Team:Aberdeen Scotland/Biobrick Related

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<li>3.Transfer of Transformants into fresh plates to prevent the depletion of nutrients.<br></ul>
<li>3.Transfer of Transformants into fresh plates to prevent the depletion of nutrients.<br></ul>
<br>
<br>
-
<h2>Week1 (14th – 18th of June)</h2><br>
+
<h2>Week 1 (14th – 18th of June)</h2><br>
<ul><li>1.Confirmation of the identity of Biobricks YFP (pSB2k3 + E2030)<br>
<ul><li>1.Confirmation of the identity of Biobricks YFP (pSB2k3 + E2030)<br>
<li>2.Plasmid purification (Miniprep) of desired BioBrick constructs:<br>
<li>2.Plasmid purification (Miniprep) of desired BioBrick constructs:<br>
Line 25: Line 25:
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul>
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul>
<br>
<br>
-
<h2>Week2 (21st – 25th of June)</h2><br>
+
<h2>Week 2 (21st – 25th of June)</h2><br>
-
1.Design of Primers for construction of Biobricks<br>
+
<ul><li>1.Design of Primers for construction of Biobricks<br>
-
i.MS2 protein<br>
+
<ul><li>i.MS2 protein<br>
-
ii.MS2 loops: 5’leader<br>
+
<li>ii.MS2 loops: 5’leader<br>
-
iii.N peptide (1)<br>
+
<li>iii.N peptide (1)<br>
-
iv.N peptide (2)<br>
+
<li>iv.N peptide (2)<br>
-
v.Bbox – 5’leader<br>
+
<li>v.Bbox – 5’leader<br></ul>
-
2.Due to unclear results obtained in the prior digestion we repeated the digestion of BioBricks:<br>
+
<li>2.Due to unclear results obtained in the prior digestion we repeated the digestion of BioBricks:<br>
-
i.BBa_I716101 plasmid and BBa_J04450 (RFP)<br>
+
<ul><li>i.BBa_I716101 plasmid and BBa_J04450 (RFP)<br>
-
ii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br>
+
<li>ii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul>
<br>
<br>
-
<h2>Week3 (28th – 2nd July)</h2><br>
+
<h2>Week 3 (28th – 2nd July)</h2><br>
-
1.Bgl Brick Preparation:<br>
+
<ul><li>1.Bgl Brick Preparation:<br>
-
i.Digestion 1, of BglBrick 1, using EcoR1 and Xho1 – higher volume than protocol since concentration of plasmid was very low.  <br>
+
<ul><li>i.Digestion 1, of BglBrick 1, using EcoR1 and Xho1 – higher volume than protocol since concentration of plasmid was very low.  <br>
-
ii.Enzymes then heat inactivated<br>
+
<li>ii.Enzymes then heat inactivated<br>
-
iii.Vector then alkaline phosphatase treated, with enzymes again heat inactived after treatment.<br>
+
<li>iii.Vector then alkaline phosphatase treated, with enzymes again heat inactived after treatment.<br></ul></ul>
<br>
<br>
-
<h2>Week4 (5th – 9th of July)</h2><br>
+
<h2>Week 4 (5th – 9th of July)</h2><br>
-
1.Miniprep of BglBrick 1<br>
+
<ul><li>1.Miniprep of BglBrick 1<br>
-
2.Digestion of BglBrick using EcoR1 and Xho1 – due to low yield when previously digested.<br>
+
<li>2.Digestion of BglBrick using EcoR1 and Xho1 – due to low yield when previously digested.<br>
-
3.PCR 1 reaction of inserts (to use for BglBrick cloning)<br>
+
<li>3.PCR 1 reaction of inserts (to use for BglBrick cloning)<br>
-
4.Transformation of BglBrick into one shot competent cells E.coli cells, to try and improve plasmid propagation – no improved propagation observed.<br>
+
<li>4.Transformation of BglBrick into one shot competent cells E.coli cells, to try and improve plasmid propagation – no improved propagation observed.<br></ul>
<br>
<br>
-
<h2>Week5 (12th – 16th of July)</h2><br>
+
<h2>Week 5 (12th – 16th of July)</h2><br>
-
1.Further Bgl Brick preparation:<br>
+
<ul><li>1.Further Bgl Brick preparation:<br>
-
i.Miniprep of BglBrick<br>
+
<ul><li>i.Miniprep of BglBrick<br>
-
ii.Digestion of BglBrick<br>
+
<li>ii.Digestion of BglBrick<br>
-
iii.Enzyme heat inactivation<br>
+
<li>iii.Enzyme heat inactivation<br>
-
iv.Alkaline phosphatase treatment.<br>
+
<li>iv.Alkaline phosphatase treatment.<br></ul>
-
2.Digestion 1 of PCR products using EcoR1 and Xho1<br>
+
<li>2.Digestion 1 of PCR products using EcoR1 and Xho1<br>
-
3.Ligation 1 of PCR products (inserts 1) and vector BglBrick<br>
+
<li>3.Ligation 1 of PCR products (inserts 1) and vector BglBrick<br>
-
i.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.<br>
+
<ul><li>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.<br></ul>
-
4.Transformation 1, of cloned products into XL1 Blue subcloning – grade cells E.coli cells.<br>
+
<li>4.Transformation 1, of cloned products into XL1 Blue subcloning – grade cells E.coli cells.<br>
-
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.<br>
+
<li>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.<br>
-
6.PCR of E.coli colonies from BglBrick 1 – Did not work.<br>
+
<li>6.PCR of E.coli colonies from BglBrick 1 – Did not work.<br>
-
7.Design and order of mOrange primers for Bio-brick testing<br>
+
<li>7.Design and order of mOrange primers for Bio-brick testing<br>
-
8.Background reading for  Bgl-Brick vector and Bgl-bricking<br>
+
<li>8.Background reading for  Bgl-Brick vector and Bgl-bricking<br>
-
9.Mini-prep, digest and gel electrophoresis of Bgl-Brick vector and pRS415 vector <br>
+
<li>9.Mini-prep, digest and gel electrophoresis of Bgl-Brick vector and pRS415 vector <br></ul>
<br>
<br>
-
<h2>Week6 (19th – 23rd of July)</h2><br>
+
<h2>Week 6 (19th – 23rd of July)</h2><br>
-
1.More Bgl Brick preparation since previous involved Nanodrop concentration determination but was decided this analysis is unrealiable:<br>
+
<ul><li>1.More Bgl Brick preparation since previous involved Nanodrop concentration determination but was decided this analysis is unrealiable:<br>
-
i.Miniprep of BglBrick<br>
+
<ul><li>i.Miniprep of BglBrick<br>
-
ii.Digestion of BglBrick<br>
+
<li>ii.Digestion of BglBrick<br>
-
iii.Enzyme heat inactivation<br>
+
<li>iii.Enzyme heat inactivation<br>
-
iv.Alkaline phosphatase treatment.<br>
+
<li>iv.Alkaline phosphatase treatment.<br></ul>
-
2.Background reading and planning for PCR <br>
+
<li>2.Background reading and planning for PCR <br>
-
3.Background reading and planning of alkaline phosphatase and ligation reaction for Bgl-bricking <br>
+
<li>3.Background reading and planning of alkaline phosphatase and ligation reaction for Bgl-bricking <br></ul>
<br>
<br>
-
<h2>Week7 (26th – 30th of July)</h2><br>
+
<h2>Week 7 (26th – 30th of July)</h2><br>
-
1.PCR of E.coli colonies from BglBrick 2 – positive PCR.<br>
+
<ul><li>1.PCR of E.coli colonies from BglBrick 2 – positive PCR.<br>
-
2.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br>
+
<li>2.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br>
-
3.Transformation 3, of cloned products (inserts 1) into subcloning efficiency DH5α E.coli cells.<br>
+
<li>3.Transformation 3, of cloned products (inserts 1) into subcloning efficiency DH5α E.coli cells.<br>
-
4.PCR of Transformation 3 colonies which were then also plated, to confirm required insert presence.<br>
+
<li>4.PCR of Transformation 3 colonies which were then also plated, to confirm required insert presence.<br>
-
5.Second stage digest , (PvuII) of pRS415 and gel electrophoeris for Bgl-bricking  <br>
+
<li>5.Second stage digest , (PvuII) of pRS415 and gel electrophoeris for Bgl-bricking  <br>
-
6.Master plate of Bgl-brick<br>
+
<li>6.Master plate of Bgl-brick<br>
-
7.PCR of mORange<br>
+
<li>7.PCR of mORange<br>
-
8.Transformation of BY4741 ΔTrp with pRS415 and mOrange (homologous recombination)<br>
+
<li>8.Transformation of BY4741 ΔTrp with pRS415 and mOrange (homologous recombination)<br></ul>
<br>
<br>
-
<h2>Week8 (2nd – 7th of August)</h2><br>
+
<h2>Week 8 (2nd – 7th of August)</h2><br>
-
1.Confirmation of BglBricks produced:<br>
+
<ul><li>1.Confirmation of BglBricks produced:<br>
-
i.Plasmid purification (miniprep) of E.coli colonies from Transformation 3 colonies.<br>
+
<ul><li>i.Plasmid purification (miniprep) of E.coli colonies from Transformation 3 colonies.<br>
-
ii.Digestion of plasmid with<br>
+
<li>ii.Digestion of plasmid with<br>
-
iii. Ecor1 and Xhol1<br>
+
<li>iii. Ecor1 and Xhol1<br>
-
iv.Ecor1 – since insert was not being observed on the gel when cut with two enzymes.<br>
+
<li>iv.Ecor1 – since insert was not being observed on the gel when cut with two enzymes.<br></ul>
-
2.Further Bgl Brick preparation as we ran out:<br>
+
<li>2.Further Bgl Brick preparation as we ran out:<br>
-
i.6 X Miniprep of BglBrick<br>
+
<ul><li>i.6 X Miniprep of BglBrick<br>
-
ii.Combined all 6 from step i. To obtain a higher concentration of BglBrick using Qiagen PCR kit (check name of kit).<br>
+
<li>ii.Combined all 6 from step i. To obtain a higher concentration of BglBrick using Qiagen PCR kit (check name of kit).<br>
-
iii.Digestion of BglBrick using EcoR1 and Xho1<br>
+
<li>iii.Digestion of BglBrick using EcoR1 and Xho1<br>
-
iv.Digestion had to be done twice as some uncut vector remained after the first cut.<br>
+
<li>iv.Digestion had to be done twice as some uncut vector remained after the first cut.<br>
-
v.Enzyme heat inactivation<br>
+
<li>v.Enzyme heat inactivation<br>
-
vi.Alkaline phosphatase treatment.<br>
+
<li>vi.Alkaline phosphatase treatment.<br></ul>
-
3.Tested BY4741 pRS415 mOrange transformants <br>
+
<li>3.Tested BY4741 pRS415 mOrange transformants <br>
-
4.Read through protocol for PCR colony screening <br>
+
<li>4.Read through protocol for PCR colony screening <br></ul>
<br>
<br>
<h2>Week 9 (9th – 14th of August)</h2><br>
<h2>Week 9 (9th – 14th of August)</h2><br>
-
1.Digestion 1, of E.coli colonies from BglBrick 2 using EcoR1 and Xho1.<br>
+
<ul><li>1.Digestion 1, of E.coli colonies from BglBrick 2 using EcoR1 and Xho1.<br>
-
2.Digestion 2, of E.coli colonies from BglBrick 2 using EcoR1, since digest with two enzymes showed no insert band on the gel<br>
+
<li>2.Digestion 2, of E.coli colonies from BglBrick 2 using EcoR1, since digest with two enzymes showed no insert band on the gel<br>
-
3.Digestion 2, of PCR products using EcoR1 and Xho1<br>
+
<li>3.Digestion 2, of PCR products using EcoR1 and Xho1<br>
-
4.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br>
+
<li>4.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br>
-
5.Transformation 2, of cloned products into XL1 Blue competent – grade E.coli cells.<br>
+
<li>5.Transformation 2, of cloned products into XL1 Blue competent – grade E.coli cells.<br>
-
6.PCR Colony screening of mOrange  transformants<br>
+
<li>6.PCR Colony screening of mOrange  transformants<br>
-
7.Bgl-bricking transformation experiment<br>
+
<li>7.Bgl-bricking transformation experiment<br>
-
8.Tested BY4741 pRS415 mOrange transformants using fluorometer<br>
+
<li>8.Tested BY4741 pRS415 mOrange transformants using fluorometer<br>
-
9.Mini-prep Bgl-brick vectors<br>
+
<li>9.Mini-prep Bgl-brick vectors<br></ul>
 +
<br>
 +
<h2>Week 10 (15th – 21st of August)</h2><br>
 +
<ul><li>1. Repeated: PCR of inserts chosen to submit into biobricks.<br>
 +
<li>2. pSB1C3 plasmid preparation:
 +
<li>i.Digestion of linear plasmid with EcoR1 and Pst1
 +
<li>ii.Enzyme heat inactivation <br>
 +
<li>3. Digestion of PCR products using EcoR1, Dpn1 and Pst1<br>
 +
<li>4. Ligation 1 of PCR products and pSB1C3 plasmid. <br>
 +
<li>5. Transformation 1, (1:1, plasmid:vector) of cloned products into XL1 Blue competent E.coli cells – grade cells E.coli cells.<br>
 +
<li>6. Transformation 2, (1:3, plasmid:vector)of cloned products into XL1 Blue competent E.coli cells. <br>
 +
<li>7. PCR of E.coli colonies<br></ul>
 +
<br>
 +
<h2>Week 11 (22nd – 29th of August)</h2><br>
 +
<ul><li>1. Design and order of primers for Bio-brick testing <br>
 +
<li>2. Sent Biobricks to iGEM registry of parts <br>
 +
<li>3.FACS analysis of dose response of <br></ul>
 +
 
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Latest revision as of 20:38, 27 October 2010

University of Aberdeen - ayeSwitch - iGEM 2010

Contents

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)


  • 1. Design and order of primers for Bio-brick testing
  • 2. Sent Biobricks to iGEM registry of parts
  • 3.FACS analysis of dose response of





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