Revision as of 16:22, 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 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, then miniprep them.

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.

Table 1.

Name of the primer	Sequence	Melting Temperature (^oC)
Latherin no HIS tag Froward	5'-GTGTGTGAATTCGCGGCCGCTTCTAGAGCGACGACGACAAGGCCATGGC-3'	76
Latherin with HIS tag Forward	5'-GTGTGTGAATTCGCGGCCGCTTCTAGAGGAAGGAGATATACATATGAGC GATAAAATTATTCACC-3'	72
Latherin Reverse	5'-GTGTGTCTGCAGCGGCCGCTACTAGTATTATTAAACGCTCAGATCCACG TTCGCAC-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'-GTGTGTGAATTCGCGGCCGCTTCTAGAGAGGAGGATTACAAAATGTTAAT ATTAGATGGGGACCTACTAAAGGAC-3'	74
Ranaspumin-2 with HIS tag Forward	5'-GTGTGTGAATTCGCGGCCGCTTCTAGAGAAGAAGGAGATATACCATGGGC AGCAG-3'	74
Ranaspumin-2 Reverse	5'-GTGTGTCTGCAGCGGCCGCTACTAGTATTATTAGGATCCTAATATCCATC ATCATCATCATCG-3'	72

Results:

References:

Mackenzie et al., 2009. Ranaspumin-2: structure and function of a surfactant protein from the foam nests of a tropical frog. Biophysical Journal, 96, pp. 4984-4992

Kaplan, J., 2010. Biofilm dispersal: Mechanisms, Clinical Implications, and Potential Therapeutic Uses. Journal of Dental Research, 89(3), pp.205-218.

Kennedy, M., 2011. Latherin and other biocompatible surfactant proteins. Biochemical Society Transactions, 39, pp. 1017-1022.

Beeley et al., 1986. Isolation and characterization of latherin, a surface-active protein from horse sweat. Biochemical Journal, 235, pp. 645-650.

Tamayo et al., 2005. The EAL domain Protein VieA is a cyclic diguanylate phosphodiesterase. The Journal of Biological Chemistry, 280(39), pp. 33324-33330.

Pugsley, A., 1983. Obligatory coupling of Colicin release and lysis in Mitomycin-treated Col+ Escherichia coli. Journal of General Microbiology, 129, pp. 1921-1928.

Konisky, J., 1982. Colicins and other bacteriocins with established modes of action. Annual Review of Microbiology, 36, pp. 125-144.