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Team:Paris Bettencourt/Notebook/Eliminate Smell
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Revision as of 14:35, 19 August 2014
Paris Bettencourt 2014
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Eliminate Smell
Notebook
June
June 23rd
Goal: Design plasmids that express agaA construct
Procedure:
Using Geneious, we first created the agaA construct:
- Promoter BBa_J23108 from the Anderson's promoter collection
- RBS from the RBS Calculator of Salis lab specific for E coli
- BioBrick Prefix + Scar from the iGEM Parts webpage
- agaA sequence codon optimized for E coli 12 (IDT online tool). We checked with Generious that there were no restriction sites for EcoRI, SpeI, ZbaI and PstI.
- Histidine tag: 6 Histidines in C-terminal were added
- Stop codon
- BioBrick scar + BioBrick suffix from the iGEM Parts webpage
To amplify this construct, we created two oligos:
| Name | Sequence (5'->3') | Notes | Binding position |
| oPB.001 | TATAGAATTCGCGGCCGCTTCTAGAGTGACAGCTAGCTCAGTCCTAGG | agaA forward for BioBrick vector | 1->23 |
| oPB.002-BAD PRIMER | GAAGCATCATCACCATCACCACTGATACTAGTAGCGGCCGCTGCAGTTA | agaA REVERSE for BioBrick vector- NOT REVERSED | 1272->1296 |
| oPB.005 | TTAACTGCAGCGGCCGCTACTAGTATCAGTGGTGATGGTGATGATGCTTC | agaA reverse for BioBrick vector | 1272->1296 |
* We noticed the 16/06/2014 that oPB.002 was NOT reversed and we designed oPB.005
Tip: When creating oligos: we add 4 nt at the beginning and the end composed by AT, to make sure the enzyme will bind properly.
We chose two backbones for the construct:
- pSB1C3 from the iGEM Parts to create a BioBrick.
- pEC-XC99E, a E coli-C glutamicum shuttle plasmid
We designed two plasmids:
1. pPB.001: Biobrick submission of agaA expression construct
1) Plasmid pSB1C3: High copy BioBrick assembly plasmid. Constitutive expression. To use in E coli
2) agaA construct
2.pPB.002:agaA expression construct. Shuttle vector E coli-C glutamicum
1) Plasmid pEC-XC99E modified: Ptrc (IPTG inducible promoter) and lacI will be removed by PCR. Primers will be oPB.003 -that includes a PstI site- and
oPB.004 -that includes a XbaI site.
| oPB.003 | 5'-CTGCAGATGCAAGCTTGGCTGTTTTGGCG-3' | PstI site + agaA forward for pEC-XC99E |
| oPB.004 | 5'-TCTAGACACCACCCTGAATTGACTCTCTTC-3' | XbaI site + aga reverse for pEC-XC99E |
2) agaA construct
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