Team:Groningen/5 July 2010


iGEM Groningen 2010

pushing coatings into a greener future

Week 27

Laura Searching information about biofilm modeling.

Arend Jan

The pMASK-EH construct is cut with EcoRI and SpeI to get the fragments containing the ChpE and ChpH genes and ligate them in to the expression plasmid. A mix of both genes will be used for ligation and the clones will be checked afterwards for which gene they contain.

- 10ul plasmid pNZ8901-bbs
- 1.5ul buffer Tango
- 0.5ul SpeI
- 3ul MQ
- perform digestion
- add 1.88ul buffer Tango
- add 0.5ul EcoRI

- 17.5ul plasmid pMASK-EH
- 2ul buffer Tango
- 0.5ul SpeI
- perform digestion
- add 2.5ul buffer Tango
- add 0.5ul EcoRI

Mix of fragments containing ChpE and ChpH genes at ~300bp
Restriction of expression backbone with EcoRI and SpeI

Ligation of ChpE and ChpH into pNZ8901-bbs:

- 2/5ul backbone
- 14/11ul insert (ChpE+H)
- 2ul T4 buffer
- 2ul T4 ligase

Colonies were picked, grown, and prepped for restriction checks (next week).


Tested the degradation of Chaplins isolated from Streptomyces coelicolor, according to protocol, in TY medium, spent TY medium (were B. sub 168 had grown in overnight) and by supernatant after Bacillus cells were lysed. Analyzed by SDS-PAGE which indicated that the chaplins were not degraded after a 24hr incubation period (data not shown). Test was repeated with more controls by Peter in the next week.

Surfaces coating with biofilm

To see how easily bacillus sub. can coat surfaces, 
different materials were placed in liquid TY medium with 0.5% glucose.

Taking 12 wells plates adding 2ml liquid TY medium with 0.5% glucose. 
Inoculate wells in duplo with 20 microlitre of overnight culture of 
bacillus strains (ROK deg degU and sp0o). Than adding sterilized materials to the wells. 

Plastic: since the wells plates are made of plastic nothing was added to these wells
Wood: untreated 1 cm diameter beech wood approx 2 cm cylindrical pieces, one piece per well
Stainless steel: 3 mm thick approx 1,5cm by 3 cm pieces of stainless steel
Steel: 8mm steel nails
Ceramics: Broken up uneven pieces of red ceramics with a diameter averaging 1,5 cm
Glass: standard microscopy cover slip

The 12-wells plates were grown for four days at 37 degrees. 
Plastic: Some biofilm formation but no higher than 2 mm above 
the liquid medium surface.

Wood: There was relative strong growth on the wood, especially the DegU strain grows 
very well on wood , while not covering the liquid/air interface. 
Rok also grew quite well but covered the hole well no only the wood.

Ceramics: Strong growth of Rok and SpoO

Glass: Similar coating as in plastic

Stainless steel: No growth wat so ever in any strain

Steel: Some growth in al the wells but, oxidation of the metal over the four days 
resulted in dark brown coloured indistinguishable wells.



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