Lab Diary concerning Biobricks

Preparation week

• 1.Preparation / rescue of iGEM DNA samples:
  
  • i.BBa_E2050 – mOrange fluorescent protein, KanR.
    
  • ii.BBa_J63005 – ADH1 promoter, AmpR.
    
  • iii.BBa_E2030 – yEYFP (yellow fluorescent protein), KanR.
    
  • iv.BBa_I716101 with J04450 (plasmid), AmpR.
    
  • v.BBa_I716101 with P1010 (plasmid with CcdB), AmpR
    
• 2.Transformation of rescued iGEM DNA samples into XL1 – Blue Subcloning – Grade Competent Cells.
  
• 3.Transfer of Transformants into fresh plates to prevent the depletion of nutrients.
  

Week 1 (14th – 18th of June)

• 1.Confirmation of the identity of Biobricks YFP (pSB2k3 + E2030)
  
• 2.Plasmid purification (Miniprep) of desired BioBrick constructs:
  
  • i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)
    
  • ii.BBa_I716101 plasmid and BBa_J04450 (RFP)
    
  • iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)
    
• 3.BioBricks digestion – gel electrophoresis, to verify plasmid sizes, of:
  
  • i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)
    
  • ii.BBa_I716101 plasmid and BBa_J04450 (RFP)
    
  • iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)
    

Week 2 (21st – 25th of June)

• 1.Design of Primers for construction of Biobricks
  
  • i.MS2 protein
    
  • ii.MS2 loops: 5’leader
    
  • iii.N peptide (1)
    
  • iv.N peptide (2)
    
  • v.Bbox – 5’leader
    
• 2.Due to unclear results obtained in the prior digestion we repeated the digestion of BioBricks:
  
  • i.BBa_I716101 plasmid and BBa_J04450 (RFP)
    
  • ii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)
    

Week 3 (28th – 2nd July)

• 1.Bgl Brick Preparation:
  
  • i.Digestion 1, of BglBrick 1, using EcoR1 and Xho1 – higher volume than protocol since concentration of plasmid was very low.
    
  • ii.Enzymes then heat inactivated
    
  • iii.Vector then alkaline phosphatase treated, with enzymes again heat inactived after treatment.
    

Week 4 (5th – 9th of July)

• 1.Miniprep of BglBrick 1
  
• 2.Digestion of BglBrick using EcoR1 and Xho1 – due to low yield when previously digested.
  
• 3.PCR 1 reaction of inserts (to use for BglBrick cloning)
  
• 4.Transformation of BglBrick into one shot competent cells E.coli cells, to try and improve plasmid propagation – no improved propagation observed.
  

Week 5 (12th – 16th of July)

• 1.Further Bgl Brick preparation:
  
  • i.Miniprep of BglBrick
    
  • ii.Digestion of BglBrick
    
  • iii.Enzyme heat inactivation
    
  • iv.Alkaline phosphatase treatment.
    
• 2.Digestion 1 of PCR products using EcoR1 and Xho1
  
• 3.Ligation 1 of PCR products (inserts 1) and vector BglBrick
  
  • Used Nanodrop to determine concentration, we have concluded the data obtained in this way is unreliable, which is why this procedure may not have been successful.
    
• 4.Transformation 1, of cloned products into XL1 Blue subcloning – grade cells E.coli cells.
  
• 5.Transformation 2, of cloned products into XL1 Blue competent E.coli cells. Since transformation 1 gave poor efficiency, a more competent cell was tried.
  
• 6.PCR of E.coli colonies from BglBrick 1 – Did not work.
  
• 7.Design and order of mOrange primers for Bio-brick testing
  
• 8.Background reading for Bgl-Brick vector and Bgl-bricking
  
• 9.Mini-prep, digest and gel electrophoresis of Bgl-Brick vector and pRS415 vector
  

Week 6 (19th – 23rd of July)

• 1.More Bgl Brick preparation since previous involved Nanodrop concentration determination but was decided this analysis is unrealiable:
  
  • i.Miniprep of BglBrick
    
  • ii.Digestion of BglBrick
    
  • iii.Enzyme heat inactivation
    
  • iv.Alkaline phosphatase treatment.
    
• 2.Background reading and planning for PCR
  
• 3.Background reading and planning of alkaline phosphatase and ligation reaction for Bgl-bricking
  

Week 7 (26th – 30th of July)

• 1.PCR of E.coli colonies from BglBrick 2 – positive PCR.
  
• 2.Ligation 2, of PCR products (inserts 1) and vector BglBrick
  
• 3.Transformation 3, of cloned products (inserts 1) into subcloning efficiency DH5α E.coli cells.
  
• 4.PCR of Transformation 3 colonies which were then also plated, to confirm required insert presence.
  
• 5.Second stage digest , (PvuII) of pRS415 and gel electrophoeris for Bgl-bricking
  
• 6.Master plate of Bgl-brick
  
• 7.PCR of mORange
  
• 8.Transformation of BY4741 ΔTrp with pRS415 and mOrange (homologous recombination)
  

Week 8 (2nd – 7th of August)

• 1.Confirmation of BglBricks produced:
  
  • i.Plasmid purification (miniprep) of E.coli colonies from Transformation 3 colonies.
    
  • ii.Digestion of plasmid with
    
  • iii. Ecor1 and Xhol1
    
  • iv.Ecor1 – since insert was not being observed on the gel when cut with two enzymes.
    
• 2.Further Bgl Brick preparation as we ran out:
  
  • i.6 X Miniprep of BglBrick
    
  • ii.Combined all 6 from step i. To obtain a higher concentration of BglBrick using Qiagen PCR kit (check name of kit).
    
  • iii.Digestion of BglBrick using EcoR1 and Xho1
    
  • iv.Digestion had to be done twice as some uncut vector remained after the first cut.
    
  • v.Enzyme heat inactivation
    
  • vi.Alkaline phosphatase treatment.
    
• 3.Tested BY4741 pRS415 mOrange transformants
  
• 4.Read through protocol for PCR colony screening
  

Week 9 (9th – 14th of August)

• 1.Digestion 1, of E.coli colonies from BglBrick 2 using EcoR1 and Xho1.
  
• 2.Digestion 2, of E.coli colonies from BglBrick 2 using EcoR1, since digest with two enzymes showed no insert band on the gel
  
• 3.Digestion 2, of PCR products using EcoR1 and Xho1
  
• 4.Ligation 2, of PCR products (inserts 1) and vector BglBrick
  
• 5.Transformation 2, of cloned products into XL1 Blue competent – grade E.coli cells.
  
• 6.PCR Colony screening of mOrange transformants
  
• 7.Bgl-bricking transformation experiment
  
• 8.Tested BY4741 pRS415 mOrange transformants using fluorometer
  
• 9.Mini-prep Bgl-brick vectors
  

Week 10 (15th – 21st of August)

• 1. Repeated: PCR of inserts chosen to submit into biobricks.
  
• 2. pSB1C3 plasmid preparation: i.Digestion of linear plasmid with EcoR1 and Pst1 ii.Enzyme heat inactivation
  
• 3. Digestion of PCR products using EcoR1, Dpn1 and Pst1
  
• 4. Ligation 1 of PCR products and pSB1C3 plasmid.
  
• 5. Transformation 1, (1:1, plasmid:vector) of cloned products into XL1 Blue competent E.coli cells – grade cells E.coli cells.
  
• 6. Transformation 2, (1:3, plasmid:vector)of cloned products into XL1 Blue competent E.coli cells.
  
• 7. PCR of E.coli colonies
  

Week 11 (22nd – 29th of August)

3.FACS analysis of dose response of
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