Date: 23/09/24
Members: Carmen
Retransformation of pMQ3A 3x
pMQ3C continued
plates from 19/09
DH10B @30C - lawns! pMQ3A and pMQ3C's have brown pigment diffused into agar but 3Cs are darker. could be more of same pigment? 3A colonies are closer to normal cream color, 3Cs colonies are markedly brown (along with agar coloration)
RARE @30C - very little growth, 1-2 colonies per plate at most. No agar coloration, but colony morphology varies (some big/small, one looks black/brown) could be contamination or mutants?
DH10B @37 - more lawns! colonies are small but expected when colonies are crowded. 3A has agar coloration (similar to @30C), 3Cs have more darker coloration and colonies are dark brown. accumulation of something?
RARE @37C - only 1x single colony on 3A and 3C_20 plates. nothing on rest.
pus250 controls wokreek fine for all 4 sets (purple lots of large healthy colonies)
still no growth on plates from 16/09, thrown out
RARE strain is clearly struggling maybe accumulating too many aldehydes, leading to toxicity?
sticking to DH10B for now due to time constraints
Induction
scrape ⅔ of each DH10B @37 plate into 1mL LB and measure OD600
for each plate resuspension, inoculate 5mL Lb-Tyr-Km at 3x different starting ODS: 0.05 and 0.1
confirm initial OD
incubate overnight at 37C
will induce with cumate tomorrow morning, these cells seem to grow slower with our plasmids (compare growth with pMQ's vs pus250)
X=(measured OD/target OD)
uL cells to add = (volume of media uL/ X)
scraped cells were very resistant to resuspending fully so let the resuspension mix sit for ~1 min so largest chunks could settle and used 100uL of supernatant + 900uL LB for OD measurements.
in 37C shaking incubator at ~3:40pm
Date: 24/09/24
Members: Carmen
ODs of overnight from 23/09
1/10 dilutions of cultures in Lb-Tyr for readings, x by 10 for “true” measurements
all way overgrown (aiming for 0.5). transferred 500uL of each into 5mL fresh Lb-Tyr-Km for outgrowth before induction
after outgrowth, transferred 2.5mL of each into new 50mL falcons for induction - rest left as uninduced controls.
induced with cumate (find concentration = 100uM) at 1:50pm, put into 37C shaking incubator overnight
Date: 24/09/2
Members: Carmen, Faiza, Jenny
repeat of retransformation of pMQ3A and pMQ3Cs
repeat of 19/09 to confirm that strains have not been mixed up, except only at 37C, omitting 30C set
R1: RARE + pMQ3A
R2: RARE + pMQ3C_11
R3: RARE + pMQ3C_15
R4: RARE + pMQ3C_20
R5: RARE + pus250
D1: DH10B + pMQ3A
D2: DH10B + pMQ3C_11
D3: DH10B + pMQ3C_15
D4: DH10B + pMQ3C_20
D5: DH10B + pus250
2uL of each plasmid transformed into 20uL of each competent cell type. recovered in Lb 1mL for 1 hour. pelleted cells at 16000G 2min, removed 800uL supernatant so 200uL leftover cells, resuspended and 100uL each plated onto Lb-Km.
10x plates in 37C incubator overnight , only patrf 1x 100uL so colony density should be the same as last time
Date: 25/09/24
Members: Carmen
Overnight Induction Results
excessive cumate was fine. don't need to redo
pMQ3A: more typical saturated E.coli culture color, but uninduced look slightly darker/denser?
pMQ3Cs: all are very dark brown, almost black! such an enormous difference compared to 3A. uninduced also looks slightly darker than induced
pus250: no noticeable difference between induced and uninduced(good and expected) both are dusky like color
Chemical analysis prep
falcons shaken to fully distribute, then 1mL of each transferred t sterile 1.5mL tubes and spiun down at max speed (21k G) for 10 minutes.
remaining cultures in falcon tubes out in 4C fridge
after spindown, 700uL of supernatant from each tube very carefully transferred to new sterile 1.5mL tubes, put into orange freezer box and into iGEM freezer drawer
Observations:
Smell
pus250 cultures smell like normal E.coli cultures
pMQ3A seems to smell less acrid than typical cultures
pMQ3Cs definitely seem to smell almost sweeter or at least far less bad than typical cultures
asked Ruby W. for opinion on culture smells without telling what each was. she said pus250 smelled normal, pMQ3A was not as strong as normal, and at least pMQ3C_15 (induced and uninduced) smelled “a little fruity” note: only gace pus250-I, 3A+1, and 3C_15 +I
Visual
3C cultures very dark brown but after pelletting, the supernatant is still brown but the cell pellets are similar color to 3A - the color is in the supernantant
supernatant color of 3A is basically same as pus250 (3A slightly darker yellow?)
for all 3Cs, the uninduced supernatant is noticebaky darker than induced
no cloudiness in any of the supernatants, different colors are all still clear
note: original samples induced with 500uM cumte. additional set repeated with 100uM climate- these have sticker on them. not needed unless something happens to first set
cumate working stick calcs: from 24/09
dilution solution 1mL 100%EtOH - 0.5TRIS in 50% v/v EtOH
needs to be diluted in same solution dissolved in
50mM cumate working stock: 900uL dilution solution + 100uL 0.5M cumate stock
induction: 5u: 50mM cumate in 2.5mL culture = final cumate concentration of 100uM
Date: 26/09/24
Members: Carmen
supernatant samples for chemical analysis submitted to Veronica
retransformation plates from 24/09 look the same as 19/09, confirming that strains were nit swapped -placed back into 37C incubator until tomorrow
cycled with proper cycling 30*( 5min37℃, 5min 16℃)
