Team:Newcastle/6 July 2010

Aim of this experiment

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 from 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 th 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.

Gram-positive Bacteria Chromosomal DNA Extraction Protocol

Puregene DNA isolation

1. Grow overnight 7.5ml cultures in THYB containing 30μg/ml hyaluronidase.

Cell Lysis

1. Two sets of chromosomal DNA were being done.
2. Cells go through 3600 x g centrifugation for 15 minutes to pellet.
3. The supernatant in the tubes are poured away.
4. 0.5ml of cell suspension solution is added to the eppendorf tubes. The solution in the tubes are pipetted up and down to resuspend cells. The solution is then transferred to another 1.5ml tube.
5. 50μl of lysozyme and 7.5μl of lytic enzyme solution is added into each tube. The tubes are then inverted 25 times to mix the solution.
6. We then incubate them at 37°C for 30 minutes in order to digest the cell walls. The tubes are inverted occasionally during incubation.
7. To pellet the cells, we centrifuged it for 10 minutes and poured away the supernatant after that.
8. 0.5ml of cell lysis solution is added to the pelleted cells and the solution is pipetted up and down in order to lyse the cells. The samples are then heated for about 5 minutes at 80°C.

RNase Treatment

1. 6μl of RNase A solution is added into the cell lysate.
2. The tubes are inverted 25 times in order to allow the solutions to mix. They are then incubated at 60minutes at 37°C.

Protein Precipitation

1. The samples are cooled on ice.
2. 0.5ml of protein precipitation solution is added into each tube.
3. The solutions are vortexed at high speed for about 20 seconds for the protein precipitation solution to mix properly with the cell lysate. The samples are then placed on ice for 5 minutes.
4. They are then centrifuged at 13000rpm for 1 minute for the proteins to form a tight pellet.

DNA Precipitation

1. The supernatant containing the DNA are poured into a clean eppendorf tube.
2. 0.5ml of isopropanol is then added into each tube.
3. The solutions are then mixed by inverting gently for 50 times.
4. The mixture is centrifuged at 13000rpm for 1 minute. The DNA can be seen as a small white pellet at the bottom of the tubes.
5. The supernatant is poured off and the tubes are drained on a clean absorbent paper. 0.5ml of 70% ethanol is added into the tubes and are inverted a few times to wash the DNA.
6. They are then centrifuged for 1 minute at 13000rpm. After that, ethanol is poured off leaving the DNA behind.
7. The tubes are drained on clean absorbent paper. They are allowed to air dry for 10-15 minutes.

DNA Hydration

1. 100μl of DNA hydration solution is added to each tube.
2. DNA is rehydrated by incubating the sample for one hour at 65°C and overnight at room temperature.

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

• To 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 pMutin4 with EcoR1.
• Restriction digest of pSB1AT3 with EcoR1 and Xba1.
• Run PCR on pMutin4.

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.