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Team:Glasgow/Results:dispersal
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To perform PCR on the <i>rsn-2</i>(Ranaspumin-2)and <i>lath</i>(Latherin) inserts. | To perform PCR on the <i>rsn-2</i>(Ranaspumin-2)and <i>lath</i>(Latherin) inserts. | ||
<p> | <p> | ||
| - | To | + | To perform site directed mutagenesis on <i>lath</i>, thereby removing illegal restriction sites. |
<p> | <p> | ||
To produce successful overnight liquid cultures, then miniprep them. | To produce successful overnight liquid cultures, then miniprep them. | ||
<p> | <p> | ||
| - | To | + | To ligate the amended <i>lath</i>, as well as <i>rsn-2</i> into a suitable plasmid and transform it into competent cells. |
| - | + | ||
| - | + | ||
<p> | <p> | ||
To produce successful overnight liquid cultures of <i>lath</i>, then miniprep them. | To produce successful overnight liquid cultures of <i>lath</i>, then miniprep them. | ||
Revision as of 16:05, 21 September 2011
Dispersal of the biofilm
Aims:
To perform PCR on the rsn-2(Ranaspumin-2)and lath(Latherin) inserts.
To perform site directed mutagenesis on lath, thereby removing illegal restriction sites.
To produce successful overnight liquid cultures, then miniprep them.
To ligate the amended lath, as well as rsn-2 into a suitable plasmid and transform it into competent cells.
To produce successful overnight liquid cultures of lath, then miniprep them.
To devise a successful assay of the surfactant proteins.
To digest and ligate the inserts to plasmids with ribosome binding sites, the three light-dependent promoters, and double terminators present.
To characterise the latherin and ranaspumin constructs, by testing them in a biofilm setting.
To ligate the inserts into the submission vector and submit them to the registry.
Methods:
The DNA of Latherin and Ranaspumin-2 we obtained was in plasmids. In both cases, there were two versions of the genes: one containing HIS tag and on without it. The coding sequences were excised from the plasmids and the Standard BioBricks end were added through PCR. Additionally, Latherin contained three illegal restriction sites. Therefore, we had to perform Site Directed Mutagenesis (SDM) to eliminate them. Table 1 contains the primers we used to achieve that.
| Name of the primer | Sequence | Melting Temperature (oC) |
| Latherin no HIS tag Froward |
5'-GTGTGTGAATTCGCGGCCGCTTCTAGAGCGACGACGACAAGGCCATGGC-3' | 76 |
| Latherin with HIS tag Forward |
5'-GTGTGTGAATTCGCGGCCGCTTCTAGAGGAAGGAGATATACATATGAGCGATAAAATTATTCACC-3' | 72 |
| Latherin Reverse | 5'-GTGTGTCTGCAGCGGCCGCTACTAGTATTATTAAACGCTCAGATCCACGTTCGCAC-3' | 74 |
| Latherin SDM1 Forward | 5'-CAGTTGCAGCAGACGGGTATCCTtCAGTTTAATTTCCGC-3' | 68 |
| Latherin SDM1 Reverse | 5'-GCGGAAATTAAACTGAAGGATACCCGTCTGCTGCAACTG-3' | 68 |
| Latherin SDM2 Forward | 5'-GCGGATTGCCGCTCTTCAGCTCAATCGC-3' | 66 |
| Latherin SDM2 Reverse | 5'-GCGATTGAGCTGAAGAGCGGCAATCCGC-3' | 66 |
| Latherin SDM3 Forward | 5'-GCAACCTGGATCTTCAGCTGGTGAACAACC-3' | 64 |
| Latherin SDM3 Reverse | 5'-GGTTGTTCACCAGCTGAAGATCCAGGTTGC-3' | 64 |
| Ranaspumin-2 no HIS tag Forward |
5'-GTGTGTGAATTCGCGGCCGCTTCTAGAGAGGAGGATTACAAAATGTTAATATTAGATGGGGACCTACTAAAGGAC-3' | 74 |
| Ranaspumin-2 with HIS tag Forward | 5'-GTGTGTGAATTCGCGGCCGCTTCTAGAGAAGAAGGAGATATACCATGGGCAGCAG-3' | 74 |
| Ranaspumin-2 Reverse | 5'-GTGTGTCTGCAGCGGCCGCTACTAGTATTATTAGGATCCTAATATCCATCATCATCATCATCG-3' | 72 |
