Team:SKLMT-China/Notebook Overview

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week1-4

Team registration and brainstorm

During this time, we registrated the team SKLMT-China in iGEM. In order to verify the feasibility of our project, we discussed with professors and experts and did some human practice work.

week5

Find promoters with various strength in the light of transcriptom references. Take the sequence from the upstream of a specific gene.

Number	Promoter	Sequence 5'-3'	Length
01	PampC	ACGCCGGGGCCGCAAAGCCGCTGGGACAAACGCGGCGTACATAACCAGAACCCAGGGAAACCAACCAATC	70
02	ParaA	CTACGACGGCTTCCTGACCCTATAGCTCTAGCAACACCCCCGATTGTTTTGTCTCTGGAGGATCACAGTA	70
03	PfabD	TGTCAGTTTCTTGCGTCCACCACGGTGTGACGTCAATTCTCCGACGACAAGATCATTAGGGGCTTGTTAC	70
04	PccmD	AGGTGCTCAAGCGTGAAGCCCGGACCAGTTGGGTCAAGGCCGAAGTCCAACGCAGCCTGGAGGCCGCCCG	70
05	PfliA	CGGCGAACCCACGCGGGCATCTGGAGTTTTTTGTCGAGCGGCTAGTGCATCAAACAGCGGGACCGGTGCT	70
06	PltR	GGCTGTCATGATCTATTTGTAATTTGCCTTTACAAAAATAAGACACTAAAAATTCTAAAAGGATTTAGGA	70
07	PproC	GCGCTGTTTGGTGCCCGCGACTACAGTCAGTCCTGATGCGACTGACCCCGACCTCTTGTAAGGACCTGTC	70
08	PrecA	GCTGTGGAATAATACTGGCTACTTATACAGGTGTTGGCCGTCAGGCCTTATTGATTACGTGAGGACTTTA	70
09	PrpoD	TGAGGTCATAGCTCGGGTATAATCCTCGGCTTGTTTTTTGCCCGCCAAGACCTTCAGTGGATAGGGTGTT	70
10	PrpoS	AGGCTCCAGCGTTGCCAGGGATAAAGGCGCCGCTTGAGCCTGAGGTCGAACTCACCAAAGGACTATAACA	70
11	PdnaA	TAAGTGGGCGGCTGATCGCTACAATGGCCGCTTGTTTTTGCCTCACCGGCTTTCAACTTAGGGGATATCC	70
12	Ppol	ACCTTGTCCCTTTGTCGTCAACGGTCCCGCTATTCTAGCGCCGGACCCGCCGAAAAGGTTGATCCCGCTC	70
13	P16s	CGAGAAATCAAAGATGTAACCAACGATTGCTGAGCCAAGTTTAGGGTTTTCTCAAAACCCAAAGATGTTT	70
14	Psig	TGCCAAATCGATAAAGCCTCAATGACAATGCGACTAATTATCATTAGTGACACTCAGGCAGTCCGCGTCC	70
15	Patf	GTCGCCGGGCGTAAAGTGATCGCCGTTTGCGCGGCCCGATTGTCCGGCCGCCCGAGTAAGGAGTCTTATC	70
16	Pfme	ATGACCGTCTTGCAAAGGCTTGCTACGGCAAGCCTTTTTCTTTTCCGCATGTTTAACATCGAGAATTCCC	70
17	Pmem	GGTTAATCCGGGGTTTGTCGTTTCTGCCCGCCCCGCTATCATTCGCCGCATCTTTCGAGGGGTATGACCG	70
18	Pfat	CGTTTACGGCTAGCGCCCGGAGCCGGGGCTCGGGGCAGGATGCACACATCACTGACCAGAGGATGGAATC	70
19	Pami	GGTTTGGGGTTGCGCACTAACTTGCGCAGTGGCGATATAAGCGCCCTATAACAATTCCAGGGGTCGTTGT	70
20	Prib	TTCGCGCCCTAATTCGTGCGGTATTCAACAATAGTGTTGGGTGGCGGCACGCTAGCCTGAGGAATACACC	70
21	Pabc	CCCGCAAATAAAATTGCCGGCCCAGGCCGGCAGGCGCCCGGGGTCGGGCGACTTACAAGGAGCTTGTTGG	70
22	Pedo	TGAAGGCGCGCTGAGCGTGGCTGACCAGTTGATCGCCAAAGAGCGCGCAGCCAAGTAAGCGCCCGCCGCC	70
23	Ptox	AAAGACGCGGGCCTTGAGTGCTCTAACACCCAAGACCCGCTAACCACAAGCAGCTAAACAGGAGCTGAAT	70
24	Pdiv	GAGCTGACCCGGCGCACCCTCAAGCAAGGTCTGGAACTGCTGGGCCTCAAAGTCCTGGAGCAAATGTAAG	70
25	Plip	AGGTTCTGATGTTTTCCGGGCAAGGCCTGAAGTAAAAAAACCGGGGCTTCGGGCCCACGGGAGAAAAATA	70

week6

design the PAGE-purified oligonucleotides used to amplify the chloramphenicol

week7&8

Amplify the chloramphnicol and promoter segment and add vector homology arms via series PCR. Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH₂O. Quantify the elution using a NanoDrop UV spectrophotometer, store them at 4℃。

In order to obtain an promoter library suitable for cloning, promoter sequence flanked by homology arms was added a chloramphenicol selection marker. This sequence was synthesized by Sangon.

TABLE 2 Oligonucleotides used for PCR of the DNA segment containing the promoter and chloramphenicol selective marker

Primer	Sequence 5'-3'	length
Syn-primer 1	AATGAATTACAACAGTTTTTATGCAGATATCAATTAATTTGGTTATGTGTGGGAGGGCTA	60
ampC-2	TGGGTTCTGGTTATGTACGCCGCGTTTGTCCCAGCGGCTTTGCGGCCCCGGCGTATTAATGACGTTGATCGGCACGTAAG	80
araA-2	AGACAAAACAATCGGGGGTGTTGCTAGAGCTATAGGGTCAGGAAGCCGTCGTAGATTAATGACGTTGATCGGCACGTAAG	80
fabD-2	GATCTTGTCGTCGGAGAATTGACGTCACACCGTGGTGGACGCAAGAAACTGACAATTAATGACGTTGATCGGCACGTAAG	80
ccmD-2	TGCGTTGGACTTCGGCCTTGACCCAACTGGTCCGGGCTTCACGCTTGAGCACCTATTAATGACGTTGATCGGCACGTAAG	80
fliA-2	TTTGATGCACTAGCCGCTCGACAAAAAACTCCAGATGCCCGCGTGGGTTCGCCGATTAATGACGTTGATCGGCACGTAAG	80
pltR-2	AATTTTTAGTGTCTTATTTTTGTAAAGGCAAATTACAAATAGATCATGACAGCCATTAATGACGTTGATCGGCACGTAAG	80
proC-2	AGGTCGGGGTCAGTCGCATCAGGACTGACTGTAGTCGCGGGCACCAAACAGCGCATTAATGACGTTGATCGGCACGTAAG	80
recA-2	TCAATAAGGCCTGACGGCCAACACCTGTATAAGTAGCCAGTATTATTCCACAGCATTAATGACGTTGATCGGCACGTAAG	80
rpoD-2	AAGGTCTTGGCGGGCAAAAAACAAGCCGAGGATTATACCCGAGCTATGACCTCAATTAATGACGTTGATCGGCACGTAAG	80
rpoS-2	TGAGTTCGACCTCAGGCTCAAGCGGCGCCTTTATCCCTGGCAACGCTGGAGCCTATTAATGACGTTGATCGGCACGTAAG	8f0
dnaA-2	GAAAGCCGGTGAGGCAAAAACAAGCGGCCATTGTAGCGATCAGCCGCCCACTTAATTAATGACGTTGATCGGCACGTAAG	80
pol-2	TTCGGCGGGTCCGGCGCTAGAATAGCGGGACCGTTGACGACAAAGGGACAAGGtATTAATGACGTTGATCGGCACGTAAG	80
16s-2	TGAGAAAACCCTAAACTTGGCTCAGCAATCGTTGGTTACATCTTTGATTTCTCGATTAATGACGTTGATCGGCACGTAAG	80
sig-2	AGTGTCACTAATGATAATTAGTCGCATTGTCATTGAGGCTTTATCGATTTGGCAATTAATGACGTTGATCGGCACGTAAG	80
atf-2	CGGGCGGCCGGACAATCGGGCCGCGCAAACGGCGATCACTTTACGCCCGGCGACATTAATGACGTTGATCGGCACGTAAG	80
fme-2	AAACATGCGGAAAAGAAAAAGGCTTGCCGTAGCAAGCCTTTGCAAGACGGTCATATTAATGACGTTGATCGGCACGTAAG	80
mem-2	AAGATGCGGCGAATGATAGCGGGGCGGGCAGAAACGACAAACCCCGGATTAACCATTAATGACGTTGATCGGCACGTAAG	80
fat-2	CAGTGATGTGTGCATCCTGCCCCGAGCCCCGGCTCCGGGCGCTAGCCGTAAACGATTAATGACGTTGATCGGCACGTAAG	80
ami-2	TTGTTATAGGGCGCTTATATCGCCACTGCGCAAGTTAATGCGCAACCCCAAACCATTAATGACGTTGATCGGCACGTAAG	80
rib-2	TAGCGTGCCGCCACCCAACACTATTGTTGAATACCGCACGAATTAGGGCGCGAAATTAATGACGTTGATCGGCACGTAAG	80
abc-2	AAGTCGCCCGACCCCGGGCGCCTGCCGGCCTGGGCCGGCAATTTTATTTGCGGGATTAATGACGTTGATCGGCACGTAAG	80
edo-2	TTGGCTGCGCGCTCTTTGGCGATCAACTGGTCAGCCACGCTCAGCGCGCCTTCAATTAATGACGTTGATCGGCACGTAAG	80
tox-2	GCTGCTTGTGGTTAGCGGGTCTTGGGTGTTAGAGCACTCAAGGCCCGCGTCTTTATTAATGACGTTGATCGGCACGTAAG	80
div-2	GACTTTGAGGCCCAGCAGTTCCAGACCTTGCTTGAGGGTGCGCCGGGTCAGCTCATTAATGACGTTGATCGGCACGTAAG	80
lip-2	GCCCGAAGCCCCGGTTTTTTTACTTCAGGCCTTGCCCGGAAAACATCAGAACCTATTAATGACGTTGATCGGCACGTAAG	80

Set up the PCR reaction using pR6K-cm-ccdB as a template. Typically, set up the PCR reaction (50ul in total) to obtain sufficient amounts of DNA.

Component	Amount(ul)	Final
Autoclaved ddH2O	22
2×primeSTAR max premix	25	1×
Template, 50ng ul-1	1	50 ng
Syn-promoter-1 Oligo(10uM)	1	30 pmol
promoter-2 Oligo(10uM)	1	30 pmol
Total	50

Carry out the PCR in a thermal cycle following the instructions below:

Cycle number	Denaturation	Annealing	Termination
1	94℃,4min
2-31	98℃, 15s	59℃, 15s	72℃, 15s
32			72℃, 4min
33				10℃, hold

·Add vector homology arms to the PCR products purified.

·Use the purified PCR products from last step as a template. Use the PAGE-purified oligonucleotides ,Syn-promoter-3 and promoter-4 (Table3), to amplify the cm-promoter.

TABLE 3 Oligonucleotides used for PCR of the DNA segment containing the promoter and chloramphenicol selective marker

Primer	Sequence 5'-3'	length
Syn-primer 3	AATGAATTACAACAGTTTTTAT	22
ampC-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATGATTGGTTGGTTTCCCTGGGTTCTGGTTATGTACGC	76
araA-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTACTGTGATCCTCCAGAGACAAAACAATCGGGGGTG	76
fabD-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATGTAACAAGCCCCTAATGATCTTGTCGTCGGAGAATT	76
ccmD-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATCGGGCGGCCTCCAGGCTGCGTTGGACTTCGGCCTTGA	76
fliA-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATAGCACCGGTCCCGCTGTTTGATGCACTAGCCGCTCG	76
pltR-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTCCTAAATCCTTTTAGAATTTTTAGTGTCTTATTTT	76
proC-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATGACAGGTCCTTACAAGAGGTCGGGGTCAGTCGCATC	76
recA-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTAAAGTCCTCACGTAATCAATAAGGCCTGACGGCCA	76
rpoD-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATAACACCCTATCCACTGAAGGTCTTGGCGGGCAAAAA	76
rpoS-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTGTTATAGTCCTTTGGTGAGTTCGACCTCAGGCTCA	76
dnaA-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATggatatcccctaagttgaaagccggtgaggcaaaaa	76
pol-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATgagcgggatcaaccttttcggcgggtccggcgctag	76
16s-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATaaacatctttgggttttgagaaaaccctaaacttgg	76
sig-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATggacgcggactgcctgagtgtcactaatgataatta	76
atf-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATgataagactccttactcgggcggccggacaatcggg	80
fme-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATgggaattctcgatgttaaacatgcggaaaagaaaaa	76
mem-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATcggtcatacccctcgaaagatgcggcgaatgatagc	76
fat-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATgattccatcctctggtcagtgatgtgtgcatcctgc	76
ami-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATacaacgacccctggaattgttatagggcgcttatat	76
rib-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATggtgtattcctcaggctagcgtgccgccacccaaca	76
abc-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATccaacaagctccttgtaagtcgcccgaccccgggcg	76
edo-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATggcggcgggcgcttacttggctgcgcgctctttggc	76
tox-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATattcagctcctgtttagctgcttgtggttagcgggt	76
div-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATcttacatttgctccaggactttgaggcccagcagtt	76
lip-4	ATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATtatttttctcccgtgggcccgaagccccggtttttt	76

·Use the purified PCR products from step2 as a template. set up the PCR reaction (50ul in total) to obtain sufficient amounts of DNA

Component	Amount(ul)	Final
Autoclaved ddH2O	22
2×primeSTAR max premix	25	1×
Template, 50ng ul-1	1	50 ng
Syn-promoter-3 Oligo(10uM)	1	30 pmol
promoter-4 Oligo(10uM)	1	30 pmol
Total	50

Carry out the PCR in a thermal cycle following the instructions below:

Cycle number	Denaturation	Annealing	Termination
1	94℃,4min
2-31	98℃, 15s	52~59℃, 15s, 15s	72℃, 15s
32			72℃, 4min
33				10℃, hold

·Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH₂O. Quantify the elution using a NanoDrop UV spectrophotometer.

week9

Transform the plasmid pBBR1-kan-amp-ccdb-BAD-GFP-firefly into GB05 red gyrA462 via electroporation. Do Mini-Prep and screen correct plasmid by restriction analysis

week10

construct the plasmid pBBR1-kan-cm-promoter-firefly via liner circular recombination in GB05 red gyrA462.

Do Mini-Prep and screen correct recombinants by restriction analysis.

week11

Transform the plasmid pBBR1-kan-amp-ccdb-BAD-GFP-firefly into P.fluorescence pf-5 via electroporation.

·Use a syringe needle (25-Gauge 5/8, 0.5 mm × 16 mm) to puncture the cap of a 1.5-ml microcentrifuge tube for aeration and add 1.0 ml LB. Inoculate the medium in the 1.5-ml tube with the several colonies of P. fluorescence pf-5 cells and incubate at 30 °C overnight with shaking at 950 r.p.m. in an Eppendorf thermomixer.

·Puncture the cap of a 1.5-ml microcentrifuge tube, add 1.4 ml of LB inoculating with 40 ul of the overnight culture.

·Incubate the mixture at 30 °C for 2.5h with shaking at 950 r.p.m. in an Eppendorf thermomixer. At this stage, the OD600 should be between 0.35 and 0.40.

· Put the cells on ice (and keep them on the ice whenever possible).

·Spin the cells down at 9,000 rpm for 30 sec in the cooling centrifuge at 2°C.

·Discard the supernatant by decanting. Discard as much supernatant as possible.

· Resuspend the pellet in 1 ml of ice-cold 10% sucrose buffer (containing 2mM HEPES) by pipetting.

· Spin the cells at 10,000 rpm for 30 sec in the cooling centrifuge at 2°C.

·Discard the supernatant by decanting. Discard as much supernatant as possible.

· Resuspend the pellet in 1 ml of ice-cold sucrose buffer (containing 2mM HEPES) by pipetting.

· Spin the cells down at 11,000 rpm for 30 sec in the cooling centrifuge at 2°C.

· Discard the supernatant by decanting and leave about 30 µl.

· Add DNA to the cells and pipette the mixture into the chilled 1 mm electroporation cuvette.

· Set the electroporator to 1250 V, 10 µF, 600 Ohms. (This setting belongs to an Eppendorf® Electroporator 2510 using an electroporation cuvette with a gap of 1 mm. Other devices can be used, but the voltage has to be fixed at 1350V and the length of the pulse should be 5 ms.)

·Carefully knock the cuvette on the table to remove air bubbles and dry the metallic sides of the cuvette with a tissue. Do not touch the metallic sides with your hands.

·Place the cuvette into the holder of the electroporator, insert, and push the “pulse” button twice.

·Add 1 ml LB medium without antibiotics to the cuvette. Resuspend the cells carefully by pipetting up and down and pipette back into the reaction tube (avoid air bubbles in the suspension).

·Recover the culture at 37 °C in an Eppendorf thermomixer with shaking at 950 r.p.m. for 1 h 40min.

· Spin down the cells by centrifugation at 8,000g for 1 min at RT.

· Discard the supernatant by decanting, leaving about 100 ul of LB medium. Resuspend the cells by pipetting up and down.

· Streak the entire mixture with a sterile 10ul loop on one LB agar plate containing 15 ug ml⁻¹ chloramphenicol and 10ug ml⁻¹kanamycin.

week12

Chracterize the strength of different promoters by luciferase firefly assay

preparation of the overnight p.fluorescence culture transformed successfully

Inoculate the medium in the 1.5-ml tube with the several colonies of p.fluorescence cells that has been transformed successfully and incubate at 30 °C overnight with shaking at 950 r.p.m. in an Eppendorf thermomixer.

·Puncture the cap of a 1.5-ml microcentrifuge tube, add 1.8 ml of LB containing 5ug ml−1 chloramphenicol and inoculate with 150 ul of the overnight culture.

·Incubate the mixture at 30 °C for 2h with shaking at 950 r.p.m. in an Eppendorf thermomixer.

·Adjust the mixture to OD600 0.2

Preparation of bacterial cell lysate

·Add 10ul of 1M K2HPO4(Ph7.8) and 20mM EDTA to 90ul of transformed bacteria in a new 1.5ml microcentrifuge tube.

·Quick-freeze on dry ice, and then equilibrate to room temperature by placing the tube in room temperature water.

·Add 300ul freshly prepared lysis mix (1 volume of freshly prepared lysozyme and 2volumes of 2×CCLR with 5mg/ml BSA). Mix and incubate for 10 minutes at room temperature.

· Mix 20ul of cell lysate with 100ul of luciferase assay reafent and measure the light produced.

Measrue the light produced using GloMaxTM 96 Microplate Luminometer

Colleborate with seven Chinese iGEM teams to characterize the promoter strength under differdent conditions.

week13

modeling

Digest Pseudomonas putida S16 genome with SpeI restriction enzyme.

The purpose of this work is to achieve nicotine degradation in Pseudomonas fluorescences with the nicotine degradation gene cluster of Pseudomonas putida S16. Besides, we want to test if our promoter library could really work in regulating gene expression. The expression of nicotine degration gene cluster will be detected by the change of nicotine’s content through HPLC.

·Digest 50ug of S16 genome DNA with SpeI restriction enzyme in 400ul and incubate it at 37℃. Mix the following in a 1.5-ml microcentrifuge tube:

·Incubate the mix in 37℃ for 3h

·Add 400ul of phenol-chloroform-isoamyl alcohol mixture(25:24:1)to the 400ul digestion mixture from Step.1, mix it thoroughly by inversion to create an emulsion and centrifuge it at 9400r for 10min at RT.

·Using an end-cut wide-bore 1ml tip, gently pipette 300ul of the aqueous phase(top layer)into a new 1.5ml microcentrifuge tube.

·Add 20ul(1/15 volumes) of 3M sodium acetate(PH 7.5)and gently invert to mix.

· Add 800ul(2.5 volumes) of absolute ethanol and gently invert to mix.

· Centrifuge at 9400r for 2min at RT to pellet the genomic DNA.

·Discard the supernatant and invert the tube on a paper towl to let all of the liquid drain off the pellet.

· Wash the pellet by carefully adding 1ml of 70%(vol/vol)ethanol and centrifuge it at 9400r for 1min at RT.

·Discard the supernatant and invert the tube on a paper towl to let all of liquid drain off the pellet.

·Dry the pellet without shaking on an Eppendorf thermomixer at 45℃ for 10min until no liquid is left.

·Resuspend the DNA pellet in 12ul of autoclaved ddH2O and use 2ul to measure the concentration using the NanoDrop UV spectrophotometer. The concentration is 1ug/ul.

· Keep the DNA pellet at 4℃.

week14

Preparation of the p15A-Cm-Vector

·the PAGE-purified oligonucleotides used to amplify the P15A-vector were P15A-primer-F and P15A-primer-R

TABLE 2 Oligonucleotides used for PCR of the DNA segment

·Set up the PCR reaction using p15A-cm-tetR-tetO-hyg as a template. Typically, set up the PCR reaction (50ul in total) to obtain sufficient amounts of DNA.

· Carry out the PCR in a thermal cycle following the instructions below:

·Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH2O. Quantify the elution using a NanoDrop UV spectrophotometer.

week15

Preparation of the pBBR1-km-Vector

·In order to make sure this DNA segment could replicate and be delivered to the next generation in Pseudomonas fluorescences, we need to transfer this gene cluster into a kind of shuttle vector called pBBR1-km-vector.

·Digest 50ug of pBBR1-km-hyg-ccdB plasmid with BamhI restriction enzyme in 400ul and incubate it at 37℃. Mix the following in a 1.5-ml microcentrifuge tube:

·Incubate the mix in 37℃ for 3h

·Add 400ul of phenol-chloroform-isoamyl alcohol mixture(25:24:1)to the 400ul digestion mixture from Step.1, mix it thoroughly by inversion to create an emulsion and centrifuge it at 9400r for 10min at RT.

·Using an end-cut wide-bore 1ml tip, gently pipette 300ul of the aqueous phase(top layer)into a new 1.5ml microcentrifuge tube.

· Add 20ul(1/15 volumes) of 3M sodium acetate(PH 7.5)and gently invert to mix.

· Add 800ul(2.5 volumes) of absolute ethanol and gently invert to mix.

·Centrifuge at 9400r for 2min at RT to pellet the genomic DNA.

·Discard the supernatant and invert the tube on a paper towl to let all of the liquid drain off the pellet.

· Wash the pellet by carefully adding 1ml of 70%(vol/vol)ethanol and centrifuge it at 9400r for 1min at RT.

·Discard the supernatant and invert the tube on a paper towl to let all of liquid drain off the pellet.

·Dry the pellet without shaking on an Eppendorf thermomixer at 45℃ for 10min until no liquid is left.

· Resuspend the DNA pellet in 12ul of autoclaved ddH2O and use 2ul to measure the concentration using the NanoDrop UV spectrophotometer. The concentration is 1ug/ul.

·Keep the DNA pellet at 4℃.

week16

Preparation of the amp-segment

In order to eliminate the influence of a segment similar to a part of our promoter sequence in liner-circular-recombination and two homology arms was designed. This sequence was synthesized by sangon .

The PAGE-purified oligonucleotides used to amplify the amp-biotics segment were amp-primer-F and amp-primer-R

Set up the PCR reaction using pR6k-amp-ccdB as a template. Typically, set up the PCR reaction (50ul in total) to obtain sufficient amounts of DNA.

Carry out the PCR in a thermal cycle following the instructions below:

Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH2O. Quantify the elution using a NanoDrop UV spectrophotometer.

week17

Preparation of the promoter segment

In order to test if our promoter could regulate gene expression in Pseudomonas fluorescences. Therefore we put promoter 4、5、11 in the beginning of NicA2 gene and six homology arms was designed. This sequence was synthesized by sangon..

1)the PAGE-purified oligonucleotides used to amplify the promoter segment were primer promoter4F、promoter4R、promoter5F、promoter5R、promoter11F and promoter11R .

primer	Sequence(5’-3’)
promoter4F	ttgtttttgttttatcactcatCGGGCGGCCTCCAGGCTGCGTTGGACTTCGGCCTTGA
promoter4R	ggtttacaactactacgacctaactagaaggcgccgggacTTACGCCCCGCCCTGCCAC
promoter5F
promoter5R
promoter11F	tgcggctaaagccttcatttgtttttgttttatcactcatcacggatatcccctaagtt
promoter11R	ggtttacaactactacgacctaactagaaggcgccgggacTTACGCCCCGCCCTGCCAC

Set up the PCR reaction using pSB1C3-promoter4-cm、pSB1C3-promoter5-cm and pSB1C3-promoter11-cm as a template. Typically, set up the PCR reaction (50ul in total) to obtain sufficient amounts of DNA.



Component	Amount(ul)	Final
Autoclaved ddH2O	22
2×primeSTAR max premix	25	1×
Template,50ng ul-1	1	50 ng
Promoter XF(10uM)(X=4、5、11)	1	30 pmol
Promoter XR(10uM)	1	30 pmol
Total	50

Carry out the PCR in a thermal cycle following the instructions below:



Cycle number	Denaturation	Annealing	Extension	Termination
1	94℃,4min
2-31	98℃, 15s	60℃, 15s	72℃, 10s
32			72℃, 4min
33				10℃, hold

Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH2O. Quantify the elution using a NanoDrop UV spectrophotometer.

week18

Improve pBBR1 vector with ampicillin resistance gene, and insert promoter sequence into the beginning of NicA2 gene in order to get the final plasmid---pBBR1-km-amp-cm-promoter-nic.

Set up the restriction digestion mixture and incubate at the suggested temperature for 2 h.

component	Amount(ul)	final
Autoclaved ddH2O	4.8	1×
10× Digestion buffer	1
DNA	4
Enzyme , 20 U ul−1	0.2	0.4 U ul−1
Total	10

Load the entire digestion mixture plus 2 ul of 6× loading dye in a 0.8% (wt/vol) agarose gel submerged in 1× TAE buffer. Run the gel using a field strength of ~4 V cm−1 for 30min.

Make a photo using the digital gel imaging system and check for release of the correct size fragment.

week19

Transfer the constructed plasmids----pBBR1-km-amp-cm-promoter-nic into Pseudomonas fluorescences and test their function with SDS-PAGE and Confocal Microscope.

week19

week20

week21

week22

week23

week24