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From 2012.igem.org

Contents 1 Transform WT ADP1 using natural competence 2 Transform Ec XL1-Blue cells 3 Transform competent cells of Bs strains 4 RESULTS

Transform WT ADP1 using natural competence

• Inoculate 20 μL of the O/N cultures from yesterday into 300 μL of the appropriate medium (LB or SOB), with two tubes for each medium
• Incubate with shaking on its side (using tape to keep the tube in place) at 30^o C for 2.5 hours
• Add 4 μL of ~280 ng pBAV1K/μL sample for ADP1+DNA (SOB) and ADP1+DNA (LB), and 4 μL of sterile H₂O for ADP1 control (SOB) and ADP1 control (LB)
• Incubate with shaking as above for 2 hours
• For each sample, absorb entire culture onto LB-Kan plates using a glass spreader
• Incubate at 34^o C until colonies form

Transform Ec XL1-Blue cells

• Thaw two 200-μL aliquots on ice
• Add 4 μL of pBAV1K to Ec XL1-Blue+DNA, and 4 μL of sterile H₂O to Ec XL1-Blue control
• Incubate on ice for 30 minutes
• Heat shock in 42^o C water bath for 45 seconds
• Incubate on ice for 2 minutes
• Add 500 μL of LB broth to each and pipette up and down to mix gently
• Incubate with shaking at 34^o C for 1 hour
• Inoculate 100 μL of each culture onto LB-Kan plates using a glass spreader
• Incubate at 34^o C until colonies form

Transform competent cells of Bs strains

• Thaw two 500-μL aliquots each of strains 168, PERM739, PY79, and 1A833 on ice
• Thaw four 1-mL aliquots of SpII+EGTA on ice
• Add 500 μL to each aliquot and mix gently by pipetting up and down
• Add 500 μL of the mixed cultures to 4 μL of pBAV1K for Bs 168+DNA, Bs PERM739+DNA, Bs PY79+DNA, and Bs 1A833+DNA, and to 4 μL of sterile water for Bs 168 control, Bs PERM739 control, Bs PY79 control, and Bs 1A833 control
• Incubate with shaking in culture tubes on their side at 34^o C for 1 hour
• Inoculate 100 μL of each culture as follows using a glass spreader:
  • Bs 168: LB-Kan
  • Bs PERM739: LB-Neo-Kan, LB-Kan
  • Bs PY79: LB-Kan
  • Bs 1A833: LB-Spc-Kan, LB-Kan
• Incubate at 34^o C until colonies form

Figure 1. Transformation of naturally competent strains of Ac, Ec, and Bs (pre-incubation)
File:2012-07-24 Transformation Expt..jpg
Top row: ADP1 incubated in SOB or LB
Second row, right: Ec XL1-Blue
Second row, left: Bs 168
Third row, left: Bs PY79
Fourth row: Bs PERM739 on LB-Neo-Kan or LB-Kan
Fifth row: Bs 1A833 on LB-Neo-Kan or LB-Kan

RESULTS

• Bs 168: traces of growth on periphery of both the control and LB-Kan (10 μg/mL) plates
  
• Bs PY79: same as Bs 168
  
• Bs PERM739: lawns for all 4 plates (LB-Kan and LB-Neo-Kan)
• Bs 1A833: ~100 colonies on LB-Kan control plate, ~4 colonies on LB-Kan transformation plate; no growth on either LB-Spc-Kan plates
  

• ADP1 WT: lawns with colonies at the edges of them for the transformed plates, no growth on the control plates
  
• Ec XL1-Blue: selection of colonies on transformed plate, no growth on the control plate

CONCLUSIONS: Based on the aggregate data for the cross-species transformation, the results seem to indicate that the concentration of Kan used may not be inhibitory for growth of untransformed Bs cells, or selective enough for ADP1. As such, a quick assay of LB-Kan plates of differing concentrations (10, 20, 30, 40, 50 μg/mL) will be performed to determine the optimal concentration to use in the future for selection of each strain. Until then, we will go by the concentrations used by Bryksin and Matsumura:

• for B. subtilis: 20 μg Kan/mL
• for E. coli and ADP1: 50 μg Kan/mL