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

• A combination of fragments from the PCR are chosen: the highest, second highest, third highest, lowest concentration first eluants, and the highest concentration second eluants are put together

• Reading that our T5 exonuclease concentration in Gibson assembly may be too high, we made up some mastermix with 1/5 of the T5 exo concentration and tested that in parallel with the fragments of various concentrations (2x5 = 10 Gibson reactions in total)

• Results: Successful Gibson assembly reactions. The lowered T5 exo conc seems to lower the Gibson efficiency, and the more concentrated fragments seem to produce more colonies.

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.

Large and small vector fragments, only positive control worked

CFP and B0015, only CFP came out

K143053 and YFP, only YFP came out

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

Ratiometrica-Lux: Restriction Digest of Lux vector

• Results: Digest came out exactly as expected.

Ratiometrica-Flu: PCR of Flourescent Construct DNA fragments

• Redo of PCR done at the end of July, as we are running out of fragments to Gibson

• Used existing DNA fragments as template rather than biobricks from the registry

• Results: Only the fluorescent protein fragments (eCFP and eYFP) came out clearly; the smaller fragments (B0015, K143053) might have came out but was mistaken for primer dimers, the vector fragments did not come out

Friday (17/08/12)

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

Saturday (18/08/12)

RiboSense: colonies picked in preparation for Miller assay

• 2 E. coli colonies transformed with the purified plasmid were picked and inoculated in LB with 50μg/ml Amp

• 2 colonies each from the 168 strain and crcB knockout B. subtilis containing the fluoride riboswitch were picked and inoculated in LB with 5μg/ml Chlor

RiboSense: 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.

Ribosense: 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.

Ratiometrica-Flu: 2nd attempt at PCR of fragments

• Reattempt of pJS130 and smaller fragments

• Used Velocity from Bioline, new mastermix recipe is used

• Results: failed- no wanted bands, since the positive control didn't come up, it is suggested that we need to optimise the PCR protocol for Velocity if we are to use it.

Ratiometrica-Flu: Gibson assembly of fluorescent construct

• Gibson performed using remaining DNA fragments which has been concentrated by dehydration

• We realised that though the decreased T5 exonuclease in the gibson mastermix might have lowered the Gibson efficiency in the positive control, the fragments we are trying to assemble are much smaller (~200bp); papers said this is likely to cause trouble

• We therefore attempt 2 solutions in parallel: (1) adding the T5 only when the reaction is already heated to 50°C (using a water bath); (2) using 1/5 of the amount of T5 exo (in both in parallel)

• These are all done in duplicates, resulting in 8 Gibson reactions and subsequent transformations

• Results: (Sunday) There are 2 colonies on one of the plates without either treatment

Sunday (19/08/12)

RiboSense: Miller Assay of Fluoride construct

• colonies picked from overnight and Miller assay run as directed on protocols page

• Lack of filters meant that only A450 data recorderd

Ratiometrica-Flu: 3rd attempt at PCR of fragments

• Similar PCR to the one from yesterday, except Velocity mastermix is further changed, and extension time is changed

• Results: failed- no bands, probably Velocity mastermix problem, will revert back to Phusion on next attempt