IGEM:Harvard/2007/Laboratory Notebooks/Two Component System

6/27/07 - Ordered oligos to create a FecA promoter BioBrick that can be used to recombine with GFP to create a reporter system.

The oligos ordered were as follows:

Construct 1 5’- GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatttcaccactgtaaggaaaataattcttatttc – 3’

Construct 2 5’- GTTTCTTCCTGCAGCGGCCGCTACTAGTAAGGGTAAAAAGGACAATCGAAATAAGAATTATTT – 3’

6/27/07 - Grew bacteria (FecA, FecI, FecR) in liquid culture to prepare for sequencing tomorrow, inoculation in 2 ml LB and 2 ul amp 50mg/ml 1000x. [edit]

6/28/07 - Miniprepped to prepare for sequencing, nanodropped to confirm presence of DNA, sequencing rxns:

SV001 - FecA plus VF2

SV002 - FecA plus VR

SV003 - FecR plus VF2

SV004 - FecR plus VR

SV005 - FecI plus VF2

SV006 - FecI plus VR

PCR extension of constructs 1 and 2
-3 cycles of PCR
-diluted first to 500 uM, then to 20 uM
-15 uL of water, 10 uL of DNA, 25 uL of PCR master mix for each.
-two redundant reactions, R1 and R2
-R2 may be wrong becuase of volume errors

6/28/07 Miniprep done. Sent for sequencing.

Emails sent to:

Duke:
Dr. Homme W. Hellinga
Dr. Loren L. Looger

Penn State:
Dr. Hossein Fazelinia
Dr. Costas D. Maranas
Dr. Gregory L. Moore

PCR extension of constructs 1 and 2
-3 cycles of PCR
-diluted first to 500 uM, then to 20 uM
-15 uL of water, 10 uL of DNA, 25 uL of PCR master mix for each.
-two redundant reactions, R1 and R2
-R2 may be wrong becuase of volume errors

6/29/07 Reply from George Khoury of Penn State lab under Maranas. He has agreed to assist us with computational design.

Week of 7/1/07 Continued contact with George Khoury, including a cell phone conversation w/ Ellenor on 7/3/07 and an in-person talk w/ Shaunak to be scheduled for 7/7/07. We've decided on lead (Pb) a small molecule ligand and Muc1 as a macromolecule (glycoprotein) ligand for FecA. Muc1 is a cancer antigen, a glycoprotein overexpressed on tumor cells. They are underglycolysated on cancerous cells.

7/5/07 Directed Mutagenesis of FecA and FecR to remove Pst1 restriction site.

7/6/07 Transformation of FecA' and FecR' (pending recovery of previous day's PCR product)
Also, Shaunak and George's meeting went well. Notes are under "Brainstorming"

7/9/07 -miniprep of site directed mutagenesis using QiaGen Miniprep kit
-purification of construct C1+C2 extension rxn R1 using QiaGen PCR purification protocol--labeled product 'FecA promoter biobrick'
-received E Coli AA93, plasmid pLCIRA, pGFPA' from Germany, waiting on instructions from Dr. Braun on how to plate them.
-grow E0240 in liquid culture

Weekend of 7/7/07
7/7/07: Transformation of FecA' and FecR' into cells, completing the Quickchange Kit protocol.
7/8/07: Growth of liquid cultures from mutagenesis.
7/9/07 - miniprep of site directed mutagenesis using QiaGen Miniprep kit
- Plates streaked with cells containing FecA' and FecR' from liquid cultures.
- Digestion with Pst1 performed on a portion of miniprepped plasmids and on plasmids containing FecA and FecR.
- Egel was run to determine whether the mutagenesis was successful. FecA' (A1B) and FecR' (R5B) were placed into liquid culture from previously streaked plate. FecI was grown in liquid culture. The mutagenesis looks successful.
- purification of construct C1+C2 extension rxn R1 using QiaGen PCR purification protocol--labeled product 'FecA promoter biobrick'
- received E Coli AA93, plasmid pLCIRA, pGFPA' from Germany, waiting on instructions from Dr. Braun on how to plate them.
- grow E0240 in liquid culture

7/13/07 -Have grown up German constructs in LB, will be doing plasmid minipreps on pGFPA', pLCIRA in order to electroporate into AA93.
-plasmid minipreps done, pGFPA' is 205.2 ng/ul, pLCIRA is 53.2 ng/ul
-electroporation (see general protocol)--used 1 mL of AA93 cells, 25 ng of each plasmid (pGFPA' and pLCIRA): 2 samples, T1 (time constant 5.2) and T2 (time constant 5.0)

7/17/07 - 7/18/07 Fec signal induction

Day 1:

-Prepare WL nutrient broth for use in liquid culture. Grow overnight; should be a color change from deep blue toward green or yellow.
-Make 1mM and 4mM solutions of sodium citrate and ferric citrate.
-Make a 50mM solution for dipyridyl.
-In 96-well plate, use relevant combinations of 200 uL of cells, 50 uL of sodium- or ferric-citrate, and 50 uL of dipyridyl.

Results: NO FLUORESCENCE

Day 2:
-Take 200uL samples of T1 in WL (8 used).
-Spin down half of these samples and resuspend in 200uL of LB.
-Add Carb and Chloro... to challenge. Add relevant combinations of 50uL of 4mM sodium- or ferric- citrate.
-Grow all in small liquid culture tubes for an hour.
-Add 100uL of 50mM dipyridyl to appropriate liquid cultures. Let continue growing for 40 minutes. -Transfer to 1.5 mL centerfuge tubes. Spin for 1 min at 13000rpm. Resuspend in 300uL PBS. Repeat until WL color is not visible in resuspension.
-Add 200uL of resuspensions to 96-well plate. Check for fluorescence using plate reader, fluorometric microscope etc.

Results: NO FLUORESCENCE