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Contents 1 Brief description of how we used the wiki as a tool for molecular biology work 2 History of the reaction steps of the DNA construction process 2.1 Upstream construct 2.2 Downstream construct 2.2.1 Intermediate construct 2.2.2 Backward construct with DapA E.Coli 2.2.3 Backward construct with DapA Subtilis 2.3 Construction of a recombinaison rate measurement tool

Brief description of how we used the wiki as a tool for molecular biology work

Our wiki has proven to be an invaluable tool in organizing work within iGEM Paris 2007.

Molecular biology based engineering of DNA constructs involves going through a series of standardized steps, including:

• performing PCR reactions.
• Digesting DNA.
• Ligating digested DNA products.
• Transforming competent E.coli cells with ligation reaction products.
• Screening of E.coli colonies for correct construct
• Miniprep extraction of plasmids & subsequent sequencing of constructs

then going throuh the wholeprocess again.

The nature of the work imposed by iGEM means that we had to go through this process several times, for a great number of different constructs, often being unsuccessful and going through various steps several times.

We therefore organized the wiki in order to allow us to precisely keep track of our work:

Our notebook is available on the wiki. It was mainly used for the wetlab experiments. A calendar allows following progress on a daily basis.

Each time any one of the following reactions was performed

• DNA digestion reaction
• PCR reaction
• Ligation reaction

A sheet, describing the reaction, was filled and added to the notebook. The templates of the sheets are as follow:

PCR sheet template:

PCR :
PCR Settings		Buffer (5x)	µl x			Expected size
Annealing (°C)		MgCl2 10µM	[]			{{{Size}}}
°		dNTP 10µM	[]			Success
Time Elongation		Oligo F 10µM	(number of the Fw primer)	µL		{{{Success}}}
‘		Oligo R 10µM	(number of the Rv primer)	µL		Image (click to enlarge)
Number cycles		Water	µL			[[Image:{{{Image}}}|30px]]
x		Polymerase				Band (0=ladder)
		DNA				{{{Band}}}

All the primers purchased during the corse of the summer are listed in a special page and carry individual code numbers. See primer collection.

Digestion sheet template:

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	Size	Description
Digestion code nbre		ligation or PCR products

Ligation sheet reactions template:

Ligations
Number	Insert	Insert Volume (µL)	Vector	Vector Volume (µL)	Comments	Number of colonies
ligation code nbre

In addition, each time one of these reactions was successful, the corresponding sheet line was copies into our database on the FREEZER page of the wiki.

In order to further enhance traceability, each time a unique digestion, ligation or PCR reaction was performed, it was given an individual code number: respectively Dx, Lx & Px. A given reaction product can thus be identified by: its code number & the date at which the reaction was performed. If a given reaction (for instance: digestion product Dx: digestion of plamid x by EcoRI & SpeI enzymes and extraction of the small fragment which can serve as a Forward insert) was performed several time, the reaction product would keep the same code number. These different products would be distinguished by the date.

We tried to do this for most of our work and we believe we have achieved a reasonable degree of traceability.

History of the reaction steps of the DNA construction process

In addition, we have included a graph showing the different reaction steps leading to the main constructs we generated. There is no need for you to go through this process now, as each construction history graph is added whenever a construct of interest is introduced in the description.

Upstream construct

Downstream construct

Intermediate construct

Backward construct with DapA E.Coli

Backward construct with DapA Subtilis

Construction of a recombinaison rate measurement tool

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