Team:Carnegie Mellon/Week7

From 2013.igem.org

Monday, July 15
10:30
Overnights do not have any color in the media. RFP might not have matured just yet.
11:30

Things to do:
Make top agar (with LB)
Make LB/amp plates
Figure out dilutions for CFU experiments
2x 10µL->1000µL (1:100)
2x 1µL-> 1000µL (1:1000)
100 10-2 10-4 10-7 10-10
plate 100µL on LB/Amp. Need 5 plates
Streak out new RFP/KR plates
Check RFP/ KR lysogens
KR:
grow culture with lysogenic cells until mid-log. incubate at 40ºC. Should clear
Streak out for storage
RFP:
Do cross test
grow culture with lysogenic cells until mid-log, incubate at 40ºC. Media should clear
@5:37 PM, OD600 =.238
@6:36PM, OD600= 0.615
6:36: put in 42degC incubator. check tomorrow morning
Prepped RFP and KR lysogen overnights for tomorrow.
6 ml LB, 6ul amp, induced have 15 ul IPTG

Tuesday, July 16

RFP cross-streak test seems to have worked:
After moving RFP lysogen culture (OD600 0.615) into 42degC water bath for the night, there didn’t appear to be any clearing
is there an optimal time for when OD should be checked?
inoculated new RFP and KR cultures. Wait until they’re mid-log, then stick them in 40degC and take OD intermittently. 6 ml culture so.... time points: 0 min, 30 min, 60 min, 90 min, 120 min, 150 min
KR
@1:28PM: OD600 is 1.001 (would be nicer if it was below 1, but oh well)
@1:58PM: OD600 is 1.288 (:( )
@2:28PM: OD600 is 1.375
@3:08PM: OD600 is 1.45
@3:28PM: OD600 is 1. 415
@3:58PM: OD600 is 1.410
Streaked out potential lysogens on LB-amp plate. in incubator in 249 (across from Jarvik lab)
induced RFP and KR lysogen cultures that were prepared for photobleaching aren’t producing visible amounts of protein.
check later with TECAN to make sure there is fluorescence
probably because they’re only single copy in infected lysogens
Dilutions from yesterday Sunday’s overnights:
100: Lawn
10-2: TMTC
10-4: ~1000
10-7: 1
10-10: 3
Aim for 106 cells for overnights for dilutions.

Streaked XL10 plates are not expressing protein (grew overnight at 30ºC)
Many single colonies to choose from.

Wednesday, July 17
Check plate in incubator across from Jarvik lab
Streak plates at 42ºC grew normally (not indicative) of a lysogen
Cross-streaked plate:
Phage are present (are infecting non-lysogen strains)
Lysogen streaks are inconclusive?
Cloning Bba_KillerRed
Find a backbone to use for cloning. Possibly another BioBrick from the distribution kit
Would have to transform, culture and miniprep to get feasible concentrations
Potential lysogenic RFP and KR carrying E. Coli. Incubated at 42degC overnight to determine whether they were actually lysogenic. Obviously they were not induced.

KR lysogen streak test doesn’t show any clearing when it crosses high titer KR phage line, indicating that it is a lysogen.
No clearing when potential RFP lysogen crosses line of high-titer RFP phage. Indicates that this phage is a lysogen as well.
1089: high frequency lysogeny strain. Small amount of clearing around KR phage line, but mostly unaffected by phage.
1088: lytic strain. large clear spots surrounding both KR and RFP phage lines
potential KR lysogen: No apparent clearing.
potential RFP lysogen: small amount of clearing surrounding KR phage streak.
Typhoon Cy3 image
left: KR/ RFP potential lysogens incubated at 42 degC overnight.
right: lysogen cross streak plate. Horizontal streaks are paired, each of the two starting in the opposite direction.
First two, from top: 1089 streaked left to right, 1089 streaked right to left. Next two, 1088, streaked in same direction. Evident that cells lysed by RFP phage have produced KR. Next two, KR lysogen, streaked in same direction. Last two, RFP lysogen, streaked in same direction. Two vertical streaks: leftmost is KR high titer phage stock. rightmost is RFP high titer phage stock.

On the whole, potential lysogens are displaying lysogenic characteristics, but so far haven’t been able to induce them by heat.

Thursday, July 18
Photobleaching started at 10:53am
20 minute time points for fluorescence/OD, only doing initial and 3 hour time points for CFU (lack of plates)
3 conditions- KR induced/exposed, KR uniduced/unexposed, no plasmid exposed

10-7 dilutions for CFU’s (dilute to 10-6 and plate 100µL)

Do TECAN 10am tomorrow

Friday, July 19
y=42636e-.006x

RFP Photobleaching t0.5=99 minutes (1.65 hr) (R2=.99)
KR Photobleaching t0.5=115.5 minutes (1.925 hr) (R2=.99785)

What a beautiful graph

KR-IX shows significantly fewer colonies after 3hrs photobleaching, and the colonies are very small... Need to replate control on non-ampicillin plates--they don’t have resistance. �