Systems assembly!

12 July

• ZR Miniprep Kit from all  the genes
  • AppA
  • PpsR
  • PrrA
  • PrrB
  • PrrA Promoter
  • PpsR Promoter
• Transformation of E. coli TOP10

13 July

• Colony PCR
• Taq Buffer                        5μL
• MgCl2                             5μL
• dnTP’s                             1μL
• Forward                           1μL
• Reverse                            1μL
• Taq polymerase                0.5μL
• H₂O                                 36.5μL
• TOTAL                 50μL

NEGATIVE

14 July

• Culture from 4 colonies per gene. 6hrs
• ZR Miniprep Kit
• Agarose gel electrophoresis 1%
• PCR from plasmid
• Taq Buffer                       2μL
• MgCl2                             2μL
• dnTP’s                             0.5μL
• Forward                          1μL
• Reverse                            1μL
• Taq polymerase                1μL
• H₂O                                 11.5μL
• TOTAL                  20μL

POSITIVE

• Band purification
• Plasmid digestion  only  for genes (1.5h)
• Xba1 and Pst1
• Plasmid digestion  only  for promoters (1.5h)
• EcoRI and SpeI
• Agarose gel electrophoresis 1%
• Genes ( +)
• Promoters   (-)
• Ligations
• RBS+GENES
• 20μL Reaction (see protocols)
•  AGAIN!!!!  Plasmid digestion, only  for promoters (1.5h)
• EcoRI and SpeI
• Promoters   (-)
• Agarose gel electrophoresis 4%

• This was 4 more times!!!!!!!!!!!!!

15 July

• Transformation by heat-shock

16 July

• Ligation of promoters in psB1A3
• Cultures of RBS+GENES (6hr)
• ZR Miniprep kit

17 July

• Digestion of RBS+GENS (1.5h, see protocols)
• RBS+PrrA     S+P
• RBS+PrrB     X+P
• RBS+PrrC     E+S
• DT                  E+X
• RBS+AppA   X+P
• Prom J23104         S+P
• RBS+PpsR    E+S
• Agarose gel electrophoresis 1%
• POSITIVE (+)

• Band Purification
• Ligations A
• 1A      RBS+PrrA +RBS+PrrB     
• 2A      RBS+PrrC+DT       
• 3A      Prom J23104+RBS+AppA
• 5A      RBS+PpsR+ DT
• Overnight cultures  of promoters+psB1A3

18 July

• Transformation of ligations A
• ZR Miniprep Kit
• Digestiion
• POSITIVE
• Band purification
• Ligation D
• PrrA Promoter+GFP+DT
• PpsR Promoter+GFP+DT

19 July

• Transformation of ligations D
• Cultures of ligations A (6h)
• ZR Miniprep Kit
• Digestion  (1.5h)
• 1A      S+P    
• 2A      X+P   
• 3A      S+P
• 5A      X+P
• Agarose gel electrophoresis 1%
• POSITIVE (+)
• Band purification
• Ligations C
• 1C      RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
• 2C      Prom J23104+RBS+AppA+RBS+PpsR+ DT

20 July

• Overnight cultures of ligations D (6h) (small colonies)
• Transformation of ligations C

21 July

• Cultures of Ligation C
• Miniprep Kit
• Digestion E and E+P
• Agarose gel electrophoresis 1%
• POSITIVE (+)
• ZR Miniprep Kit of ligations D
• Digestion E and X+P
• Agarose gel electrophoresis 1%
• POSITIVE (+)
• Band purification!
• NEGATIVE
• Overnight culture

22 July

• ZR Miniprep Kit ligations D
• Digestion E and E+P
• Ppsr promoter+GFP+DT            X+P
• Agarose gel electrophoresis 1%
• POSITIVE (+)
• High concentration!
• Band purification
• POSITIVE +
• Miniprep Kit ligation C
• Digestion
• PromJ23104                                                      S+P
• RBS+PrrA +RBS+PrrB +RBS+PrrC+DT         X+P
• Prom J23104+RBS+AppA+RBS+PpsR+ DT   S+P
• Agarose gel electrophoresis 1%
• High concentration! J
• Band purification
• POSITIVE +
• Ligations E
• 1E      PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
• 2E      Prom J23104+RBS+AppA+RBS+PpsR+ DT+ Ppsr promoter+GFP+DT

23 July

• Transformation of ligations E
• WE are also worked in the assembly of the butanol pathway + regulatory systems

24 July

• 1E NOTHING!!!!!!!!!!
• Again ligation!!
• Miniprep Kit ligation C
• Digestion
• PromJ23104                                                         S+P
• RBS+PrrA +RBS+PrrB +RBS+PrrC+DT            X+P
• Agarose gel electrophoresis 1%
• High concentration! J
• Band purification
• POSITIVE +
• 2E; 3 COLONIES!!... ;       Prom J23104+RBS+AppA+RBS+PpsR+ DT+ Ppsr promoter+GFP+DT
• 6h culture!
• ZR Miniprep Kit
• Digestion (1.5h)    S+P and X+P
• Agarose gel electrophoresis 1%
• POSITIVE!!!!!!!!!!!!!!!!!!!
• Band purification
• FIRST SYSTEM READY

25 July

• Transformation of ligation 1E

26 July

• 1E;  2 COLONIES!!! ; PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
  • 6h culture!
  • ZR Miniprep Kit
  • Digestion (1.5h)
  • S+P and X+P
  • Agarose gel electrophoresis 1%
    • POSITIVE!!!!!!!!!!!!!!!!!!!
    • Band Purification
    • JUST ONE MORE LIGATION
    • Overnight digestion

27 July

• ZR Miniprep Kit ligations 1D
• Digestion X+P
• PrrA promoter+GFP+DT            X+P
• Agarose gel electrophoresis 1%
• POSITIVE (+)
• High concentration!
• Band purification
• POSITIVE
• 1E PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
• ZR Miniprep Kit
• Digestion (1.5h)
• S+P and X+P
• Agarose gel electrophoresis 1%
• POSITIVE!!!!!!!!!!!!!!!!!!!
• Band Purification
• POSITIVE
• Ligation 2F:
• PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT+ PrrA promoter+GFP+DT    

28 July

• Transformation ligation 2F

29 July

• NOTHING, AGAIN!           
• ZR Miniprep Kit ligations 1D
• Digestion X+P
• PrrA promoter+GFP+DT            X+P
• Agarose gel electrophoresis 1%
• POSITIVE (+)
• High concentration!
• Band purification
• POSITIVE
• 1E PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
• ZR Miniprep Kit
• Digestion (1.5h)
• S+P and X+P
• Agarose gel electrophoresis 1%
• POSITIVE!!!!!!!!!!!!!!!!!!!
• Band Purification
• POSITIVE
• Ligation 2F

30 July

• Transformation

1 August

• 2F, 4 COLONIES    
  • 6h cultures
  • ZR Miniprep Kit
  • Digestion (1.5h)
    • E and E+P
  • Agarose gel electrophoresis 1%
    • POSITIVE!!!!!!!!!!!!!!!!!!!
• Band Purification
• POSITIVE
• SECOND SYSTEM READY

• Party! all the systems are ready…

Biobricks Final assembly to psB1C3

• From 3 of August to 15 of August, we did the following things.
  • Overnight and 6h cultures
  • ZR Miniprep Kit
  • Digestion (1.5h)
    • For ligate
    • For confirmation
  • Agarose gel electrophoresis 1%
  • Band purification
  • Ligation
  • Transformation; and again!
  • TOTAL                                  11 parts in psB1C3