Wisconsin-Madison/26 June 2009

June 26, 2009
'''Ex 17: GFP Regulation by ProU Promoter

''Test 2: Results:

Salt testing of GFP, ProU had very indicative results that the promoter is working





Problems: no GFP only, no ProU+GFP Control 0M, no DH10B: controls put in after test NEED BETTER Experimental Design 

Experimental Design: (next test - Tomorrow)

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

Inoculate: ProU+GFP, GFP only, DH10B

Ex 16: Choline, ProU, NudF, YhfR, ProU + GFP

Choline BB Plating is Decent: 3 white colonies in the lower left-hand corner (will redo along with other samples just to be safe since the pink quality is expressed with time)

Streak: Independent lanes of three white colonies

Redo Cloning: YhfR, NudF, ProU, Choline

Important: Take concentration before and after digestion.

A. PCR clean up: Taq PCR Product - Promega Wizard Genomic DNA Purification Kit

'''B. Spec 1:

NudF (560)	680 ug/mL

YhfR (560)	665 ug/mL

Choline (2k)	716 ug/mL

ProU (300)	542 ug/mL

BB (2600)	98 ug/mL

C. Digestion: DNA should be at 1ug/50uL concentration

ProU

YhfR

NudF

Choline

BB P1

BB P2

*EcoR1 Buffer and Protein

D. Purification: according to Promega Wizard Genomic DNA Purification Kit

E. Spec 2: original - speedvac

NudF (560)	2 ug/mL	-	30 ug/mL

YhfR (560)	4 ug/mL	-	27 ug/mL

Choline (2k)	1 ug/mL	-	63 ug/mL

ProU (300)	1 ug/mL	-	104 ug/mL

BB P1(2600) 4 ug/mL	-	97 ug/mL

BB P2(2600)	2 ug/mL	-	100 ug/mL

'''F. Ligation:

BB P2 + ProU

BB P1 + YhfR

BB P1 + NudF

BB P1 + Choline

BB P2

BB P1

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

'''Ex 15: Inducing various Modified (Triple) E.Coli Strain Cultures 3

Transformed Confirmed Triple DNA into following strains and plated:

1.	BL21 (DE3) == Lawn

2.	K-12 MG1655 AaraBAD == 6 colonies

3.	K-12 MG1655 (wildtype) == 4 colonies

Transformed Confirmed Double (MevT, NudF) DNA into following strains and plated (incase triple was unsuccessful):

1.	BL21 (DE3) === Lawn

2.	K-12 MG1655 AaraBAD == ~300 colonies

3.	K-12 MG1655 (wildtype) == ~300 colonies



Important: BL21 are the most competent cells we have been able to obtain - should use for transformations in cloning instead of DH10B

Inoculate: 250ml culture – 250 uL Amp, 250 uL Tet, 183.75 uL Cm – with triple transformed plasmid

1.	BL21 (DE3)

2.	MG1655 Wildtype

3.	MG1655 Delta Arabad

Ex 10: Made medium and grew cyanos 7002

Bubbling of C02 has substantially increased the growth rate of cyanobacteria

'''Ex 18:



WHAT DOES THIS MEAN???

'''EX 10: Cyanobacteria

cyano freezer stock test and attempt

- 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