Team:BCCS-Bristol/Notebook/Week 12

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===Bioscaffold Tests===
===Bioscaffold Tests===
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*During week 11 tests for mutation success for GFP illegal Bioscaffold sitewere carried out and successful mutants were identified to proceed with the bioscaffold tests
*The entire construct AraC-RBS-FhuA-Bioscaffold-GFP-Terminator was inserted in a pSB2K3 plasmid. pSB2k3 has no restriction enzyme recognition sequences for the Bioscaffold specific enzymes.
*The entire construct AraC-RBS-FhuA-Bioscaffold-GFP-Terminator was inserted in a pSB2K3 plasmid. pSB2k3 has no restriction enzyme recognition sequences for the Bioscaffold specific enzymes.
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*Different liquid cultures carrying the entire construct were harvested by centrifugation and DNA was miniprepped.
*Different liquid cultures carrying the entire construct were harvested by centrifugation and DNA was miniprepped.
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*To test for mutation success for GFP illegal Bioscaffold site, the DNA was tested with Single Digest (XbaI), Single Digest (BpuEI)and Double Digest. As a control premutated DNA was also digested. Incubations were for 60min/37oC.
 
*Successful mutants were identified. Also prolonged incubation leads to BpuEI having star activity. As a result we decided to incubate for 45min. (Bioscaffold enzymes are time-saver qualified and can also be used for 5min incubation).
*Successful mutants were identified. Also prolonged incubation leads to BpuEI having star activity. As a result we decided to incubate for 45min. (Bioscaffold enzymes are time-saver qualified and can also be used for 5min incubation).
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*Samples were sent for sequencing to confirm if Bioscaffold application was successful.
*Samples were sent for sequencing to confirm if Bioscaffold application was successful.
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[[Image:BCCS_Bioscaffold_gels2.jpg|center|200px|frame| The ladder is a standard 1kb NEB DNA marker. Lanes run in pairs of four illustrating Uncut, Single Digest (BpuEI), Single Digest (BseRI) and Double Digest of plasmid DNA carrying the entire AraC-RBS-FhuA-Bioscaffold-GFP-Terminator construct (left) and the product post application of the Bioscaffold as outlined in text (right). Band shift are seen on samples on the gel still carrying the Bioscaffold but no band shifts are seen on the gel from samples post-scaffold application indicating collapse of the scaffold. ]]
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{{:Team:BCCS-Bristol/NotebookHeader}}
{{:Team:BCCS-Bristol/NotebookHeader}}

Revision as of 23:10, 12 October 2009

BCCS-Bristol
iGEM 2009


Bioscaffold Tests

  • During week 11 tests for mutation success for GFP illegal Bioscaffold sitewere carried out and successful mutants were identified to proceed with the bioscaffold tests
  • The entire construct AraC-RBS-FhuA-Bioscaffold-GFP-Terminator was inserted in a pSB2K3 plasmid. pSB2k3 has no restriction enzyme recognition sequences for the Bioscaffold specific enzymes.
  • Different liquid cultures carrying the entire construct were harvested by centrifugation and DNA was miniprepped.


  • Successful mutants were identified. Also prolonged incubation leads to BpuEI having star activity. As a result we decided to incubate for 45min. (Bioscaffold enzymes are time-saver qualified and can also be used for 5min incubation).
  • The Bioscaffold was tested by following its application instructions: 
 Step1: Restrict DNA with BseRI to remove stop codons and DNA scar.
 Step2: Ligate to convert the remaining stop codon into tyrosine aminoacid (From TA to TAC).
 Step3: Restrict Digest with BpuEI to collapse bioscaffold. 
 Step4: Ligate to obtain a seemingly scarless fusion.

All incubations were for 60min/37oC. After each step, the relevant enzymes were inactivated and DNA purification was implemented.

Aliquots from each step were kept and run on an agarose gel to assess Bioscaffold viability.

  • Transformations with XL-1 Blue cells from each restriction-ligation pair and subsequent restreak and liquid cultures were made. Cells were harvested by centrifugation and DNA plasmid obtained by minipreps.
  • Aliquots from each step were assessed by BpuEI single digest, BseRI single digest and Double Digest. Preliminary results show bioscaffold to be successful.
  • Samples were sent for sequencing to confirm if Bioscaffold application was successful.


The ladder is a standard 1kb NEB DNA marker. Lanes run in pairs of four illustrating Uncut, Single Digest (BpuEI), Single Digest (BseRI) and Double Digest of plasmid DNA carrying the entire AraC-RBS-FhuA-Bioscaffold-GFP-Terminator construct (left) and the product post application of the Bioscaffold as outlined in text (right). Band shift are seen on samples on the gel still carrying the Bioscaffold but no band shifts are seen on the gel from samples post-scaffold application indicating collapse of the scaffold.


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