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Multiple Cloning Site Biobrick

We wanted to test multiple genes with the same promoter, RBS and terminator to characterise PDE, latherin, latherin with HIS tag, ranaspumin and ranaspumin with HIS tag , which are some of our novel biobricks. To ligate one of these constructs together requires three or four (three if you’re using the 3A method) restriction and ligation reactions. So to test all five under the same promoter, RBS and terminator would require at the very least 15 restriction and ligation reactions. However using our multiple cloning site (MCS) biobrick it is possible to create the promoter, RBS, MCS and terminator construct in three-four ligations before inserting each gene into the MCS with just one extra restriction and ligation step. So it would be possible to have all five constructs made in just nine restriction and ligation reactions, saving a huge amount of time. And for each extra gene you want to test this would only require one extra restriction and ligation rather than three or four.

This is great for characterisation of a large number of biobricks. We suggest that if you plan to use the MCS biobrick, when you are adding biobrick ends to your PCR primers or sequence for synthesis , add one of the restriction sites included in our MCS before and after the whole construct. As long as you ensure the gene is still in-frame then you can easily insert it into your standard construct for testing. When it comes to submission, just restrict the novel biobrick out of the characterisation construct and into ligate your submission vector . Restricting with X+S only just your gene will drop out. Restricting with E+P will give fragments for your promoter+ RBS and terminator as well as your gene however the gene will usually be much larger than the fragments allowing for gel purification of only the restricted gene.

Figure 1: Multiple cloning site (MCS) biobrick. The MCS has a range of the most commonly used restriction sites. Once the Promoter+RBS+MCS+Terminator construct has been made this can be used to easily insert a range of genes.

Table 1: Number of restriction/ligations required to built multiple constructs with and without the MCS. when using lower numbers of genes in the Promoter+RBS+MCS+Terminator construct not much time is saved, however as the number of genes you are using increase it becomes much more time effective.