Davidson Missouri W/Web tool

The gene-flipping mechanism that powers our computer requires the positioning of HixC sites within reporter genes' coding sequences. Through the use of PCR, a gene can be split into two pieces with BioBrick ends attached at the extremities. This allows for the ligation with a HixC site in the middle. Our team has developed a web-based tool to automate the generation of these PCR primers. It also predicts the resultant DNA sequences for the two intermediary parts, the final part, and the resultant polypeptide. By using this program we hope to make the gene-splitting process faster and to remove the potential for human error. The tool can be accessed at http://gcat.davidson.edu/iGEM07/genesplitter.html.

Tutorial

What follows here is an explanation of how to use the web tool and what it does.

The First Page

The initial page begins with an overview of what the tool does and how to use it. There are 3 inputs required from the user:

1. The DNA sequence for the gene to split.
2. The amino acid number where the insertion is desired. The HixC site will be inserted immediately after the given amino acid.
3. The choice for an extra nucleotide. Inserting a HixC site amounts to an addition of 38 bases. Unless a 39th base is added, a frameshift will occur since 38 is not a multiple of 3. The nucleotide choice results in either a glutamate or aspartate.

Once the user has entered this information, he or she can then click the 'submit' button to initiate the calculations.

The Output

Once the user has submitted the information a new page opens. Before any calculations are done, some information is provided to allow the user to verify that the tool is analyzing the correct sequence. The first output is the original sequence. This should be identical to the sequence provided by the user. The next two sequences show the DNA sequences that lie before and after the insertion point. The tool then restates the insertion point, by amino acid number.

The first calculations are the 4 primers for the two PCR reactions. The display is color-coded so that the restriction enzyme sites in the BioBrick ends are easily distinguishable. After the BioBrick ends, between 20 and 25 bases should follow. These bases should function as PCR primers to the given gene. Their length is optimized to match melting temperatures as much as possible. This is done using Proligo's formula from their own web page. Note that they use two formulas: a simple one for sequences of size less than 20, and a more complicated one for larger sequences. Below the primer sequences are their computed melting temperatures and GC content, as well as their lengths (excluding BioBrick ends).

Once the primers have been displayed, the tool provides some additional useful information. The two intermediate parts, with BioBrick ends, are shown. This is useful if it is necessary to verify the PCR results by sequencing. The DNA sequences for the scars and the HixC site are provided, as well as which amino acids these will produce (along with the user-inputted 39th base). The final product is then displayed, with the inserted HixC site and BioBrick ends at the extremities. Also shown are what becomes translated (the final product without the BioBrick ends), and the translation itself.