IGEM:Harvard/2007/Laboratory Notebooks/Two Component System: Difference between revisions
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'''6/28/07''' | |||
Miniprep done. Sent for sequencing. | |||
Emails sent to: | |||
Duke:<br> | |||
Dr. Homme W. Hellinga<br> | |||
Dr. Loren L. Looger<br> | |||
Penn State:<br> | |||
Dr. Hossein Fazelinia<br> | |||
Dr. Costas D. Maranas<br> | |||
Dr. Gregory L. Moore<br> | |||
PCR extension of constructs 1 and 2<br> | |||
-3 cycles of PCR<br> | |||
-diluted first to 500 uM, then to 20 uM<br> | |||
-15 uL of water, 10 uL of DNA, 25 uL of PCR master mix for each.<br> | |||
-two redundant reactions, R1 and R2<br> | |||
-R2 may be wrong becuase of volume errors<br> | |||
'''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'''<br> | |||
-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.<br> | |||
- 7/5/07: Directed Mutagenesis of FecA and FecR to remove Pst1 restriction site.<br> | |||
- 7/6/07: Transformation of FecA' and FecR' (pending recovery of previous day's PCR product)<br> | |||
Also, Shaunak and George's meeting went well. Notes are under "Brainstorming" | |||
Revision as of 14:22, 9 July 2007
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