• This week we tried to reclone pSB1A2_T7_adhA into the pSB1C3 backbone.
• Because the recloning didn't worked well till now, we decided to restrict the used backbone pSB1C3_RFP with EcoRI, XbaI, SpeI and PstI to minimize the chance of religation.
• pSB1C3_T7_adhA we cut with EcoRI and PstI
• Clean up and Ligation as explained in the BioBrick Assembly
• Transformation with electrocompotetent cells

• Colony PCR (rev_pB1C3_adhA, fw_adhA_pSB1C3)
• Annealing temperature: 65 °C
• Bands as expected (~1200 bp)
• Liquid cultures for a restriction digest were prepared.
• Plasmid isolation of pSB1C3_T7_adhA
• Restriction digestion with EcoRI and PstI
• Bands as expected (backbone: ~2,2 kb and insert: ~1,2 kb)

• This week we tried to combine the alsS_ilvC_ilvD_kivD construt with the T7 promotor.
• BioBrick Assembly (Suffix)
• Backbone pSB1A2_T7 (digested with SpeI, PstI)
• pSB1A2_T7
• Insert pSB1C3_alsS_ilvC_ilvD_kivD (digested with XbaI, PstI)
• alsS_ilvC_ilvD_kivD
• Colony PCR (fw_ilvD_kivD, rev_pSB1C3_kiVD)
• Annealing temperature: 65 °C
• Bands as expected (~ 7500 bp)
• Liquid culture for a restriction digest was prepared.
• Plasmid isolation of pSB1A2-T7_alsS_ilvC_ilvD_kivD
• Restriction digestion with EcoRI and PstI
• Bands as expected (backbone: ~2,2 kb and insert: ~7500 kb)
• For the protein expression analysis of AlsS, IlvC, IlvD and kivD we made a cultivation. Samples were taken like explained in the cell lysis for a SDS-PAGE Protocol. Protein expression was induced when the culture reached a OD₆₀₀ 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples two and four, 21 and 23 hours later. Of these samples, we made a SDS Page
  
  SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD
  The sizes of the included proteins are ~61 Da for the AlsS,
  ~54 Da for the IlvC, ~65 Da for the IlvD and ~62 Da KivD

• pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA
• This week we tried to combine the alsS_ilvC_ilvD_kivD_adhA construt with the T7 promotor.
• BioBrick Assembly (Suffix)
• Backbone pSB1A2_T7 (digested with SpeI, PstI)
• pSB1A2_T7
• Insert pSB1C3_alsS_ilvC_ilvD_kivD_adhA (digested with XbaI, PstI)
• alsS_ilvC_ilvD_kivD_adhA
• Colony PCR (fw_ilvD_kivD, rev_pSB1C3_kiVD)
• Annealing temperature: 65 °C
• Bands as expected (~ 17500 bp)
• Liquid culture for a restriction digest was prepared.
• Plasmid isolation of pSB1A2-T7_alsS_ilvC_ilvD_kivD_adhA
• Restriction digestion with EcoRI and PstI
• Bands as expected (backbone: ~2,2 kb and insert: ~8500 kb)
• For the protein expression analysis of AlsS, IlvC, IlvD, KivD and AdhA we made a cultivation. Samples were taken like explained in the cell lysis for a SDS-PAGE Protocol. Protein expression was induced when the culture reached a OD₆₀₀ 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples two, four, six, 22 and 24 hours later. Of these samples, we made a SDS Page
  
  SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA
  The sizes of the included proteins are ~61 Da for the AlsS, ~54 Da for the IlvC,
  ~65 Da for the IlvD, ~62 Da for KivD and ~36 Da for AdhA


• Restriction digestion as a control

Agarose gel control restriction digestion from several constructs with the restriction. IB stands for pSB1C3_alsS_ilvC_ilvD_kivD. As Ladder we used a 1kb Ladder.

This week we made to cultivation for the proof of the isobutanol production.
The first cultivation was at 37°C and the second one at 30°C. During the cultivations we took severel samples for GC-MS analysis