Wisconsin-Madison/26 June 2009

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Calendar

April
MTWTFSS
    [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/1_April_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/2_April_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/3_April_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/4_April_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/5_April_2009&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/6_April_2009&action=edit 6] [http://2009.igem.org/Wisconsin-Madison/7_April_2009 7] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/8_April_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/9_April_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/10_April_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/11_April_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/12_April_2009&action=edit 12]
[http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/13_April_2009&action=edit 13] [http://2009.igem.org/Wisconsin-Madison/14_April_2009 14] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/15_April_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/16_April_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/17_April_2009&action=edit 17] [http://2009.igem.org/Wisconsin-Madison/18_April_2009 18] [http://2009.igem.org/Wisconsin-Madison/19_April_2009 19]
[http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/20_April_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/21_April_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/22_April_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/23_April_2009&action=edit 23] [http://2009.igem.org/Wisconsin-Madison/24_April_2009 24] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/25_April_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/26_April_2009&action=edit 26]
[http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/27_April_2009&action=edit 27] [http://2009.igem.org/Wisconsin-Madison/28_April_2009 28] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/29_April_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/30_April_2009&action=edit 30]
May
MTWTFSS
        [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/1_May_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/2_May_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/3_May_2009&action=edit 3]
[http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/4_May_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/5_May_2009&action=edit 5] [http://2009.igem.org/Wisconsin-Madison/6_May_2009 6] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/7_May_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/8_May_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/9_May_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/10_May_2009&action=edit 10]
[http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/11_May_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/12_May_2009&action=edit 12] [http://2009.igem.org/Wisconsin-Madison/13_May_2009 13] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/14_May_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/15_May_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/16_May_2009&action=edit 16] [http://2009.igem.org/Wisconsin-Madison/17_May_2009 17]
[http://2009.igem.org/Wisconsin-Madison/18_May_2009 18] [http://2009.igem.org/Wisconsin-Madison/19_May_2009 19] [http://2009.igem.org/Wisconsin-Madison/20_May_2009 20] [http://2009.igem.org/Wisconsin-Madison/21_May_2009 21] [http://2009.igem.org/Wisconsin-Madison/22_May_2009 22] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/23_May_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/24_May_2009&action=edit 24]
[http://2009.igem.org/Wisconsin-Madison/25_May_2009 25] [http://2009.igem.org/Wisconsin-Madison/26_May_2009 26] [http://2009.igem.org/Wisconsin-Madison/27_May_2009 27] [http://2009.igem.org/Wisconsin-Madison/28_May_2009 28] [http://2009.igem.org/Wisconsin-Madison/29_May_2009 29] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/30_May_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/31_May_2009&action=edit 31]
June
MTWTFSS
[http://2009.igem.org/Wisconsin-Madison/1_June_2009 1] [http://2009.igem.org/Wisconsin-Madison/2_June_2009 2] [http://2009.igem.org/Wisconsin-Madison/3_June_2009 3] [http://2009.igem.org/Wisconsin-Madison/4_June_2009 4] [http://2009.igem.org/Wisconsin-Madison/5_June_2009 5] [http://2009.igem.org/Wisconsin-Madison/6_June_2009 6] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/7_June_2009&action=edit 7]
[http://2009.igem.org/Wisconsin-Madison/8_June_2009 8] [http://2009.igem.org/Wisconsin-Madison/9_June_2009 9] [http://2009.igem.org/Wisconsin-Madison/10_June_2009 10] [http://2009.igem.org/Wisconsin-Madison/11_June_2009 11] [http://2009.igem.org/Wisconsin-Madison/12_June_2009 12] [http://2009.igem.org/Wisconsin-Madison/13_June_2009 13] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/14_June_2009&action=edit 14]
[http://2009.igem.org/Wisconsin-Madison/15_June_2009 15] [http://2009.igem.org/Wisconsin-Madison/16_June_2009 16] [http://2009.igem.org/Wisconsin-Madison/17_June_2009 17] [http://2009.igem.org/Wisconsin-Madison/18_June_2009 18] [http://2009.igem.org/Wisconsin-Madison/19_June_2009 19] [http://2009.igem.org/Wisconsin-Madison/20_June_2009 20] [http://2009.igem.org/Wisconsin-Madison/21_June_2009 21]
[http://2009.igem.org/Wisconsin-Madison/22_June_2009 22] [http://2009.igem.org/Wisconsin-Madison/23_June_2009 23] [http://2009.igem.org/Wisconsin-Madison/24_June_2009 24] [http://2009.igem.org/Wisconsin-Madison/25_June_2009 25] [http://2009.igem.org/Wisconsin-Madison/26_June_2009 26] [http://2009.igem.org/Wisconsin-Madison/27_June_2009 27] [http://2009.igem.org/Wisconsin-Madison/28_June_2009 28]
[http://2009.igem.org/Wisconsin-Madison/29_June_2009 29] [http://2009.igem.org/Wisconsin-Madison/30_June_2009 30]
July
MTWTFSS
    [http://2009.igem.org/Wisconsin-Madison/1_July_2009 1] [http://2009.igem.org/Wisconsin-Madison/2_July_2009 2] [http://2009.igem.org/Wisconsin-Madison/3_July_2009 3] [http://2009.igem.org/Wisconsin-Madison/4_July_2009 4] [http://2009.igem.org/Wisconsin-Madison/5_July_2009 5]
[http://2009.igem.org/Wisconsin-Madison/6_July_2009 6] [http://2009.igem.org/Wisconsin-Madison/7_July_2009 7] [http://2009.igem.org/Wisconsin-Madison/8_July_2009 8] [http://2009.igem.org/Wisconsin-Madison/9_July_2009 9] [http://2009.igem.org/Wisconsin-Madison/10_July_2009 10] [http://2009.igem.org/Wisconsin-Madison/11_July_2009 11] [http://2009.igem.org/Wisconsin-Madison/12_July_2009 12]
[http://2009.igem.org/Wisconsin-Madison/13_July_2009 13] [http://2009.igem.org/Wisconsin-Madison/14_July_2009 14] [http://2009.igem.org/Wisconsin-Madison/15_July_2009 15] [http://2009.igem.org/Wisconsin-Madison/16_July_2009 16] [http://2009.igem.org/Wisconsin-Madison/17_July_2009 17] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/18_July_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/19_July_2009&action=edit 19]
[http://2009.igem.org/Wisconsin-Madison/20_July_2009 20] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/21_July_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/22_July_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/23_July_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/24_July_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/25_July_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/26_July_2009&action=edit 26]
[http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/27_July_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/28_July_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/29_July_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/30_July_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/31_July_2009&action=edit 31]
August
MTWTFSS
          [http://2009.igem.org/Wisconsin-Madison/1_August_2009 1] [http://2009.igem.org/Wisconsin-Madison/2_August_2009 2]
[http://2009.igem.org/Wisconsin-Madison/3_August_2009 3] [http://2009.igem.org/Wisconsin-Madison/4_August_2009 4] [http://2009.igem.org/Wisconsin-Madison/5_August_2009 5] [http://2009.igem.org/Wisconsin-Madison/6_August_2009 6] [http://2009.igem.org/Wisconsin-Madison/7_August_2009 7] [http://2009.igem.org/Wisconsin-Madison/8_August_2009 8] [http://2009.igem.org/Wisconsin-Madison/9_August_2009 9]
[http://2009.igem.org/Wisconsin-Madison/10_August_2009 10] [http://2009.igem.org/Wisconsin-Madison/11_August_2009 11] [http://2009.igem.org/Wisconsin-Madison/12_August_2009 12] [http://2009.igem.org/Wisconsin-Madison/13_August_2009 13] [http://2009.igem.org/Wisconsin-Madison/14_August_2009 14] [http://2009.igem.org/Wisconsin-Madison/15_August_2009 15] [http://2009.igem.org/Wisconsin-Madison/16_August_2009 16]
[http://2009.igem.org/Wisconsin-Madison/17_August_2009 17] [http://2009.igem.org/Wisconsin-Madison/18_August_2009 18] [http://2009.igem.org/Wisconsin-Madison/19_August_2009 19] [http://2009.igem.org/Wisconsin-Madison/20_August_2009 20] [http://2009.igem.org/Wisconsin-Madison/21_August_2009 21] [http://2009.igem.org/Wisconsin-Madison/22_August_2009 22] [http://2009.igem.org/Wisconsin-Madison/23_August_2009 23]
[http://2009.igem.org/Wisconsin-Madison/24_August_2009 24] [http://2009.igem.org/Wisconsin-Madison/25_August_2009 25] [http://2009.igem.org/Wisconsin-Madison/26_August_2009 26] [http://2009.igem.org/Wisconsin-Madison/27_August_2009 27] [http://2009.igem.org/Wisconsin-Madison/28_August_2009 28] [http://2009.igem.org/Wisconsin-Madison/29_August_2009 29] [http://2009.igem.org/Wisconsin-Madison/30_August_2009 30]
[http://2009.igem.org/Wisconsin-Madison/31_August_2009 31]
September
MTWTFSS
  [http://2009.igem.org/Wisconsin-Madison/1_September_2009 1] [http://2009.igem.org/Wisconsin-Madison/2_September_2009 2] [http://2009.igem.org/Wisconsin-Madison/3_September_2009 3] [http://2009.igem.org/Wisconsin-Madison/4_September_2009 4] [http://2009.igem.org/Wisconsin-Madison/5_September_2009 5] [http://2009.igem.org/Wisconsin-Madison/6_September_2009 6]
[http://2009.igem.org/Wisconsin-Madison/7_September_2009 7] [http://2009.igem.org/Wisconsin-Madison/8_September_2009 8] [http://2009.igem.org/Wisconsin-Madison/9_September_2009 9] [http://2009.igem.org/Wisconsin-Madison/10_September_2009 10] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/11_September_2009&action=edit 11] [http://2009.igem.org/Wisconsin-Madison/12_September_2009 12] [http://2009.igem.org/Wisconsin-Madison/13_September_2009 13]
[http://2009.igem.org/Wisconsin-Madison/14_September_2009 14] [http://2009.igem.org/Wisconsin-Madison/15_September_2009 15] [http://2009.igem.org/Wisconsin-Madison/16_September_2009 16] [http://2009.igem.org/Wisconsin-Madison/17_September_2009 17] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/18_September_2009&action=edit 18] [http://2009.igem.org/Wisconsin-Madison/19_September_2009 19] [http://2009.igem.org/Wisconsin-Madison/20_September_2009 20]
[http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/21_September_2009&action=edit 21] [http://2009.igem.org/Wisconsin-Madison/22_September_2009 22] [http://2009.igem.org/Wisconsin-Madison/23_September_2009 23] [http://2009.igem.org/Wisconsin-Madison/24_September_2009 24] [http://2009.igem.org/Wisconsin-Madison/25_September_2009 25] [http://2009.igem.org/Wisconsin-Madison/26_September_2009 26] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/27_September_2009&action=edit 27]
[http://2009.igem.org/Wisconsin-Madison/28_September_2009 28] [http://2009.igem.org/Wisconsin-Madison/29_September_2009 29] [http://2009.igem.org/Wisconsin-Madison/30_September_2009 30]
October
MTWTFSS
      [http://2009.igem.org/Wisconsin-Madison/1_October_2009 1] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/2_October_2009&action=edit 2] [http://2009.igem.org/Wisconsin-Madison/3_October_2009 3] [http://2009.igem.org/Wisconsin-Madison/4_October_2009 4]
[http://2009.igem.org/Wisconsin-Madison/5_October_2009 5] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/6_October_2009&action=edit 6] [http://2009.igem.org/Wisconsin-Madison/7_October_2009 7] [http://2009.igem.org/Wisconsin-Madison/8_October_2009 8] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/9_October_2009&action=edit 9] [http://2009.igem.org/Wisconsin-Madison/10_October_2009 10] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/11_October_2009&action=edit 11]
[http://2009.igem.org/Wisconsin-Madison/12_October_2009 12] [http://2009.igem.org/Wisconsin-Madison/13_October_2009 13] [http://2009.igem.org/Wisconsin-Madison/14_October_2009 14] [http://2009.igem.org/Wisconsin-Madison/15_October_2009 15] [http://2009.igem.org/Wisconsin-Madison/16_October_2009 16] [http://2009.igem.org/Wisconsin-Madison/17_October_2009 17] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/18_October_2009&action=edit 18]
[http://2009.igem.org/Wisconsin-Madison/19_October_2009 19] [http://2009.igem.org/Wisconsin-Madison/20_October_2009 20] [http://2009.igem.org/Wisconsin-Madison/21_October_2009 21] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/22_October_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/23_October_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/24_October_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/25_October_2009&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/26_October_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/27_October_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/28_October_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Wisconsin-Madison/29_October_2009&action=edit 29] [http://2009.igem.org/Wisconsin-Madison/30_October_2009 30] [http://2009.igem.org/Wisconsin-Madison/31_October_2009 31]

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


0626 graph.jpg


0626 flour.jpg


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)

0626 plat 1.jpg


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

0626 plat 2.jpg


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

0626 plat 3.jpg


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:

0626 ladder.jpg 0626 gel.jpg

WHAT DOES THIS MEAN???


EX 10: Cyanobacteria

cyano freezer stock test and attempt

Retrieved from "http://2009.igem.org/Wisconsin-Madison/26_June_2009"