Team:LMU-Munich/Data/Knockout

From 2012.igem.org

Revision as of 19:50, 26 September 2012

• Home
• Team
• Project
  • Why Beadzillus?
  • Bacillus Introduction
  • Bacillus BioBrickBox
  • GerminationSTOP
  • Sporobeads
  • Data
  • Applications
• Extras
  • Attributions
  • Collaborations
  • Registry
  • Bacillus Strains
  • Special: Inverter
  • Achievements
• Safety
  • Project Safety
  • Release for application
• Lab Notebook
  • Weekly Journal
  • Protocols
• Human Practice
  • SynBio Day
  • Students Practical Course
  • CAS SynBio Conference
  • Berlin Conference
• Sponsors

Knockouts of germination genes

We incubated our mutant strains in Difco sporulation media (DSM) at 37°C, shaking at 230RPM for 18-24 hours to induce sporulation. We heated 1 milliliter of each of the DSM cultures at 80°C for one hour to kill vegetative cells, and used 1 milliliter at room temperature (allowing living cells to survive). From here, we quantified the cells and spores per milliliter of each culture using a Neubauer cell counting chamber. Then on LB-agar, we plated different dilutions of the cultures, with heated and unheated cultures on separate plates. The purpose of plating both heated and unheated cultures was to demonstrate the viability of the unheated vegetative cells in each culture. This makes evident that the lack of colonies on heated plates is not due to the inability of the strain's vegetative cells to grow, but from the inability of spores to germinate into vegetative cells capable of growing. For all germination mutants, vegetative cells were able to grow and form colonies. Plates were kept at 37°C for minimally 24 hours. We then quantified the colonies on each plate, and used this to check for cell growth and calculate the spore germination rate for each mutant strain. For details on this germination assay, see Protocols. The germination assays were carried out several times, and yielded similar results each time. The results are shown in Fig. 1.

Fig 1: Germination rates of B. subtilis mutant stains. All germination rates are relative to wild type (W168), our positive control. The mutant spo0A::tet is unable to form spores, and should therefore show no germination (our negative control). Mutants used were: W168: wild-type strain spo0A::tet: a sporulation-deficient strain B29: cwlD::kan, sleB::mls B30: gerD::cm, sleB::mls B32: cwlJ::spec, cwlD::kan B40: cwlD::kan, sleB::mls, cwlJ::spec B41: cwlD::kan, sleB::mls, gerD::cm B42: cwlD::kan, cwlJ::spec, gerD::cm B43: gerD::cm, sleB::mls, cwlJ::spec B46: cwlD::kan, cwlJ::spec, gerD::cm, sleB::mls B47: gerD::cm, sleB::mls, cwlJ::spec, cwlD::kan

The next figure showing the LB-agar plates from the germination assays (see below) demonstrates the inability of our mutant spores to germinate.
The plates below demonstrate the procedure of plating for the germination assay. Plates here include those containing heated and unheated cultures, and show both germinable spores (B42) and spores unable to germinate (B47). These plating experiments allowed us to quantify the number of ingerminable spores in each culture.

Plates of germination mutants. Plates contained heated (to kill vegetative cells and leave only spores to germinate) cultures and unheated (where colonies may come from germinated spores and the vegetative cells) cultures. The mutant spo0A::tet is unable to form spores, and should therefore show no germination (our negative control). The mistake of photographing empty plates against a background of spots was realized after plates had been discarded. Presence of colonies is noted below, for clarity. Mutants used were: WT168: Wild-type 168; Heated plate: colonies present; Unheated plate: colonies present spo0A::tet: A sporulation-deficient strain; Heated plate: no colonies; Unheated plate: colonies present B42: cwlD::kan, cwlJ::spec, gerD::cm; Heated plate: colonies present; Unheated plate: colonies present B47: gerD::cm, sleB::mls, cwlJ::spec, cwlD::kan; Heated plate: no colonies; Unheated plate: colonies present

The table below shows the maximum number of spores from heated cultures plated which yielded zero colonies. In the case of B30, the germination assay was performed only once, and as visible in the above graph "Mutant Germination Rates," contained viable, germinable spores. The numbers shown here are the highest number of spores plated, excluding B30. This means that we do not have data for higher concentrations of spores which would suggest any spore germinability. The mutant strain B42 contained germinable spores when 4x10⁷ were plated, but contrarily, zero germinable spores were found when 9.83x10⁷ spores were plated in another round of the germination assay.

Germinable spores per mutant.