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.

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)

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)

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.

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.

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

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

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)

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

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

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