Team:Paris Bettencourt/Notebook/Eliminate Smell

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Paris Bettencourt 2014

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

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July

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August

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September

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October

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@@ Line 289: / Line 289: @@
       <p>To amplify this construct, we created two oligos:</p>
 <TABLE BORDER>
-<TR><b><TD>Name</TD><TD>Sequence (5'->3')</TD><TD>Notes</TD><TDBinding position</b></TD></TR>
+<TR><b><TD>Name</TD><TD>Sequence (5'->3')</TD><TD>Notes</TD><TD>Binding position</b></TD></TR>
 <TR><TD><b>oPB.001</b></TD><TD>TATAGAATTCGCGGCCGCTTCTAGAGTGACAGCTAGCTCAGTCCTAGG</TD><TD>agaA forward for BioBrick vector</TD><TD>1->23</TD></TR>
 <TR><TD><b>oPB.002-BAD PRIMER</b></TD><TD>GAAGCATCATCACCATCACCACTGATACTAGTAGCGGCCGCTGCAGTTA</TD><TD>agaA REVERSE for BioBrick vector- NOT REVERSED</TD><TD>1272->1296</TD></TR>
 <TR><TD><b>oPB.005</b></TD><TD>TTAACTGCAGCGGCCGCTACTAGTATCAGTGGTGATGGTGATGATGCTTC</TD><TD>agaA reverse for BioBrick vector</TD><TD>1272->1296</TD></TR>
 </TABLE>
+<p><i>* We noticed the 16/06/2014 that oPB.002 was NOT reversed and we designed oPB.005 </i></p>
+</br>
+<p><b>Tip:</b> When creating oligos: we add 4 nt at the beginning and the end composed by AT, to make sure the enzyme will bind properly.</p>
+</br>
+<p>We chose two backbones for the construct:</p>
+<ul><li>pSB1C3 from the iGEM Parts to create a BioBrick.</li>
+<li>pEC-XC99E, a E coli-C glutamicum shuttle plasmid</li></ul>
+</br>
+<p>We designed two plasmids: </p>
+ <p><b>    1. pPB.001:</b> Biobrick submission of agaA expression construct</p>
+</br>
+<p><u>1) Plasmid pSB1C3: </u>High copy BioBrick assembly plasmid. Constitutive expression. To use in E coli</p>
+ </br>
+<p><u>2) agaA construct</u></p>
 					<h5>Date 2</h5>