Team:BCCS-Bristol/Notebook/Week 12

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Revision as of 22:45, 12 October 2009

BCCS-Bristol
iGEM 2009

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.

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).

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.

Week Zero	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9	Week 10	Week 11	Week 12

@@ Line 15: / Line 15: @@
 *The Bioscaffold was tested by following its application instructions:
-   Step1: Restrict DNA with BseRI to remove stop codons 5'-TAA TAA-3' from protein coding gene (only 5'-TA-3' left) and DNA Scar. Incubate 60min/37oC. Heat Inactivate enzymes and purify DNA with QIAGEN PCR purification kit.
+   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). Heat Inactivate ligase enzyme and purify DNA with QIAGEN PCR purification kit.
+   Step2: Ligate to convert the remaining stop codon into tyrosine aminoacid (From TA to TAC).
-   Step3: Restrict Digest with BpuEI to collapse bioscaffold. Heat Inactivate enzymes and purify DNA with QIAGEN PCR purification kit.
+   Step3: Restrict Digest with BpuEI to collapse bioscaffold.
-   Step4: Ligate to obtain a seemingly scarless fusion.Heat Inactivate ligase enzyme and purify DNA with QIAGEN PCR purification kit.
+   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.