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Team:BCCS-Bristol/Notebook/Week 12
From 2009.igem.org
BCCS-Bristol
iGEM 2009
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 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. 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. Step3: Restrict Digest with BpuEI to collapse bioscaffold. Heat Inactivate enzymes and purify DNA with QIAGEN PCR purification kit. Step4: Ligate to obtain a seemingly scarless fusion.Heat Inactivate ligase enzyme and purify DNA with QIAGEN PCR purification kit.
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.
