Team:Goettingen/week21-2

V09_17

• Experiment:
  In order to clone the flhDC, FliC and 18K-RFP constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. After measuring the concentration of the purified fragments, an overnight ligation was initiated.
  

• Experiment: We prepared overnight cultures of different promoter-gene constructs in two different E. coli strains (MG1655 & BL21) in order to test wether the motility is influenced. The following constructs were used:
  
  - 20E_flhDC
  - 18C_flhDC
  - 20I_flhdc
  - 20E_motA
  - 18C_motA
  - 20E_motB
  - 18C_motB
  

V09_18

• Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
  
• Observation and Results:
  Text
  

• Experiment:
  TEXT
  

• Experiment:
  Chemical transformation was done according to the standard protocol. The following constructs were used:
  - pSB1C3-FliC into E. coli (DH10B)
  pSB1C3-flhDC into E. coli (DH10B)
  PSB1C3-RFP into E. coli (MG1655)
  
  
  

V09_19

• Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
  
• Observation and Results:
  Text
  

• Experiment:
  For the chemical retransformation the standard protocol was followed. The following constructs were transformed:
  - pSB1C3-18C-motA (DH10B)
  - pSB1C3-18C-motB (DH10B)
  - pSB1C3-18C-yhjH (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20I-motA (DH10B)
  - pSB1C3-20I-motB (DH10B)
  - pSB1C3-20I-yhjH (DH10B)
  - pSB1C3-RFP (BL21)
  
  

• Experiment:
  Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.
  - pSB1C3-flhDC
  - pSB1C3-fliC
  - pSB1C3-18K-RFP
  
  
  

V09_20

• Experiment:
  Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the protocol.
  
  - pSB1C3-flhDC
  - pSB1C3-fliC
  - pSB1C3-18K-RFP
  
  
• Observation and Results:
  All three the pSB1C3-18K-RFP clones host the correctly inserted gene in the plasmid. The pSB1C3-flhDC / fliC clones do not host the correctly inserted gene.
  

• Experiment:
  In order to get more fliC, the already quikchanged fliC was amplified via PCR. The PCR product was analyzed via gel-electrophoresis and subsequently purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.
  

• Experiment:
  In order to clone the flhDC, FliC constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol.
  

• Experiment:
  Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.
  
  - pSB1C3-18C-motA (DH10B)
  - pSB1C3-18C-motB (DH10B)
  - pSB1C3-18C-yhjH (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20I-motA (DH10B)
  - pSB1C3-20I-motB (DH10B)
  - pSB1C3-20I-yhjH (DH10B)
  - pSB1C3-RFP (BL21)
  
  

V09_21

• Experiment:
  The restriction products were separated on a gel in order to verify the digestions success and purify the desired fragments. After ligation of the genes into pSB1C3, we transformed the ligated plasmids into E. coli DH10B according to the protocol.
  
  

• Experiment:
  Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the protocol.
  
• Observation and Results:
  All three the pSB1C3-18K-RFP clones host the correctly inserted gene in the plasmid. The pSB1C3-flhDC / fliC clones do not host the correctly inserted gene.
  

• Experiment:
  TEXT