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

week14

constract our software tool

week15

week16

week17

week18

week19

week19

week20

week21

week22

week23

week24

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