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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

[https://static.igem.org/mediawiki/2014/0/09/PPB001.png pPB.001]

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July

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August

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September

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