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 artificial 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

Using 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

Using 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(1-25)-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.

week12

Chracterize the strength of different promoters by luciferase firefly assay.

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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