Team:Carnegie Mellon/Week10


Killer Red

Monday, August 5

Plates from last week:
KR/RFP streaks were left at RT all weekend but no observable color was present. This might mean that temperature is a big factor in the protein folding. To test this, re-streaks of the plates will be put at 30º and 37º overnight to see if there is a difference in color.
The lysogen plates were subject to the same conditions. If we don’t see a change in the color in the plasmid expression, it is less likely that we will see a color change in a lower-copy lysogen strain, which is grown at 30ºC. Therefore, these plates are to undergo the same streaks and conditions that the plasmid plates are subject to.
Redo the XL10 streaking

Backbones to use for cloning:
Plate 2: 5N (BBa_K817000) 162 bp
add 10µL dH2O to well and pipet
Transform, miniprep, and digest with XbaI and SpeI

Start 2x3mL overnights of the plasmid cultures and incubate at 37ºC. Tomorrow morning, spin down the cells and resuspend in SM (pH 7.4) and see if the SM proteins are brighter (store overnight at 4ºC and analyze on the TECAN)

Andrew is making the cultures and plates to test for the temperature sensitivity of the RFP and KR... Cultures made: (all 3 mL with 3uL amp): 2x RFP XL10 w/IPTG, 2x KR XL10 w/IPTG, RFP lysogen, KR lysogen, 1088, 1089. Lysogens in 30deg shaker, all others in 37deg shaker.

Plates streaked: 2 each of RFP XL10, KR XL10, RFP lysogen, KR lysogen.... half in 30deg incubator, half in 37deg incubator.

Tuesday August 6

At 8:50-- none of the 30deg plates are red... cultures look happy, RFP XL10 cultures are very red At 10:30--37deg RFP XL10 plate is pink (others are not)

To test effect of pH on maturation, 1.5mL of XL10 cultures (RFP/KR) were resuspended in SM (pH 7.4) buffer. For comparison, 1.5mL of the same cultures were left in LB. These were kept in microcentrifuge tubes and put in a 37ºC incubator starting at 11:20am. The initial resuspension of the RFP in SM seem more vibrant than its partners in LB. Note: the LB is from the overnight culture, which is likely to be acidic. Since These chromophores are fluorescent when they are deprotonated, higher pH ranges are ideal for these proteins. 37ºC is optimal for av-derived chromophores (like KillerRed) and mRFP1 (even though it is derived from DsRed). At 2pm, the cells were moved to 4ºC, spun down, 750µL of respective media removed and mixed to allow oxygen in. Let sit overnight at 4ºC to check for pH effect on fluorescence intensity

Resuspend cells in 10mM MgSO4 (Keep the volume the same to maintain OD600). The MgSO4 is currently 1M, so prepare 10mM dilution with ddH2O.
Resuspended RFP and KR XL10, RFP and KR lysogens, 1088 and 1089

To remaining 2 mL of the four KR and RFP cultures: added 4uL IPTG and placed in 37deg shaker.

Wednesday, August 7

Transformation of the backbone wasn’t successful. We’re going to try with more DNA.
We TECANed some cultures... our lysogens are producing protein (maybe)!!!

Some of us aren’t convinced since the peaks are very wide so we are running controls on the Tecan (without the FP’s). Excitation AND emissions scans are going to be performed to confirm that the emission is from the KillerRed/RFP. Also excitation should be at 560nm so see the entire emission peak without feedback from the laser.

Promising results from this morning seem to be background :..(

Made 3mL cultures with 3uL ampicillin, induced with 6uL IPTG: 2xKR-IX, KR, RFP-IX, RFP, 1089-X, 1089. Placed in 37deg shaker. Will do photobleaching tomorrow...

Thursday, August 8

Digestion of PCR product and Backbones started at 12:07pm
Photobleaching started at 12pm:
Lysed KillerRed Induced/Exposed by resuspsending 1.5mL of cells in 3mL 1M MgSO4, pipetting up and down vigorously and adding 200µL of chloroform to the solution, after shaking to solubilize the membranes without denaturing the proteins. 1µL of 25mM HCy5 was added for detection of superoxide. However, the dye is green, which is different than the original color of the reduced dye, which had a blue tint. If the Cy5 dye is present, proper detection might be the destruction of the chromophore, instead of its production due to the presence of the superoxide radicals. Time points for the two KillerRed IX and 1088 X are to be 0, 90 minutes and 180 minutes. The rest of them only need the 0 and 180 minute time points (to save LB/amp plates)

Made 10^-6 dilutions of all samples; plated 100uL for 10^-7 overall. Placed 15 plates in 37deg incubator at 7:15 pm:
RFP-IX at t=0, 3 hrs
RFP at t=0, 3 hrs
KR-IX at t=0, 1.5, 3 hrs
KR at t=0 1.5, 3 hrs
1089-X at t=0, 1.5, 3hrs
1089 at t=0, 3 hrs

Friday, August 9

Plates from photobleaching showed a significant number of colonies on all of the plates. Individual counts will determine the significance of the photobleaching.
...Plates are all TMTC with no apparent patterns in colony morphology.

MachT1 cells were transformed with the ligated KillerRed/Backbone. 4 colonies were chosen to be sequenced. Colonies are grown for 6 hours (from 10:15am), then miniprepped and shipped for sequencing.