Team:Cambridge/Lab book/Week 8

Monday (13/08/12)

Ratiometrica-Lux: Miniprep of LuxBrick plasmid

• Miniprepped is done using miniprep kit supplied by Cambio
• Plasmids are miniprepped from 3 cultures

Ratiometrica-Lux: PCR of lux vector

• With split primers, similar conditions to last week (except no longer using E. coli colonies), each half in triplicates
• Results: Half of the vector (Fragment B) came out (gel 1), positive control worked (gel 2 lane 5)

Ratiometrica-Lux: Arabinose induction of Lux E. coli

• E. coli with the lux plasmid (K325909) O/N liquid cultures are induced with 3mM arabinose
• They did not produce visible bioluminescence after 7 hours. We suspect it to be a problem associated either with the concentration of cells in the culture or the amount of arabinose we added.

Tuesday (14/08/12)

Gibson assembly of positive control

• Fragments from Tom's PCR from last week are used: the PSB4K5 backbone (in triplicates), and sfGFP from sfGFP-ampR (in triplicates)

• Protocol changed slightly: 0.5 μl of each DNA fragment solution mixed in with master mix to make up 4 μl total volume (1μl DNA and 3μl master mix).

Transformation of e.coli with positive control DNA

• Chemically competent e.coli cells transformed with plasmid DNA produced by Gibson assembly step.

• Transformants plated out on 50 μg/ml kanomycin plates. Incubated overnight at 37 °C.

Ratiometrica-Lux: Arabinose Induction of Lux E. coli

• After our failed liquid culture induction, we made LB agar plates with 3mM arabinose and the appropriate antibiotics.
• Cells are plated out on the arabinose plates and incubated at 37°C overnight.
• Colonies on plates show bioluminescence after overnight incubation

Wednesday (15/08/12)

PCR of positive control fragments

• A replication of last week's PCR,except each fragment is done 5 times to generate more fragments so that the positive control could be used for future experiments

• Results: successful amplification of all fragments.

Extraction of positive control DNA

• Positive control DNA from gel excised and purified.

• Additional elution steps used to concentrate resultant solution: eluants are recycled at least once into the spin column

• Verified with nanodropper - final DNA concentrations are around 20ng/ul for first eluants, and 15ng/ul for second eluants; this is twice as concentrated as before

Gibson assembly of positive control DNA

Transformation of positive control DNA into e.coli

• Chemically competent e.coli transformed with positive control Gibson products made previously.

• Transformants plated out onto 50 μg/ml kanomycin plates and incubated at 37 °C overnight.

Characterization of fluoride riboswitch construct

• Strain of bacillus lacking fluoride transporter system tested. Concentrations used:

• 0mM - 0.5mM - 1mM - 2.5mM - 5mM - 10mM - 20mM - 30mM

Thursday (16/08/12)

Characterization of fluoride riboswitch construct

The results of our fluoride assay.

• Eppindorfs containing the bacillus with the fluoride riboswitch removed from incubator and imaged.

• Now this definitely works, we will try quantifying this riboswitch with an ONPG assay and the plate reader.

PCR ran by paul - insert photos/discuss

• Only positive control and two sets of triplicates worked
• YFP and CFP extracted successfully

Friday (17/08/12)

• Team meet up today! No lab work was done.

Saturday (18/08/12)

PCR of Fluoride biobrick format

Gels from PCR of Fluoride BB and PJS130 fragments. Top: Lanes 2-4: Gibson compatable Fluoride biobrick fragment. Lanes 5-7: Ligation compatable Fluoride biobrick fragment. Lane 8: PJS130 fragment A. Bottom: Lanes 1-2: PJS130 fragment A. Lanes 3-5: PJS 130 fragment B. Lane 6: Positive control. Lane 7: Negative control.

• Two sets of primers used: one to allow insertion into backbone by ligation, and one to allow insertion by Gibson assembly.

• Cycle settings:

• Melting - 98 °C - 10 seconds

• Annealing - 58 °C - 30 seconds

• Elongation - 72 °C - 100 seconds

• Products run on gel. Fragments produced of correct size, however they overlapped with the primer dimer band due to the small size of the fragments.

• DNA extracted and purified.

PCR of Mg2+ riboswitch construct vector

• Separate reactions for

• Cycle settings:

• Melting - 98 °C - 10 seconds

• Annealing - 58 °C - 30 seconds

• Elongation - 72 °C - 100 seconds

• Products run on gel. No fragments produced. Positive control worked however, so master mix clearly works.

• Given this PCR has worked in the past, will try to re-run with the same settings and hope for success in the future.