Team:Carnegie Mellon/Week8

From 2013.igem.org

Monday, July 22
Plated Negative control onto LB plates (10:45am). Since growing at 30ºC, extra time is needed for overnights.

CFUs

Condition
CFU at T0
CFU at T3
Killer Red Induced Exposed
78x107
16x107 *
Killer Red
76x107
58x107
XL10 Exposed
120x107
0x107

• Colony morphology is notably different between the two time points. The colonies take much longer to grow, indicating some sort of damage has taken place.
  

Once the XL10 plates grow, we can determine if the damage is from the light or from the KillerRed

Plated 15 potential lysogens each of KR and RFP phage. One set at 30degC, other set at 42degC.

Tuesday, July 23 Plates from last Thursday’s experiment:
XL10 T0 has several colonies while the T3 plate has no visible colonies. This might indicate excessive UV radiation from the lamp is causing damage. � To test whether KR produces radicals:
Grow 7 mL overnights (KR IX and XL10)
Take A260 measurement of 1mL of culture
Lyse cells
Spin down 2x3 mL of culture and resuspend in 500µL Buffer P1
Pipet up and down vigorously until viscous (vortex if needed)
take 1mL and test A260 (absorbance should have increased greatly)
[Spin down and remove supernatant]??
Combine in a glass culture tube
Add 4mL (slowly) SM buffer (RNase A activity might be hindered by Mg2+?)
Take out 200µL and test for presence of KillerRed by analyzing for Ex/Em spectra in a Tecan.
Add (10µM?) of HCy5 dye (MW: 793 g/mol) to the samples
Positive control: Add 5 mL SM and bring to 10µM Fluorescein and 1µM HCy5 (Fluorescein should be in excess). 10:1 ratio will virtually guarantee 100% conversion if every fluorescein produces 1 superoxide molecule.
Photobleach samples for 3 hours (400 nm longpass filter) taking 20 minute time points (200µL apiece)
Analyze on Tecan.

Buffers:
P1:
50 mM Tris-HCl (pH 8.0)
10 mM EDTA
SM:
5.8 g of NaCl → .1 M
2.0 g of MgSO4·7H2O (1mM)
50.0 ml of 1 M Tris-HCl (pH 7.5) (50 mM)
5.0 ml of 2% (w/v) gelatin
Add deionized H2O to final volume of 1L
When combining these buffers, salts might precipitate but proteins should stay intact (Add slowly anyway)
approximate pH of solution: 7.5<pH<8.0

Two possible KR lysogens were identified: KR8 and KR11 both grew at 30C but not 42C.
restreaked both out on fresh LB amp plates
double check that they were induced:
try to get them to grow in 42C again.
cross-streak test:
1089, 1088, KR11, KR8 across KR hi-titer stock
Still no RFP lysogens that have not grown at 42.
Picked 14 more colonies of RFP lysogen. One plate in 42C, other in 30C.
Prof. Jarvik says we might not be getting any induction because 1) the cells are for some reason resistant to the phage or 2) S100 mutation affects function of phage protein that degrades cell walls. Phage and KR might be accumulating, but the cell doesn’t burst.

Wednesday, July 24
Can’t induce lysogens with heat- phage has S100 mutation and host doesn’t have supE or supF.
Started RFP10, KR11 and 1089 cultures (not induced) to catch at mid-log
add chloroform
see if we get some phage that can infect.
Later: Start RFP10, KR11 and 1089 and induce
grow overnight
use TECAN to check for KR
(if KR/RFP not obvious, lyse cells, try again)
Dyes should be available tomorrow (Thursday July 25th) morning

Thursday, July 25

HCy5 solution is 25mM in 200µL
TECAN reserved for 11am on Friday to test for the presence of KR and RFP in the lysogens
Lysogens have phage in them- confirmed by infecting 1088 with chloroformed lysogens and inducing w/ IPTG. Resulted in lots of plaques.
Used Typhoon [Cy3] to image- no fluorescence detectable for either KR or RFP

Overnight cultures from yesterday are in fridge until TECAN time tomorrow. Neither RFP or KR are making cultures visibly red.
Do overnights of same lysogens uninduced for tomorrow
Prep more 1088 and 1089 for infections in case

Model suggests nanomolar concentrations of Cy5 will be attained on the order of minutes.

Picked up fluoresceinamine:
347g/mol
--- 16 mg = 4.61e-5 mol in 1.5 mL = .0307 mol/L = 30.7 mM
2mg in 1.5mL=.0038M= 3.8 mM*

Friday, July 25

diluted 2 mg fluoresceinamine in 3 mL → 1.9 mM

TECAN results- no fluorescence detected in either RFP or KR lysogens, even after lysing cells open with chloroform

Filter for lamp: 400LP is probably the best option. There’s a peak from the mercury arc at 405 nm.