From 2011.igem.org

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. The primers for Latherin and Ranaspumin-2

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

The two other protein we used for the biofilm dispersal, Colicin E2 (BBa_K131000) and T4 Endolysin (BBa_112806), were obtained from Registry.

An assay was devised for the surfactant proteins: A DNAse is used to lyse the cells containing the surfactant in liquid culture. The culture is then shaken and should produce considerably more bubbles than if the surfactants were not present.

The DNA of c-di-GMP specific Phosphodiesterase (PDE) was in the form of a plasmid. The coding sequences were excised from the plasmids and the Standard BioBricks end were added through PCR. Additionally, it contained two illegal restriction sites. Therefore, we had to perform Site Directed Mutagenesis (SDM) to eliminate them. Table 2 contains the primers we used to achieve that.

Table 2. The primers for Phosphodiesterase

Name of the primer	Sequence	Melting Temperature (^oC)
PDE Forward	5'-GTGTGTGAATTCGCGGCCGCTTCTAGAGCGAGGTCGAGCCCATCGTGC-3'	77
PDE Reverse	5'-GTGTGTCTGCAGCGGCCGCTACTAGTATTATTACTACGGATTTCCGGTTCCCGCG-3'	75
PDE SDM1 Forward	5'-CAGCCAGGCCGACCTGCAACGCCTGCGCG-3'	73
PDE SDM1 Reverse	5'-CGCGCAGGCGTTGCAGGTCGGCCTGGCTG-3	73
PDE SDM2 Forward	5'-CGCCCTGCTTTGCAGCCGTGCCAAGG-3'	67
PDE SDM2 Reverse	5'-CGCAAAGGCGGTCTTCAGTACTTCATTGGTG-3'	64

To determine the rate of diffusion through a biofilm of each of the molecules and calculate the attainable spatial resolution, we used those following equations:

Equation 1

where:
P (r,t) – number of molecules at the certain distance after a certain time
A – total number of molecules in the system
r – distance from point of origin (mm)
D – diffusion coefficient
t – time (min)

Equation 2

where:
P_f (r,t) – Final number of molecules at given distance at given time
P (r,t) – initial number of molecules according to Equation 1
λ – rate of degradation (molecule/min)
t – time passed (min)

Results:

PCR of ranaspumin and latherin was successful.

Gel showing Ranaspumin-2 after PCR.

Gel showing both the His-tagged, and non His-tagged versions of Latherin after PCR.

Site Directed Mutagenesis was attempted several times for latherin, but was unsuccessful. There are still illegal restriction sites in the insert. Transformation of latherin was therefore not possible.

Transformation of ranaspumin-2 was successful. Ranaspumin-2 was miniprepped.

An assay for the surfactants was devised but not tested, owing to time constraints (see Methods).

The results regarding the biofilm dispersal we acquired using our single-cell models and some additional values are shown in the table below. We also created models for Phosphodiesterase activity and the multi-cell models for Latherin and Colicin E2. For more information, please refer to Modelling site.

Results for modelling, base on the single-cell models

Name of the molecule	Diffusion rate (mm²/min)	The critical concentration (molecule)	Achieving Effective Concentration* (min)	The attainable spatial resolution (mm)
Ranaspumin-2	1.68*10^-4	374	28	0.09
T4 Endolysin	1.52*10^-4	3000	52	~0.01
Latherin	1.38*10^-4	173	29	0.08
Colicin E2	0.72*10^-4	1629	90	~0.01

*Due to the mode of action, for Colicin E2 and T4 Endolysin it is equal to the Critical Concentration and for Latherin and Ranaspumin-2 it is equal to 8x Critical Concentration

Figure 1,2 and 3 are showing results regarding modelled diffusion of the molecules involved in our system.

Figure.1. Relative One-dimensional Diffusion of (a)Latherin and (b)Ranaspumin-2 at certain points in time, when initial concentrations of Latherin and Ranaspumin-2 are 8x critical concentration. Threshold indicates critical concentration required for the molecule to work.

(a) Latherin

(b) Ranaspumin-2

Figure 2. Model of Colicin E2 where 21 cells are next to each other in a row. Two plots are shown: the first shows cells illuminated for 90 min, the second shows cells illuminated for 45 min. Plots show concentration of the protein after 70 min of diffusion. Threshold indicates the critical concentration needed by Colicin E2 to lyse a cell.

Figure 3. Relative One-dimensional diffusion of (a)Colicin E2 and (b)T4 Endolysin within first 60 min.

(a) Colicin E2

(b) T4 Endolysin

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.