min
16^oC	∞

4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

20μL digestion system
10× FastDigest Buffer	2μL
Kpn Ⅰ	1μL
EcoR Ⅰ	1μL
pTrc-lsrK	6μL
ddH2O	10μL
Total	20μL
Reaction condition: 37^oC for 40 min

@@ Line 1: / Line 1: @@
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a.setAttribute("type",b),c&&(a.value=c),b}}}},removeAttr:function(a,b){var c,d=0,e=b&&b.match(K);
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(i.canvas.pxratio = window.devicePixelRatio, i.tmp.retina = !0) : (i.canvas.pxratio = 1, i.tmp.retina = !1), i.canvas.w = i.canvas.el.offsetWidth * i.canvas.pxratio, i.canvas.h = i.canvas.el.offsetHeight * i.canvas.pxratio, i.particles.size.value = i.tmp.obj.size_value * i.canvas.pxratio, i.particles.size.anim.speed = i.tmp.obj.size_anim_speed * i.canvas.pxratio, i.particles.move.speed = i.tmp.obj.move_speed * i.canvas.pxratio, i.particles.line_linked.distance = i.tmp.obj.line_linked_distance * i.canvas.pxratio, i.interactivity.modes.grab.distance = i.tmp.obj.mode_grab_distance * i.canvas.pxratio, i.interactivity.modes.bubble.distance = i.tmp.obj.mode_bubble_distance * i.canvas.pxratio, i.particles.line_linked.width = i.tmp.obj.line_linked_width * i.canvas.pxratio, i.interactivity.modes.bubble.size = i.tmp.obj.mode_bubble_size * i.canvas.pxratio, i.interactivity.modes.repulse.distance = i.tmp.obj.mode_repulse_distance * i.canvas.pxratio }, i.fn.canvasInit = function () { i.canvas.ctx = i.canvas.el.getContext("2d") }, i.fn.canvasSize = function () { i.canvas.el.width = i.canvas.w, i.canvas.el.height = i.canvas.h, i && i.interactivity.events.resize && window.addEventListener("resize", function () { i.canvas.w = i.canvas.el.offsetWidth, i.canvas.h = i.canvas.el.offsetHeight, i.tmp.retina && (i.canvas.w *= i.canvas.pxratio, i.canvas.h *= i.canvas.pxratio), i.canvas.el.width = i.canvas.w, i.canvas.el.height = i.canvas.h, i.particles.move.enable || (i.fn.particlesEmpty(), i.fn.particlesCreate(), i.fn.particlesDraw(), i.fn.vendors.densityAutoParticles()), i.fn.vendors.densityAutoParticles() }) }, i.fn.canvasPaint = function () { i.canvas.ctx.fillRect(0, 0, i.canvas.w, i.canvas.h) }, i.fn.canvasClear = function () { i.canvas.ctx.clearRect(0, 0, i.canvas.w, i.canvas.h) }, i.fn.particle = function (e, a, t) { if (this.radius = (i.particles.size.random ? Math.random() : 1) * i.particles.size.value, i.particles.size.anim.enable && (this.size_status = !1, this.vs = i.particles.size.anim.speed / 100, i.particles.size.anim.sync || (this.vs = this.vs * Math.random())), this.x = t ? t.x : Math.random() * i.canvas.w, this.y = t ? t.y : Math.random() * i.canvas.h, this.x > i.canvas.w - 2 * this.radius ? this.x = this.x - this.radius : this.x < 2 * this.radius && (this.x = this.x + this.radius), this.y > i.canvas.h - 2 * this.radius ? this.y = this.y - this.radius : this.y < 2 * this.radius && (this.y = this.y + this.radius), i.particles.move.bounce && i.fn.vendors.checkOverlap(this, t), this.color = {}, "object" == typeof e.value) if (e.value instanceof Array) { var s = e.value[Math.floor(Math.random() * i.particles.color.value.length)]; this.color.rgb = hexToRgb(s) } else void 0 != e.value.r && void 0 != e.value.g && void 0 != e.value.b && (this.color.rgb = { r: e.value.r, g: e.value.g, b: e.value.b }), void 0 != e.value.h && void 0 != e.value.s && void 0 != e.value.l && (this.color.hsl = { h: e.value.h, s: e.value.s, l: e.value.l }); else "random" == e.value ? this.color.rgb = { r: Math.floor(256 * Math.random()) + 0, g: Math.floor(256 * Math.random()) + 0, b: Math.floor(256 * Math.random()) + 0 } : "string" == typeof e.value && (this.color = e, this.color.rgb = hexToRgb(this.color.value)); this.opacity = (i.particles.opacity.random ? Math.random() : 1) * i.particles.opacity.value, i.particles.opacity.anim.enable && (this.opacity_status = !1, this.vo = i.particles.opacity.anim.speed / 100, i.particles.opacity.anim.sync || (this.vo = this.vo * Math.random())); var n = {}; switch (i.particles.move.direction) { case "top": n = { x: 0, y: -1 }; break; case "top-right": n = { x: .5, y: -.5 }; break; case "right": n = { x: 1, y: -0 }; break; case "bottom-right": n = { x: .5, y: .5 }; break; case "bottom": n = { x: 0, y: 1 }; break; case "bottom-left": n = { x: -.5, y: 1 }; break; case "left": n = { x: -1, y: 0 }; break; case "top-left": n = { x: -.5, y: -.5 }; break; default: n = { x: 0, y: 0 } } i.particles.move.straight ? (this.vx = n.x, this.vy = n.y, i.particles.move.random && (this.vx = this.vx * Math.random(), this.vy = this.vy * Math.random())) : (this.vx = n.x + Math.random() - .5, this.vy = n.y + Math.random() - .5), this.vx_i = this.vx, this.vy_i = this.vy; var r = i.particles.shape.type; if ("object" == typeof r) { if (r instanceof Array) { var c = r[Math.floor(Math.random() * r.length)]; this.shape = c } } else this.shape = r; if ("image" == this.shape) { var o = i.particles.shape; this.img = { src: o.image.src, ratio: o.image.width / o.image.height }, this.img.ratio || (this.img.ratio = 1), "svg" == i.tmp.img_type && void 0 != i.tmp.source_svg && (i.fn.vendors.createSvgImg(this), i.tmp.pushing && (this.img.loaded = !1)) } }, i.fn.particle.prototype.draw = function () { function e() { i.canvas.ctx.drawImage(r, a.x - t, a.y - t, 2 * t, 2 * t / a.img.ratio) } var a = this; if (void 0 != a.radius_bubble) var t = a.radius_bubble; else var t = a.radius; if (void 0 != a.opacity_bubble) var s = a.opacity_bubble; else var s = a.opacity; if (a.color.rgb) var n = "rgba(" + a.color.rgb.r + "," + a.color.rgb.g + "," + a.color.rgb.b + "," + s + ")"; else var n = "hsla(" + a.color.hsl.h + "," + a.color.hsl.s + "%," + a.color.hsl.l + "%," + s + ")"; switch (i.canvas.ctx.fillStyle = n, i.canvas.ctx.beginPath(), a.shape) { case "circle": i.canvas.ctx.arc(a.x, a.y, t, 0, 2 * Math.PI, !1); break; case "edge": i.canvas.ctx.rect(a.x - t, a.y - t, 2 * t, 2 * t); break; case "triangle": i.fn.vendors.drawShape(i.canvas.ctx, a.x - t, a.y + t / 1.66, 2 * t, 3, 2); break; case "polygon": i.fn.vendors.drawShape(i.canvas.ctx, a.x - t / (i.particles.shape.polygon.nb_sides / 3.5), a.y - t / .76, 2.66 * t / (i.particles.shape.polygon.nb_sides / 3), i.particles.shape.polygon.nb_sides, 1); break; case "star": i.fn.vendors.drawShape(i.canvas.ctx, a.x - 2 * t / (i.particles.shape.polygon.nb_sides / 4), a.y - t / 1.52, 2 * t * 2.66 / (i.particles.shape.polygon.nb_sides / 3), i.particles.shape.polygon.nb_sides, 2); break; case "image": if ("svg" == i.tmp.img_type) var r = a.img.obj; else var r = i.tmp.img_obj; r && e() } i.canvas.ctx.closePath(), i.particles.shape.stroke.width > 0 && (i.canvas.ctx.strokeStyle = i.particles.shape.stroke.color, i.canvas.ctx.lineWidth = i.particles.shape.stroke.width, i.canvas.ctx.stroke()), i.canvas.ctx.fill() }, i.fn.particlesCreate = function () { for (var e = 0; e < i.particles.number.value; e++) i.particles.array.push(new i.fn.particle(i.particles.color, i.particles.opacity.value)) }, i.fn.particlesUpdate = function () { for (var e = 0; e < i.particles.array.length; e++) { var a = i.particles.array[e]; if (i.particles.move.enable) { var t = i.particles.move.speed / 2; a.x += a.vx * t, a.y += a.vy * t } if (i.particles.opacity.anim.enable && (1 == a.opacity_status ? (a.opacity >= i.particles.opacity.value && (a.opacity_status = !1), a.opacity += a.vo) : (a.opacity <= i.particles.opacity.anim.opacity_min && (a.opacity_status = !0), a.opacity -= a.vo), a.opacity < 0 && (a.opacity = 0)), i.particles.size.anim.enable && (1 == a.size_status ? (a.radius >= i.particles.size.value && (a.size_status = !1), a.radius += a.vs) : (a.radius <= i.particles.size.anim.size_min && (a.size_status = !0), a.radius -= a.vs), a.radius < 0 && (a.radius = 0)), "bounce" == i.particles.move.out_mode) var s = { x_left: a.radius, x_right: i.canvas.w, y_top: a.radius, y_bottom: i.canvas.h }; else var s = { x_left: -a.radius, x_right: i.canvas.w + a.radius, y_top: -a.radius, y_bottom: i.canvas.h + a.radius }; switch (a.x - a.radius > i.canvas.w ? (a.x = s.x_left, a.y = Math.random() * i.canvas.h) : a.x + a.radius < 0 && (a.x = s.x_right, a.y = Math.random() * i.canvas.h), a.y - a.radius > i.canvas.h ? (a.y = s.y_top, a.x = Math.random() * i.canvas.w) : a.y + a.radius < 0 && (a.y = s.y_bottom, a.x = Math.random() * i.canvas.w), i.particles.move.out_mode) { case "bounce": a.x + a.radius > i.canvas.w ? a.vx = -a.vx : a.x - a.radius < 0 && (a.vx = -a.vx), a.y + a.radius > i.canvas.h ? a.vy = -a.vy : a.y - a.radius < 0 && (a.vy = -a.vy) } if (isInArray("grab", i.interactivity.events.onhover.mode) && i.fn.modes.grabParticle(a), (isInArray("bubble", i.interactivity.events.onhover.mode) || isInArray("bubble", i.interactivity.events.onclick.mode)) && i.fn.modes.bubbleParticle(a), (isInArray("repulse", i.interactivity.events.onhover.mode) || isInArray("repulse", i.interactivity.events.onclick.mode)) && i.fn.modes.repulseParticle(a), i.particles.line_linked.enable || i.particles.move.attract.enable) for (var n = e + 1; n < i.particles.array.length; n++) { var r = i.particles.array[n]; i.particles.line_linked.enable && i.fn.interact.linkParticles(a, r), i.particles.move.attract.enable && i.fn.interact.attractParticles(a, r), i.particles.move.bounce && i.fn.interact.bounceParticles(a, r) } } }, i.fn.particlesDraw = function () { i.canvas.ctx.clearRect(0, 0, i.canvas.w, i.canvas.h), i.fn.particlesUpdate(); for (var e = 0; e < i.particles.array.length; e++) { var a = i.particles.array[e]; a.draw() } }, i.fn.particlesEmpty = function () { i.particles.array = [] }, i.fn.particlesRefresh = function () { cancelRequestAnimFrame(i.fn.checkAnimFrame), cancelRequestAnimFrame(i.fn.drawAnimFrame), i.tmp.source_svg = void 0, i.tmp.img_obj = void 0, i.tmp.count_svg = 0, i.fn.particlesEmpty(), i.fn.canvasClear(), i.fn.vendors.start() }, i.fn.interact.linkParticles = function (e, a) { var t = e.x - a.x, s = e.y - a.y, n = Math.sqrt(t * t + s * s); if (n <= i.particles.line_linked.distance) { var r = i.particles.line_linked.opacity - n / (1 / i.particles.line_linked.opacity) / i.particles.line_linked.distance; if (r > 0) { var c = i.particles.line_linked.color_rgb_line; i.canvas.ctx.strokeStyle = "rgba(" + c.r + "," + c.g + "," + c.b + "," + r + ")", i.canvas.ctx.lineWidth = i.particles.line_linked.width, i.canvas.ctx.beginPath(), i.canvas.ctx.moveTo(e.x, e.y), i.canvas.ctx.lineTo(a.x, a.y), i.canvas.ctx.stroke(), i.canvas.ctx.closePath() } } }, i.fn.interact.attractParticles = function (e, a) { var t = e.x - a.x, s = e.y - a.y, n = Math.sqrt(t * t + s * s); if (n <= i.particles.line_linked.distance) { var r = t / (1e3 * i.particles.move.attract.rotateX), c = s / (1e3 * i.particles.move.attract.rotateY); e.vx -= r, e.vy -= c, a.vx += r, a.vy += c } }, i.fn.interact.bounceParticles = function (e, a) { var t = e.x - a.x, i = e.y - a.y, s = Math.sqrt(t * t + i * i), n = e.radius + a.radius; n >= s && (e.vx = -e.vx, e.vy = -e.vy, a.vx = -a.vx, a.vy = -a.vy) }, i.fn.modes.pushParticles = function (e, a) { i.tmp.pushing = !0; for (var t = 0; e > t; t++) i.particles.array.push(new i.fn.particle(i.particles.color, i.particles.opacity.value, { x: a ? a.pos_x : Math.random() * i.canvas.w, y: a ? a.pos_y : Math.random() * i.canvas.h })), t == e - 1 && (i.particles.move.enable || i.fn.particlesDraw(), i.tmp.pushing = !1) }, i.fn.modes.removeParticles = function (e) { i.particles.array.splice(0, e), i.particles.move.enable || i.fn.particlesDraw() }, i.fn.modes.bubbleParticle = function (e) { function a() { e.opacity_bubble = e.opacity, e.radius_bubble = e.radius } function t(a, t, s, n, c) { if (a != t) if (i.tmp.bubble_duration_end) { if (void 0 != s) { var o = n - p * (n - a) / i.interactivity.modes.bubble.duration, l = a - o; d = a + l, "size" == c && (e.radius_bubble = d), "opacity" == c && (e.opacity_bubble = d) } } else if (r <= i.interactivity.modes.bubble.distance) { if (void 0 != s) var v = s; else var v = n; if (v != a) { var d = n - p * (n - a) / i.interactivity.modes.bubble.duration; "size" == c && (e.radius_bubble = d), "opacity" == c && (e.opacity_bubble = d) } } else "size" == c && (e.radius_bubble = void 0), "opacity" == c && (e.opacity_bubble = void 0) } if (i.interactivity.events.onhover.enable && isInArray("bubble", i.interactivity.events.onhover.mode)) { var s = e.x - i.interactivity.mouse.pos_x, n = e.y - i.interactivity.mouse.pos_y, r = Math.sqrt(s * s + n * n), c = 1 - r / i.interactivity.modes.bubble.distance; if (r <= i.interactivity.modes.bubble.distance) { if (c >= 0 && "mousemove" == i.interactivity.status) { if (i.interactivity.modes.bubble.size != i.particles.size.value) if (i.interactivity.modes.bubble.size > i.particles.size.value) { var o = e.radius + i.interactivity.modes.bubble.size * c; o >= 0 && (e.radius_bubble = o) } else { var l = e.radius - i.interactivity.modes.bubble.size, o = e.radius - l * c; o > 0 ? e.radius_bubble = o : e.radius_bubble = 0 } if (i.interactivity.modes.bubble.opacity != i.particles.opacity.value) if (i.interactivity.modes.bubble.opacity > i.particles.opacity.value) { var v = i.interactivity.modes.bubble.opacity * c; v > e.opacity && v <= i.interactivity.modes.bubble.opacity && (e.opacity_bubble = v) } else { var v = e.opacity - (i.particles.opacity.value - i.interactivity.modes.bubble.opacity) * c; v < e.opacity && v >= i.interactivity.modes.bubble.opacity && (e.opacity_bubble = v) } } } else a(); "mouseleave" == i.interactivity.status && a() } else if (i.interactivity.events.onclick.enable && isInArray("bubble", i.interactivity.events.onclick.mode)) { if (i.tmp.bubble_clicking) { var s = e.x - i.interactivity.mouse.click_pos_x, n = e.y - i.interactivity.mouse.click_pos_y, r = Math.sqrt(s * s + n * n), p = ((new Date).getTime() - i.interactivity.mouse.click_time) / 1e3; p > i.interactivity.modes.bubble.duration && (i.tmp.bubble_duration_end = !0), p > 2 * i.interactivity.modes.bubble.duration && (i.tmp.bubble_clicking = !1, i.tmp.bubble_duration_end = !1) } i.tmp.bubble_clicking && (t(i.interactivity.modes.bubble.size, i.particles.size.value, e.radius_bubble, e.radius, "size"), t(i.interactivity.modes.bubble.opacity, i.particles.opacity.value, e.opacity_bubble, e.opacity, "opacity")) } }, i.fn.modes.repulseParticle = function (e) { function a() { var a = Math.atan2(d, p); if (e.vx = u * Math.cos(a), e.vy = u * Math.sin(a), "bounce" == i.particles.move.out_mode) { var t = { x: e.x + e.vx, y: e.y + e.vy }; t.x + e.radius > i.canvas.w ? e.vx = -e.vx : t.x - e.radius < 0 && (e.vx = -e.vx), t.y + e.radius > i.canvas.h ? e.vy = -e.vy : t.y - e.radius < 0 && (e.vy = -e.vy) } } if (i.interactivity.events.onhover.enable && isInArray("repulse", i.interactivity.events.onhover.mode) && "mousemove" == i.interactivity.status) { var t = e.x - i.interactivity.mouse.pos_x, s = e.y - i.interactivity.mouse.pos_y, n = Math.sqrt(t * t + s * s), r = { x: t / n, y: s / n }, c = i.interactivity.modes.repulse.distance, o = 100, l = clamp(1 / c * (-1 * Math.pow(n / c, 2) + 1) * c * o, 0, 50), v = { x: e.x + r.x * l, y: e.y + r.y * l }; "bounce" == i.particles.move.out_mode ? (v.x - e.radius > 0 && v.x + e.radius < i.canvas.w && (e.x = v.x), v.y - e.radius > 0 && v.y + e.radius < i.canvas.h && (e.y = v.y)) : (e.x = v.x, e.y = v.y) } else if (i.interactivity.events.onclick.enable && isInArray("repulse", i.interactivity.events.onclick.mode)) if (i.tmp.repulse_finish || (i.tmp.repulse_count++, i.tmp.repulse_count == i.particles.array.length && (i.tmp.repulse_finish = !0)), i.tmp.repulse_clicking) { var c = Math.pow(i.interactivity.modes.repulse.distance / 6, 3), p = i.interactivity.mouse.click_pos_x - e.x, d = i.interactivity.mouse.click_pos_y - e.y, m = p * p + d * d, u = -c / m * 1; c >= m && a() } else 0 == i.tmp.repulse_clicking && (e.vx = e.vx_i, e.vy = e.vy_i) }, i.fn.modes.grabParticle = function (e) { if (i.interactivity.events.onhover.enable && "mousemove" == i.interactivity.status) { var a = e.x - i.interactivity.mouse.pos_x, t = e.y - i.interactivity.mouse.pos_y, s = Math.sqrt(a * a + t * t); if (s <= i.interactivity.modes.grab.distance) { var n = i.interactivity.modes.grab.line_linked.opacity - s / (1 / i.interactivity.modes.grab.line_linked.opacity) / i.interactivity.modes.grab.distance; if (n > 0) { var r = i.particles.line_linked.color_rgb_line; i.canvas.ctx.strokeStyle = "rgba(" + r.r + "," + r.g + "," + r.b + "," + n + ")", i.canvas.ctx.lineWidth = i.particles.line_linked.width, i.canvas.ctx.beginPath(), i.canvas.ctx.moveTo(e.x, e.y), i.canvas.ctx.lineTo(i.interactivity.mouse.pos_x, i.interactivity.mouse.pos_y), i.canvas.ctx.stroke(), i.canvas.ctx.closePath() } } } }, i.fn.vendors.eventsListeners = function () { "window" == i.interactivity.detect_on ? i.interactivity.el = window : i.interactivity.el = i.canvas.el, (i.interactivity.events.onhover.enable || i.interactivity.events.onclick.enable) && (i.interactivity.el.addEventListener("mousemove", function (e) { if (i.interactivity.el == window) var a = e.clientX, t = e.clientY; else var a = e.offsetX || e.clientX, t = e.offsetY || e.clientY; i.interactivity.mouse.pos_x = a, i.interactivity.mouse.pos_y = t, i.tmp.retina && (i.interactivity.mouse.pos_x *= i.canvas.pxratio, i.interactivity.mouse.pos_y *= i.canvas.pxratio), i.interactivity.status = "mousemove" }), i.interactivity.el.addEventListener("mouseleave", function (e) { i.interactivity.mouse.pos_x = null, i.interactivity.mouse.pos_y = null, i.interactivity.status = "mouseleave" })), i.interactivity.events.onclick.enable && i.interactivity.el.addEventListener("click", function () { if (i.interactivity.mouse.click_pos_x = i.interactivity.mouse.pos_x, i.interactivity.mouse.click_pos_y = i.interactivity.mouse.pos_y, i.interactivity.mouse.click_time = (new Date).getTime(), i.interactivity.events.onclick.enable) switch (i.interactivity.events.onclick.mode) { case "push": i.particles.move.enable ? i.fn.modes.pushParticles(i.interactivity.modes.push.particles_nb, i.interactivity.mouse) : 1 == i.interactivity.modes.push.particles_nb ? i.fn.modes.pushParticles(i.interactivity.modes.push.particles_nb, i.interactivity.mouse) : i.interactivity.modes.push.particles_nb > 1 && i.fn.modes.pushParticles(i.interactivity.modes.push.particles_nb); break; case "remove": i.fn.modes.removeParticles(i.interactivity.modes.remove.particles_nb); break; case "bubble": i.tmp.bubble_clicking = !0; break; case "repulse": i.tmp.repulse_clicking = !0, i.tmp.repulse_count = 0, i.tmp.repulse_finish = !1, setTimeout(function () { i.tmp.repulse_clicking = !1 }, 1e3 * i.interactivity.modes.repulse.duration) } }) }, i.fn.vendors.densityAutoParticles = function () { if (i.particles.number.density.enable) { var e = i.canvas.el.width * i.canvas.el.height / 1e3; i.tmp.retina && (e /= 2 * i.canvas.pxratio); var a = e * i.particles.number.value / i.particles.number.density.value_area, t = i.particles.array.length - a; 0 > t ? i.fn.modes.pushParticles(Math.abs(t)) : i.fn.modes.removeParticles(t) } }, i.fn.vendors.checkOverlap = function (e, a) { for (var t = 0; t < i.particles.array.length; t++) { var s = i.particles.array[t], n = e.x - s.x, r = e.y - s.y, c = Math.sqrt(n * n + r * r); c <= e.radius + s.radius && (e.x = a ? a.x : Math.random() * i.canvas.w, e.y = a ? a.y : Math.random() * i.canvas.h, i.fn.vendors.checkOverlap(e)) } }, i.fn.vendors.createSvgImg = function (e) { var a = i.tmp.source_svg, t = /#([0-9A-F]{3,6})/gi, s = a.replace(t, function (a, t, i, s) { if (e.color.rgb) var n = "rgba(" + e.color.rgb.r + "," + e.color.rgb.g + "," + e.color.rgb.b + "," + e.opacity + ")"; else var n = "hsla(" + e.color.hsl.h + "," + e.color.hsl.s + "%," + e.color.hsl.l + "%," + e.opacity + ")"; return n }), n = new Blob([s], { type: "image/svg+xml;charset=utf-8" }), r = window.URL || window.webkitURL || window, c = r.createObjectURL(n), o = new Image; o.addEventListener("load", function () { e.img.obj = o, e.img.loaded = !0, r.revokeObjectURL(c), i.tmp.count_svg++ }), o.src = c }, i.fn.vendors.destroypJS = function () { cancelAnimationFrame(i.fn.drawAnimFrame), t.remove(), pJSDom = null }, i.fn.vendors.drawShape = function (e, a, t, i, s, n) { var r = s * n, c = s / n, o = 180 * (c - 2) / c, l = Math.PI - Math.PI * o / 180; e.save(), e.beginPath(), e.translate(a, t), e.moveTo(0, 0); for (var v = 0; r > v; v++) e.lineTo(i, 0), e.translate(i, 0), e.rotate(l); e.fill(), e.restore() }, i.fn.vendors.exportImg = function () { window.open(i.canvas.el.toDataURL("image/png"), "_blank") }, i.fn.vendors.loadImg = function (e) { if (i.tmp.img_error = void 0, "" != i.particles.shape.image.src) if ("svg" == e) { var a = new XMLHttpRequest; a.open("GET", i.particles.shape.image.src), a.onreadystatechange = function (e) { 4 == a.readyState && (200 == a.status ? (i.tmp.source_svg = e.currentTarget.response, i.fn.vendors.checkBeforeDraw()) : (console.log("Error pJS - Image not found"), i.tmp.img_error = !0)) }, a.send() } else { var t = new Image; t.addEventListener("load", function () { i.tmp.img_obj = t, i.fn.vendors.checkBeforeDraw() }), t.src = i.particles.shape.image.src } else console.log("Error pJS - No image.src"), i.tmp.img_error = !0 }, i.fn.vendors.draw = function () { "image" == i.particles.shape.type ? "svg" == i.tmp.img_type ? i.tmp.count_svg >= i.particles.number.value ? (i.fn.particlesDraw(), i.particles.move.enable ? i.fn.drawAnimFrame = requestAnimFrame(i.fn.vendors.draw) : cancelRequestAnimFrame(i.fn.drawAnimFrame)) : i.tmp.img_error || (i.fn.drawAnimFrame = requestAnimFrame(i.fn.vendors.draw)) : void 0 != i.tmp.img_obj ? (i.fn.particlesDraw(), i.particles.move.enable ? i.fn.drawAnimFrame = requestAnimFrame(i.fn.vendors.draw) : cancelRequestAnimFrame(i.fn.drawAnimFrame)) : i.tmp.img_error || (i.fn.drawAnimFrame = requestAnimFrame(i.fn.vendors.draw)) : (i.fn.particlesDraw(), i.particles.move.enable ? i.fn.drawAnimFrame = requestAnimFrame(i.fn.vendors.draw) : cancelRequestAnimFrame(i.fn.drawAnimFrame)) }, i.fn.vendors.checkBeforeDraw = function () { "image" == i.particles.shape.type ? "svg" == i.tmp.img_type && void 0 == i.tmp.source_svg ? i.tmp.checkAnimFrame = requestAnimFrame(check) : (cancelRequestAnimFrame(i.tmp.checkAnimFrame), i.tmp.img_error || (i.fn.vendors.init(), i.fn.vendors.draw())) : (i.fn.vendors.init(), i.fn.vendors.draw()) }, i.fn.vendors.init = function () { i.fn.retinaInit(), i.fn.canvasInit(), i.fn.canvasSize(), i.fn.canvasPaint(), i.fn.particlesCreate(), i.fn.vendors.densityAutoParticles(), i.particles.line_linked.color_rgb_line = hexToRgb(i.particles.line_linked.color) }, i.fn.vendors.start = function () { isInArray("image", i.particles.shape.type) ? (i.tmp.img_type = i.particles.shape.image.src.substr(i.particles.shape.image.src.length - 3), i.fn.vendors.loadImg(i.tmp.img_type)) : i.fn.vendors.checkBeforeDraw() }, i.fn.vendors.eventsListeners(), i.fn.vendors.start() }; Object.deepExtend = function (e, a) { for (var t in a) a[t] && a[t].constructor && a[t].constructor === Object ? (e[t] = e[t] || {}, arguments.callee(e[t], a[t])) : e[t] = a[t]; return e }, window.requestAnimFrame = function () { return window.requestAnimationFrame || window.webkitRequestAnimationFrame || window.mozRequestAnimationFrame || window.oRequestAnimationFrame || window.msRequestAnimationFrame || function (e) { window.setTimeout(e, 1e3 / 60) } }(), window.cancelRequestAnimFrame = function () { return window.cancelAnimationFrame || window.webkitCancelRequestAnimationFrame || window.mozCancelRequestAnimationFrame || window.oCancelRequestAnimationFrame || window.msCancelRequestAnimationFrame || clearTimeout }(), window.pJSDom = [], window.particlesJS = function (e, a) { "string" != typeof e && (a = e, e = "particles-js"), e || (e = "particles-js"); var t = document.getElementById(e), i = "particles-js-canvas-el", s = t.getElementsByClassName(i); if (s.length) for (; s.length > 0;) t.removeChild(s[0]); var n = document.createElement("canvas"); n.className = i, n.style.width = "100%", n.style.height = "100%"; var r = document.getElementById(e).appendChild(n); null != r && pJSDom.push(new pJS(e, a)) }, window.particlesJS.load = function (e, a, t) { var i = new XMLHttpRequest; i.open("GET", a), i.onreadystatechange = function (a) { if (4 == i.readyState) if (200 == i.status) { var s = JSON.parse(a.currentTarget.response); window.particlesJS(e, s), t && t() } else console.log("Error pJS - XMLHttpRequest status: " + i.status), console.log("Error pJS - File config not found") }, i.send() };
+    </script>
+    <script>
+        var rem = Number($('html').css('font-size').replace('px', ''));
+        function log(value) { console.log(value); }
+        function particles() { $('#particles-js').toggle(); }
+    </script>
      <!--separatrix-->
      <style>
@@ Line 80: / Line 109: @@
          }
-         table {
+         #notebook  table {
              font-size: 1rem;
              padding: 0;
          }
-         table caption {
+         #notebook  table caption {
              border-top: 0.1rem solid;
              border-bottom: 0.1rem solid;
@@ Line 94: / Line 123: @@
          }
-         table tr {
+         #notebook  table tr {
              margin: 0;
              padding: 0;
@@ Line 100: / Line 129: @@
          }
-         table td {
+         #notebook  table td {
              border: none;
              margin: 0;
@@ Line 722: / Line 751: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/e/e9/T--NKU_China--notebook-1-5-1.png">
+                                     <img src="../image/Notebook/notebook-1-5-1.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 801: / Line 830: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/d/de/T--NKU_China--notebook-1-5-2.jpeg">
+                                     <img src="../image/Notebook/notebook-1-5-2.jpeg">
                                      <figcaption>
                                          <div class="p"><span>Selection of single-crossover clones by PCR</span></div>
@@ Line 898: / Line 927: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/3/3d/T--NKU_China--notebook-1-5-3.png">
+                                     <img src="../image/Notebook/notebook-1-5-3.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of double-crossover clones by PCR</span></div>
@@ Line 991: / Line 1,020: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/9/93/T--NKU_China--notebook-1-5-4.png">
+                                     <img src="../image/Notebook/notebook-1-5-4.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of single-crossover clones by PCR</span></div>
@@ Line 1,075: / Line 1,104: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-1-5-5.png">
+                                     <img src="../image/Notebook/notebook-1-5-5.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of &Delta;<i>luxS</i> clones by PCR</span></div>
@@ Line 1,280: / Line 1,309: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/7/72/T--NKU_China--notebook-1-5-6.png">
+                                     <img src="../image/Notebook/notebook-1-5-6.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 1,322: / Line 1,351: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/9/91/T--NKU_China--notebook-1-5-7.png">
+                                     <img src="../image/Notebook/notebook-1-5-7.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 1,501: / Line 1,530: @@
                                      </div>
                                      <figure>
-                                         <img src="https://static.igem.org/mediawiki/2016/d/dc/T--NKU_China--notebook-1-5-8.png">
+                                         <img src="../image/Notebook/notebook-1-5-8.png">
                                          <figcaption>
                                              <div class="p"><span>PCR cloning product of promoter <i>C2</i> and gene <i>luxS</i></span></div>
@@ Line 1,688: / Line 1,717: @@
                                      </div>
                                      <figure>
-                                         <img src="https://static.igem.org/mediawiki/2016/c/c9/T--NKU_China--notebook-1-5-9.png">
+                                         <img src="../image/Notebook/notebook-1-5-9.png">
                                          <figcaption>
                                              <div class="p"><span>PCR cloning product of promoter <i>C2</i> and gene <i>luxS</i></span></div>
@@ Line 1,729: / Line 1,758: @@
                                      </div>
                                      <figure>
-                                         <img src="https://static.igem.org/mediawiki/2016/f/fb/T--NKU_China--notebook-1-5-10.png">
+                                         <img src="../image/Notebook/notebook-1-5-10.png">
                                          <figcaption>
                                              <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 1,877: / Line 1,906: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/e/e0/T--NKU_China--notebook-6-13-1.png">
+                                     <img src="../image/Notebook/notebook-6-13-1.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by colony PCR</span></div>
@@ Line 1,915: / Line 1,944: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/e/eb/T--NKU_China--notebook-6-13-2.png">
+                                     <img src="../image/Notebook/notebook-6-13-2.png">
                                      <figcaption>
                                      </figcaption>
@@ Line 2,007: / Line 2,036: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/3/3a/T--NKU_China--notebook-6-13-3.png">
+                                     <img src="../image/Notebook/notebook-6-13-3.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of gene <i>luxS</i></span></div>
@@ Line 2,152: / Line 2,181: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/4/49/T--NKU_China--notebook-6-13-4.png">
+                                     <img src="../image/Notebook/notebook-6-13-4.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by colony PCR</span></div>
@@ Line 2,193: / Line 2,222: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/3/35/T--NKU_China--notebook-6-13-5.png">
+                                     <img src="../image/Notebook/notebook-6-13-5.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 2,267: / Line 2,296: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-6-13-6.png">
+                                     <img src="../image/Notebook/notebook-6-13-6.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by colony PCR</span></div>
@@ Line 2,337: / Line 2,366: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/1/17/T--NKU_China--notebook-6-13-7.png">
+                                     <img src="../image/Notebook/notebook-6-13-7.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by colony PCR</span></div>
@@ Line 2,363: / Line 2,392: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/c/c2/T--NKU_China--notebook-6-13-8.png">
+                                     <img src="../image/Notebook/notebook-6-13-8.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by colony PCR</span></div>
@@ Line 2,370: / Line 2,399: @@
                                  </figure>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/7/7a/T--NKU_China--notebook-6-13-9.png">
+                                     <img src="../image/Notebook/notebook-6-13-9.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 2,430: / Line 2,459: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/a/ad/T--NKU_China--notebook-6-13-10.png">
+                                     <img src="../image/Notebook/notebook-6-13-10.png">
                                      <figcaption>
                                          <div class="p"><span>OD<sub>600</sub> of different groups at specific times</span></div>
@@ Line 2,436: / Line 2,465: @@
                                  </figure>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/0/00/T--NKU_China--notebook-6-13-11.png">
+                                     <img src="../image/Notebook/notebook-6-13-11.png">
                                      <figcaption>
                                          <div class="p"><span>Growth curve of GR286 and GR286&Delta;<i>luxS</i></span></div>
@@ Line 2,442: / Line 2,471: @@
                                  </figure>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/a/a9/T--NKU_China--notebook-6-13-12.png">
+                                     <img src="../image/Notebook/notebook-6-13-12.png">
                                      <figcaption>
                                          <div class="p"><span>Growth curve of pWH-<i>luxS</i>, pWH-<i>luxS</i> + xyl and pWH1520</span></div>
@@ Line 2,448: / Line 2,477: @@
                                  </figure>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/6/6b/T--NKU_China--notebook-6-13-13.png">
+                                     <img src="../image/Notebook/notebook-6-13-13.png">
                                      <figcaption>
                                          <div class="p"><span>Growth curve of pHT-<i>lsrACDB</i> and pHT-01</span></div>
@@ Line 2,461: / Line 2,490: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/a/a4/T--NKU_China--notebook-6-13-14.png">
+                                     <img src="../image/Notebook/notebook-6-13-14.png">
                                      <figcaption>
                                          <div class="p"><span>Fluorescence intensity of different groups at specific times</span></div>
@@ Line 2,467: / Line 2,496: @@
                                  </figure>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-15.png">
+                                     <img src="../image/Notebook/notebook-6-13-15.png">
                                      <figcaption>
                                          <div class="p"><span>Relative fluorescence intensity of different groups</span></div>
@@ Line 2,493: / Line 2,522: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-16.png">
+                                     <img src="../image/Notebook/notebook-6-13-16.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 2,507: / Line 2,536: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/f/f1/T--NKU_China--notebook-6-13-17.png">
+                                     <img src="../image/Notebook/notebook-6-13-17.png">
                                      <figcaption>
                                          <div class="p"><span>Fluorescence intensity of BB170 and co-culture medium</span></div>
@@ Line 2,813: / Line 2,842: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/f/f7/T--NKU_China--notebook-14-17-01.png">
+                                     <img src="../image/Notebook/notebook-14-17-01.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of gene <i>luxS</i></span></div>
@@ Line 2,884: / Line 2,913: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/6/66/T--NKU_China--notebook-14-17-02.png">
+                                     <img src="../image/Notebook/notebook-14-17-02.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of gene <i>mtn</i></span></div>
@@ Line 2,962: / Line 2,991: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/e/e4/T--NKU_China--notebook-14-17-03.png">
+                                     <img src="../image/Notebook/notebook-14-17-03.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 3,008: / Line 3,037: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/8/83/T--NKU_China--notebook-14-17-04.png">
+                                     <img src="../image/Notebook/notebook-14-17-04.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 3,116: / Line 3,145: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/b/bc/T--NKU_China--notebook-14-17-05.png">
+                                     <img src="../image/Notebook/notebook-14-17-05.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of gene <i>luxS</i></span></div>
@@ Line 3,295: / Line 3,324: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/6/65/T--NKU_China--notebook-14-17-06.png">
+                                     <img src="../image/Notebook/notebook-14-17-06.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 3,340: / Line 3,369: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/c/ca/T--NKU_China--notebook-14-17-07.png">
+                                     <img src="../image/Notebook/notebook-14-17-07.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 3,448: / Line 3,477: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/b/b6/T--NKU_China--notebook-14-17-08.png">
+                                     <img src="../image/Notebook/notebook-14-17-08.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of segment <i>lsrACDB1</i>,<i>lsrACDB1</i>2</span></div>
@@ Line 3,462: / Line 3,491: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/e/e1/T--NKU_China--notebook-14-17-09.png">
+                                     <img src="../image/Notebook/notebook-14-17-09.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 3,507: / Line 3,536: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/5/5d/T--NKU_China--notebook-14-17-10.png">
+                                     <img src="../image/Notebook/notebook-14-17-10.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 3,643: / Line 3,672: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/6/69/T--NKU_China--notebook-14-17-11.png">
+                                     <img src="../image/Notebook/notebook-14-17-11.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of segment <i>lsrACDBFG</i>1,<i>lsrACDBFG</i>2, <i>lsrACDBFG</i>3</span>
@@ Line 3,656: / Line 3,685: @@
                              <div class="flex-container">
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/9/97/T--NKU_China--notebook-14-17-12.png">
+                                     <img src="../image/Notebook/notebook-14-17-12.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 3,701: / Line 3,730: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/3/33/T--NKU_China--notebook-14-17-13.png">
+                                     <img src="../image/Notebook/notebook-14-17-13.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 3,811: / Line 3,840: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/5/5c/T--NKU_China--notebook-14-17-14.png">
+                                     <img src="../image/Notebook/notebook-14-17-14.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of gene <i>lsrFG</i></span></div>
@@ Line 3,992: / Line 4,021: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/5/5b/T--NKU_China--notebook-14-17-15.png">
+                                     <img src="../image/Notebook/notebook-14-17-15.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 4,038: / Line 4,067: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/b/b0/T--NKU_China--notebook-14-17-16.png">
+                                     <img src="../image/Notebook/notebook-14-17-16.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 4,116: / Line 4,145: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/8/85/T--NKU_China--notebook-14-17-17.png">
+                                     <img src="../image/Notebook/notebook-14-17-17.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of gene <i>lsrK</i></span></div>
@@ Line 4,297: / Line 4,326: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/c/cb/T--NKU_China--notebook-14-17-18.png">
+                                     <img src="../image/Notebook/notebook-14-17-18.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 4,343: / Line 4,372: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/5/56/T--NKU_China--notebook-14-17-19.png">
+                                     <img src="../image/Notebook/notebook-14-17-19.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 4,421: / Line 4,450: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/8/85/T--NKU_China--notebook-14-17-17.png">
+                                     <img src="../image/Notebook/notebook-14-17-17.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of gene <i>lsrK</i></span></div>
@@ Line 4,602: / Line 4,631: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/7/78/T--NKU_China--notebook-14-17-20.png">
+                                     <img src="../image/Notebook/notebook-14-17-20.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 4,648: / Line 4,677: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/e/e2/T--NKU_China--notebook-14-17-21.png">
+                                     <img src="../image/Notebook/notebook-14-17-21.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>
@@ Line 4,747: / Line 4,776: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/2/24/T--NKU_China--notebook-14-17-22.png">
+                                     <img src="../image/Notebook/notebook-14-17-22.png">
                                      <figcaption>
                                          <div class="p"><span>PCR cloning product of gene <i>lsrK</i></span></div>
@@ Line 4,928: / Line 4,957: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/3/30/T--NKU_China--notebook-14-17-23.png">
+                                     <img src="../image/Notebook/notebook-14-17-23.png">
                                      <figcaption>
                                          <div class="p"><span>Selection of positive clones by PCR</span></div>
@@ Line 4,973: / Line 5,002: @@
                                  </div>
                                  <figure>
-                                     <img src="https://static.igem.org/mediawiki/2016/2/22/T--NKU_China--notebook-14-17-24.png">
+                                     <img src="../image/Notebook/notebook-14-17-24.png">
                                      <figcaption>
                                          <div class="p"><span>Restriction enzyme digestion verification</span></div>