Team:SKLMT-China/Notebook Overview

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.

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

design the PAGE-purified oligonucleotides used to amplify the chloramphenicol

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
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
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.

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

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.

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.

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.

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℃.

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.

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℃.

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.

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=4511)

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.

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 ul1

0.2

0.4 U ul1

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.

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

Standard part construction

1.Transforme the plasmid backbone, pSB1C3, into E.coli GB05red- gyrA462 .

2.Amplify linear amp-ccdB vector via PCR using PAGE-purified oligonucleotides containing 80-nt homology arms flanking the target DNA segment, R6K-amp-ccdB, and 20-nt standard PCR primers at the 3’ end.

Name

Sequence (5’→3’)

Length

R6K-amp-ccdb -1

AAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATTATATTCCCCAGAACATCA

 

80bp

 

R6K-amp-ccdb -2

TAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTGTTTATTTTTCTAAATA

 

80bp

 

Set the PCR system as foolws:

Component

Amount(ul)

Final

Autoclaved ddH2O

22

 

2×primeSTAR max premix

25

Template,50ng ul-1

1

50 ng

Syn-promoter-1 Oligo(10uM)

1

30 pmol

promoter-2 Oligo(10uM)

1

30 pmol

 

Total

50

 

 Fig1.  Agarose Gel of PCR production, the linear amp-ccdB vector.

3.Transform the linear fragment into E. coli GB05red- gyrA462thathas containe the plasmid pSB1C3-cm, where the recombination will be achieved be Redab after arabinose induction.

4.Prepare the pSB1C3-cm-amp-ccdb through mini-prep.

5.Screen correct recombinants by restriction analysis.

Component

Amount

ddH20

4.8ul

Cust smart

1ul

DNA

4ul

Restriction enzyme

0.2ul

Total

10ul

 Fig.2 correct recombination by DraI restriction analysis.

6.Amplify linear pSB1C3-cm-promoter vector via PCR using PAGE-purified oligonucleotides containing 40-nt homology arms flanking the target DNA segment, pSB1C3-cm-amp-ccdb, and 20-nt standard PCR primers containing part of promoter sequence at the 3’ end. What’s more, each pair of primers have 10-nt homology arms.

Oligonucleotides used in this step are as below:



Name

Sequence (5’→3’)

Length

09 SB1C3-PproD-1

CAAAAAACAAGCCGAGGATTATACCCGAGCTATGACCTCACTCTAGAAGCGGCCGCGAA

59

09 SB1C3-PproD-2

TTGTTTTTTGCCCGCCAAGACCTTCAGTGGATAGGGTGTTTACTAGTAGCGGCCGCTGC

59

11 SB1C3-PdnaA-1

CAAAAACAAGCGGCCATTGTAGCGATCAGCCGCCCACTTACTCTAGAAGCGGCCGCGAA

59

11 SB1C3-PdnaA-2

CTTGTTTTTGCCTCACCGGCTTTCAACTTAGGGGATATCCTACTAGTAGCGGCCGCTGC

59

12 SB1C3-Ppol-1 

CGCTAGAATAGCGGGACCGTTGACGACAAAGGGACAAGGTCTCTAGAAGCGGCCGCGAA

59

12 SB1C3-Ppol-2

TATTCTAGCGCCGGACCCGCCGAAAAGGTTGATCCCGCTCTACTAGTAGCGGCCGCTGC

59

13 SB1C3-P16s-1 

ACTTGGCTCAGCAATCGTTGGTTACATCTTTGATTTCTCGCTCTAGAAGCGGCCGCGAA

59

13 SB1C3-P16s-2

TGAGCCAAGTTTAGGGTTTTCTCAAAACCCAAAGATGTTTTACTAGTAGCGGCCGCTGC

59

14 SB1C3-Psig-1 

TAATTAGTCGCATTGTCATTGAGGCTTTATCGATTTGGCACTCTAGAAGCGGCCGCGAA

59

14 SB1C3-Psig-2 

CGACTAATTATCATTAGTGACACTCAGGCAGTCCGCGTCCTACTAGTAGCGGCCGCTGC

59

15 SB1C3-Patf-1 

ATCGGGCCGCGCAAACGGCGATCACTTTACGCCCGGCGACCTCTAGAAGCGGCCGCGAA

59

15 SB1C3-Patf-2

GCGGCCCGATTGTCCGGCCGCCCGAGTAAGGAGTCTTATCTACTAGTAGCGGCCGCTGC

59

17 SB1C3-memp-1

GATAGCGGGGCGGGCAGAAACGACAAACCCCGGATTAACCCTCTAGAAGCGGCCGCGAA

59

17 SB1C3-memp-2

CCCCGCTATCATTCGCCGCATCTTTCGAGGGGTATGACCGTACTAGTAGCGGCCGCTGC

59

19 SB1C3-Pami-1 

TTATATCGCCACTGCGCAAGTTAGTGCGCAACCCCAAACCCTCTAGAAGCGGCCGCGAA

59

19 SB1C3-Pami-2

GGCGATATAAGCGCCCTATAACAATTCCAGGGGTCGTTGTTACTAGTAGCGGCCGCTGC

59

21 SB1C3-Pabc-1

CGGGCGCCTGCCGGCCTGGGCCGGCAATTTTATTTGCGGGCTCTAGAAGCGGCCGCGAA

59

21 SB1C3-Pabc-2

CAGGCGCCCGGGGTCGGGCGACTTACAAGGAGCTTGTTGGTACTAGTAGCGGCCGCTGC

59

     

22 SB1C3-Pedo-1

TTTGGCGATCAACTGGTCAGCCACGCTCAGCGCGCCTTCACTCTAGAAGCGGCCGCGAA

59

22 SB1C3-Pedo-2

GATCGCCAAAGAGCGCGCAGCCAAGTAAGCGCCCGCCGCCTACTAGTAGCGGCCGCTGC

59

23 SB1C3-Ptox-1

GCGGGTCTTGGGTGTTAGAGCACTCAAGGCCCGCGTCTTTCTCTAGAAGCGGCCGCGAA

59

23 SB1C3-Ptox-2

CAAGACCCGCTAACCACAAGCAGCTAAACAGGAGCTGAATTACTAGTAGCGGCCGCTGC

59

24 SB1C3-Pdiv-1

GCAGTTCCAGACCTTGCTTGAGGGTGCGCCGGGTCAGCTCCTCTAGAAGCGGCCGCGAA

59

24 SB1C3-Pdiv-2

CTGGAACTGCTGGGCCTCAAAGTCCTGGAGCAAATGTAAGTACTAGTAGCGGCCGCTGC

59

25 SB1C3-Plip-1

TTTTTTTACTTCAGGCCTTGCCCGGAAAACATCAGAACCTCTCTAGAAGCGGCCGCGAA

59

25 SB1C3-Plip-2

AGTAAAAAAACCGGGGCTTCGGGCCCACGGGAGAAAAATATACTAGTAGCGGCCGCTGC

59

PCR process is as below:

Cycle number

Denaturation

Annealing

Extension

Termination

1

94℃,4min

 

 

 

2-31

98℃, 15s

59℃, 15s

72℃, 15s

 

32

 

 

72℃, 4min

 

33

 

 

 

10℃, hold

Set the PCR system as below:

Component

Amount(ul)

Final

Autoclaved ddH2O

22

 

2×primeSTAR max premix

25

Template,50ng ul-1

1

50 ng

Syn-promoter-1 Oligo(10uM)

1

30 pmol

promoter-2 Oligo(10uM)

1

30 pmol

 

Total

50

 

 Figure4  Agarose Gel of PCR production, the linear pSB1C3-cm-promoter vector.

Electroporate linear pSB1C3-cm-promoter vector into E. coli GB05dir cells, and enhance linear-linear homologous recombination.

Screen correct recombinants by restriction analysis.

 Fig4.  Screening of correct recombinants by AvaI restriction analysis.