Wisconsin-Madison/27 June 2009

June 27, 2009
'''Ex 17: GFP Regulation by ProU Promoter '''Experimental Design: 0.0-1.0M in increments of 0.2 concentrations of (4 wells of each concentration) 1. Wild Type E.Coli 2. GFP only 3. ProU + GFP

''(Plan to include the SAM synthase (METK) to boost gsSDMT function, and eventually getting rid of the GFP and evaluating solely on growth) ''4. ProU + GFP + gsSDMT ''5. ProU + GFP + gsSDMT + MetK 6. Salt Free LB – start (2 wells of each concentration) 7. Water (2 wells of each concentration)

1. Grow four different cultures till OD600=0.4 to 0.6 2. Induce cultures with various concentrations of salt in the plate reader 3. Let the plate reader shake and incubate overnight 4. Take OD and excitation/emission value at 501/511nm during the entire time course

''Test 3:

'''Conc of salt (M): 0.0 / 0.2 / 0.4 / 0.6 / 0.8 / 1.0

DH10B

GFP only ProU+GFP

Salt Free LB

Water

C1 has extra 8uL water

D8 appears twice

H2 appears twice

H3, G8, A4 have too little water

A4, F6, H2, C4, B2 - GFP

D12, F4 – GFP+ProU

'''Ex 15: Inducing Various Modified (triple) E.Coli Strains - Forgot to use terrific Broth in seed cultures - Tet from Rebecca was actually tet 10 not Tet 100, added 10x too much/little in seed cultures Making new seed colonies: 5ml culture – 5 uL Amp, 5 uL Tet (tet100), 3.68 uL Cm – with triple transformed plasmid

1.	BL21 (DE3)

2.	MG1655 Wildtype

3.	MG1655 Delta Arabad

4.	DH10B

5.	Control: Empty Vectors pBad33 and pTrc99 (100uL, amp 1uL Cm)

'''Ex 16:

F. Transformation: of YhfR, NudF, ProU, Choline, P1, P2 into BL21 (DE3) (extremely high competency)

MiniPrep of Choline Colonies A-C

Ran on 1% Agarose Gel

Undigested (A, B, C), 1kb Ladder, Digested (A, B, C)

- Results

- Digested Choline should be 2000bp

'''Ex 10: Cyanobacteria

Tested natural transformation on PCC 7002 and PCC 7942 (1mL of culture and DNA from one mini prep each, plated .5mL of 7942 and made 1mL liquid culture, made 2mL liquid culture of 7002) - NP - inoculated stock cultures of Synechococcus sp PCC 7002 (10:1 in media A) with aeration - NP - made two types of freezer stocks of PCC 7942, both 25% glycerol, the first 600uL culture to 400uL of 60% glycerol, the second 50mL culture centrifuged at 4000rpm for 10 mins and resuspended in 1mL then 666uL glycerol - NP

- tested freezer stock made on June 22 - NP

- digested ProU, nudF, yhfR, and peamt and ligated overnight - NC - inoculated DH10B, GFP, PFP and ProU, Bl21(3x), MG1655(3x), and MG1655 delta arabad(3x) - NC - developed next assay for salt tolerance - NC - triple transformation of Bl21, MG1655, and MG1655 delta arabad all worked! - NC - choline transformation from ligation worked - NC

- added 8mL to 2mL 6803 cultures to achieve 10mL cultures - NP