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Team:Penn/Notebook/DrugDelivery
From 2012.igem.org
Contents |
Week 1
6/07/2012
Wet Lab Today we transformed both versions of cph8 from 2012 Distribution.
Dry Lab We also placed order for the following new BioBricks:
- BBa_K207001 - PhyB-DBD Fusion
- BBa_K422012 - Pif3 (Aar1 C part)
- BBa_K422013 - PhyB (Aar1 C part)
- BBa_K592006 - pFixK2
- BBa_K592004 - YF1
- BBa_K592005 - FixJ
- BBa_K592000 - Cph8
- BBa_K365000 - Pif3
In addition, we brainstormed and developed a new idea to use the PYP/GCN system to activate a gene.
6/08/2012
Wet Lab No colonies appeared in the transformation of both cph8 a/b. We suspect that the failure of the transformatiosns were due to bad sequences.
Week 2
6/11/2012
Wet Lab We performed PCR to obtain mCherry from the NAS 151 plasmid. Following this we ran a 1% gel and purified the product which resulted in a 30ns/λ yield.
6/12/2012
Wet Lab We digested mCherry PCR product with restriction enzymes BamHI and NotI. Then we column purified mCherry and ligated into NAS152 backbone. Afterwards we transformed NAS152-mCherry into DH5alpha cell lines. This was then poured 25 LB-Kan plates.
6/13/2012
Dry Lab Read papers to research the project.
6/14/2012
Dry Lab Continued to read papers.
6/15/2012
Dry Lab Met with Mark Goulian to develop the project. Also obtained pDawn and pDusk plasmids.
Week 3
6/18/2012
Wet Lab Did a test cut of pDawn and pDusk plasmids using Xma1. Ran an analytical gel. Then cut the two plasmids for ligation using BamHI and NotI . Finally ran and purified the gel.
Dry Lab We ordered and picked up PCR purification kit. Additional components were also ordered.
6/19/2012
Wet Lab Completed the ligation protocol for pDawn and pDusk plasmids with mChery. Then transformed the pDawn-mCherry and pDusk-mCherry constructs.
Dry Lab Emailed out corporations for sponsorships.
6/20/2012
Wet Lab Today we picked 2 colonies of pDawn-mCherry and then innoculated the colonies in 5 mL of LB and 50 ug/mL of Kan.
Dry Lab Started by researching DARPin binding domains and linkers. Then finalized biobrick and synthesis orders.
6/21/2012
Nothing known.
6/22/2012
Wet Lab Repeated a miniprep on pDawn and then did a test cut. Also did a miniprepped pET26b and used it to make a glycerol stock. Lastly, digested pET-26b with BamH1 and Not1
Dry Lab Sent in synthesis orders!
Week 4
6/25/2012
Wet Lab Did a column purification, ligation, and transformation of pET26b-mCherry
Dry Lab Actually sent out final gene synthesis order. Also reviewed pDawn protocol and TetR sequences
6/26/2012
No idea. There is a to-do list but nothing noting what was actually accomplished.
6/27/2012
Wet Lab Did a test-cut of pET-26b-mCherry using ClaI and HindIII with colonies C2,C3,C4, and C5. Took C2 and transformed them in to B221. Then we miniprepped 4x TB culture of pDawn-mCherry. After that we transformed the product in to BL-21/
6/28/2012
Wet Lab Today we picked colonies at 11:30, then inociated in a 5 mL LB until 7:40 pm. The OD of pDawn was 1.0 and the OD of pET is 1.4. Then we diluted it down to 0.004 as well as 5x and x/5 dilutions (0.020,0.0008). At 7:40pm cultures were 3x pDawn-mCherry, 3x pET-mCherry(+IPTG), 3x pET-mCherry(-IPTG) in both dark and light.
6/29/2012
Wet Lab Wet Lab: We tested pDawn-mCherry and pET-mCherry under light and dark conditions and determined that pDawn-mCherry expressed mCherry only after light induction. We were also able to nanodrop pJT122, pJT106b, PhyB, PIF3, Cph8.
Dry Lab Opened Bio-Rad shipments.
Week 5
7/2/2012
Wet Lab Today we were able to transform the ho1 and pcyA BioBricks.
Dry Lab Worked on human practices.
7/3/2012
'Dry Lab Today we created the digital schematic. We also designed primers and developed a cloning strategy for cloning cph8 into the pDawn backbone. In addition, we worked on researching bacterial therapeutics.
7/4/2012
Happy 4th of July!
7/5/2012
Unknown
7/6/2012 - 7/7/2012
Wet Lab We took pDawn-mCherry time course. Also spent time sterilizing the incubator and TC hood.
Dry Lab Contacted FDA contacts for human practices
