Team:Penn/Notebook/DrugDelivery

From 2012.igem.org

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'''6/07/2012'''  
'''6/07/2012'''  
-
'''Wet Lab'''
+
'''''Wet Lab'''
-
 
+
''
Today we transformed both versions of cph8 from 2012 Distribution.
Today we transformed both versions of cph8 from 2012 Distribution.
-
'''Dry Lab'''
+
'''''Dry Lab'''
-
 
+
''
We also placed order for the following new BioBricks:
We also placed order for the following new BioBricks:
* BBa_K207001 - PhyB-DBD Fusion
* BBa_K207001 - PhyB-DBD Fusion
Line 22: Line 22:
'''6/08/2012'''  
'''6/08/2012'''  
-
'''Wet Lab'''
+
'''''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.
No colonies appeared in the transformation of both cph8 a/b. We suspect that the failure of the transformatiosns were due to bad sequences.
Line 29: Line 29:
'''6/11/2012'''  
'''6/11/2012'''  
-
'''Wet Lab'''
 
 +
'''''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.
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''' ==
+
'''6/12/2012'''
-
'''Wet Lab'''
+
 +
'''''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.
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''' ==
+
'''6/13/2012'''
-
'''Wet Lab'''
+
 
-
Unknown. Must consult lab notebook.
+
'''''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

Latest revision as of 22:08, 10 July 2012

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

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