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Team:Paris Bettencourt/Notebook/Eliminate Smell
From 2014.igem.org
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<div id="June"> | <div id="June"> | ||
<h4>June</h4> | <h4>June</h4> | ||
| - | <h5 id="date">June | + | <h5 id="date">June 11th</h5> |
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| - | + | ||
| - | + | <h5>June 23rd</h5> | |
| + | </br> | ||
| + | <h6>Goal: PCR agaA gBlock with oPB.001 and oPB.005</h6> | ||
| + | </br> | ||
| + | <h6>Procedure:</h6> | ||
| + | <div> | ||
| + | We are going to <strong>PCR</strong> using the | ||
| + | <a | ||
| + | href="https://www.evernote.com/Home.action?__fp=ZloDp4AdW-c3yWPvuidLz-TPR6I9Jhx8&username=iGEM-PB2014&login=true&_sourcePage=L90YhFqIo3HiMUD9T65RG_YvRLZ-1eYO3fqfqRu0fynRL_1nukNa4gH1t86pc1SP#st=p&n=f47d72e7-4213-468f-989d-2e9620b0fe66" | ||
| + | target="_blank" | ||
| + | shape="rect" | ||
| + | > | ||
| + | DreamTaq polymerase protocol | ||
| + | </a> | ||
| + | the agaA gBlock with the forward primer | ||
| + | <a | ||
| + | href="https://www.evernote.com/Home.action?__fp=IYz5qkyPGHc3yWPvuidLz-TPR6I9Jhx8&username=iGEM-PB2014&login=true&_sourcePage=FOC90vQVhF_iMUD9T65RG_YvRLZ-1eYO3fqfqRu0fynRL_1nukNa4gH1t86pc1SP#b=596d48d5-2edd-48b4-b5e9-dc81ce1b9aab&st=p&n=caecc826-a5e8-4b0b-b8a8-14a5aad1eb7c" | ||
| + | target="_blank" | ||
| + | shape="rect" | ||
| + | > | ||
| + | oPB.001 | ||
| + | </a> | ||
| + | and the reverse primer | ||
| + | <a | ||
| + | href="https://www.evernote.com/Home.action?__fp=IYz5qkyPGHc3yWPvuidLz-TPR6I9Jhx8&username=iGEM-PB2014&login=true&_sourcePage=FOC90vQVhF_iMUD9T65RG_YvRLZ-1eYO3fqfqRu0fynRL_1nukNa4gH1t86pc1SP#b=596d48d5-2edd-48b4-b5e9-dc81ce1b9aab&st=p&n=340942ec-952b-4a13-8f31-df72361990e4" | ||
| + | target="_blank" | ||
| + | shape="rect" | ||
| + | > | ||
| + | oPB.005. | ||
| + | </a> | ||
| + | These two oligos include the BioBrick prefix and suffix. The aim is to add the prefix and suffix to the agaA sequence and amplify it. | ||
| + | </div> | ||
| + | <div> | ||
| + | <br clear="none"/> | ||
| + | </div> | ||
| + | <div> | ||
| + | We will set the parameters in the machine and run the reaction overnight. | ||
| + | </div> | ||
| + | </br> | ||
| + | |||
</br> | </br> | ||
</div> | </div> | ||
Revision as of 09:07, 20 August 2014
Paris Bettencourt 2014
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Eliminate Smell
Notebook
June
June 11th
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
June 16th
Goal: Design new reverse oligo for Biobrick construction
Procedure:
We noticed oPB.002 was NOT reverse and therefore could not be used. We created oPB.005 instead:
| oPB.005 | 5'-TTAACTGCAGCGGCCGCTACTAGTATCAGTGGTGATGGTGATGATGCTTC-3' | agaA reverse for BioBrick vector |
1272->1296 |
|||
June 23rd
Goal: PCR agaA gBlock with oPB.001 and oPB.005
Procedure:
We are going to PCR using the
DreamTaq polymerase protocol
the agaA gBlock with the forward primer
oPB.001
and the reverse primer
oPB.005.
These two oligos include the BioBrick prefix and suffix. The aim is to add the prefix and suffix to the agaA sequence and amplify it.
We will set the parameters in the machine and run the reaction overnight.
July
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August
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September
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October
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