Team:Aberdeen Scotland/Biobrick Related
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Revision as of 21:40, 24 October 2010
University of Aberdeen - ayeSwitch
iGEM 2010
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
- i.BBa_E2050 – mOrange fluorescent protein, KanR.
- 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.
Week1 (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)
- i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)
- 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)
- i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)
Week2 (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
- i.MS2 protein
- 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)
- i.BBa_I716101 plasmid and BBa_J04450 (RFP)
Week3 (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.
- i.Digestion 1, of BglBrick 1, using EcoR1 and Xho1 – higher volume than protocol since concentration of plasmid was very low.
Week4 (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.
Week5 (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.
- i.Miniprep of BglBrick
- 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.
- 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
Week6 (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.
- i.Miniprep of BglBrick
- 2.Background reading and planning for PCR
- 3.Background reading and planning of alkaline phosphatase and ligation reaction for Bgl-bricking
Week7 (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)
Week8 (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.
- i.Plasmid purification (miniprep) of E.coli colonies from Transformation 3 colonies.
- 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.
- i.6 X Miniprep of BglBrick
- 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
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