Genomic DNA extraction from Bacillus subtilis strain ATCC 6633

Aim

The aim of this experiment was to extract genomic DNA from Bacillus subtilis strain ATCC 6633. This strain produces the subtilin genes, required for our Subtilin Production and Immunity BioBricks as well as the Quorum sensor biobrick designed by Team Newcastle 2008.

Materials and methods

• ara forward and reverse primers
• sac forward and reverse primers
• Pipettes
• Universal tubes
• Eppendorf tubes
• Centrifuge
• PCR (see PCR protocol from Lab training)
• Gel electrophoresis (see Gel electrophoresis from Lab training)

ara and sac are two genes that exist in the ATCC 6633 strain. ara and sac forward and reverse primers are two tests, which will be used in PCR. At the end, Gel Electrophersis can be used to distinguish the bands. If the bands from either of ara or sac show up at the expected bps against the DNA ladder, this proves that the genomic DNA is sucessfully extracted.

Protocol

Please refer to: DNA extraction of Bacillus subtilis for materials required and protocol.

Results

Results were as expected see: https://2010.igem.org/Team:Newcastle/7_July_2010

Conclusion

See: https://2010.igem.org/Team:Newcastle/7_July_2010

LacI BioBrick Construction

Aims

Use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (rfp).

Materials

• Overnight culture.
• Restriction enzymes EcoR1 and Xba1.
• PCR primers designed for lacI extraction.

Protocol

• Plasmid miniprep from overnight cultures.
• Restriction digest of pSB1AT3 with EcoR1 and Xba1.
• Amplify lacI fragment by PCR from pMutin4.
• Restriction digest of amplified fragment of pMutin4 with EcoR1 and Spe1.

Inference

• The plasmids are extracted from the transformed E. coli and digested so that they are linear molecules, ready to be run on an agarose gel. Undigested pMutin4 is used as the PCR template to amplify the lacI coding sequence.