IGEM:MIT/2006/Notebook/2006-6-12: Difference between revisions
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===T-Shirt=== | ===T-Shirt=== | ||
'''<font color="red">WE NEED A T-SHIRT DESIGN!</font>''' | '''<font color="red">WE NEED A T-SHIRT DESIGN!</font>'''<br> | ||
'''[[User:Jennyn|Jennyn]] 13:46, 12 June 2006 (EDT)''': I'd love to help with this if the team needs. | |||
==06/12/06 Repeat (from 6/09/06) Transformation Lab== | ==06/12/06 Repeat (from 6/09/06) Transformation Lab== | ||
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#**DH5&alpha (big clear tubes with green tape);, Top 10 (Invitrogen) (purple caps), Top 10 (Tom) (big clear tubes), BL21 (Invitrogen) (greenish brown caps) | #**DH5&alpha (big clear tubes with green tape);, Top 10 (Invitrogen) (purple caps), Top 10 (Tom) (big clear tubes), BL21 (Invitrogen) (greenish brown caps) | ||
#*Used 2μL of plasmid/DNA. | #*Used 2μL of plasmid/DNA. | ||
#*Heat | #*Heat shocked the BL21(DL3) cells | ||
#**42 c for 50 seconds | #**42 c for 50 seconds | ||
#*Used 2xYT | #*Used 500μL 2xYT media for DH5&alpha cells | ||
#*Used 500μL S.O.C. media for other 3 cells | |||
#*horizontal shaking for 60 minutes | |||
#*Plated 200μL cells | #*Plated 200μL cells | ||
| Line 33: | Line 36: | ||
#Invitrogen Top 10 BSMT tube fell. It was put on the microfuge for a second to get the cells off the side of the tube. | #Invitrogen Top 10 BSMT tube fell. It was put on the microfuge for a second to get the cells off the side of the tube. | ||
#For TOP10 cells use this [https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&objectId=E62FEAF0F3C8B0E215833D3E8A49BD5D&treeNodeId=289731BD016F54BEBFEAA20F29AA1796 transformation protocol], which is similar but slightly different from the what we did [[Transforming chemically competent cells | previously]] | #For TOP10 cells use this [https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&objectId=E62FEAF0F3C8B0E215833D3E8A49BD5D&treeNodeId=289731BD016F54BEBFEAA20F29AA1796 transformation protocol], which is similar but slightly different from the what we did [[Transforming chemically competent cells | previously]] | ||
==Updates on Research in Biosynthetic Pathways for Acid Production== | |||
#I emailed out the two referenced papers on this pathway: CA-->BA-->SA | |||
#Unfortunately, it seems that the gene's sequence encoding the enzyme responsible (benzoic acid 2-hydroxylase) for catalyzing the BA-->SA reaction is not in Pubmed. It seems that the last paper written on the enzyme was in 2000 and did not seem to have any information on sequence information. | |||
#Here is a piece from a paper that seems to explan the CA-->BA reaction. "Our results revealed the metabolic pathway that leads from l-phenylalanine to the major aryl metabolites produced by B. adusta. The presence of 13C-labelled trans-cinnamic acid together with PAL activity shows that trans-cinnamic acid is a key pathway intermediate. In addition, trans-cinnamic acid is an efficient precursor of benzoic acid, benzyl alcohol, and benzaldehyde. trans-Cinnamic acid can be subsequently hydroxylated to β-hydroxyphenylpropionic acid (Fig. 2, pathway 2), which in turn can be converted via a β-oxidation step to benzoic acid. This was confirmed by the presence of acetophenone as a degradation product of β-hydroxyphenylpropionic acid. Furthermore, trans-cinnamic acid is the precursor that is most efficiently converted to benzoic acid among the putative precursors which we tested. This confirms that there is a β-oxidation process in B. adusta and that benzoic acid is the major product of this process. β-Oxidation has been found previously in several other fungi (13), although apparently not in B. adusta. To our knowledge, this is the first time that β-oxidation of this nature has been found in a white rot fungus." http://www.pubmedcentral.gov/articlerender.fcgi?artid=92016 | |||
#http://www.biology.lsa.umich.edu/research/labs/pichersky/references/pub16.pdf | |||
Latest revision as of 12:56, 12 June 2006
T-Shirt
WE NEED A T-SHIRT DESIGN!
Jennyn 13:46, 12 June 2006 (EDT): I'd love to help with this if the team needs.
06/12/06 Repeat (from 6/09/06) Transformation Lab
Sources
- Note, used 1/2 of designated source
- SAMT in PET28 1.6 μg
- BAMT in PET28 2.0 μg
- BSMT in PET28 2.0 μg
Dehydrated DNA Recovery (from paper)
- Final DNA Concentration: 1ng/μL (room for better quantification)
- Add appropriate amount of TE buffer to 1.7mL Eppendorf tubes (500μL for BAMT and BSMT; 400μL for SAMT).
- Carefully cut out paper that contains DNA and place into Eppen.
- Ensure submergence with toothpick
- Wait for 10 minutes
Transformation
- Followed chemical transformation protocol listed here.
- Cell Types Transformed:
- DH5&alpha (big clear tubes with green tape);, Top 10 (Invitrogen) (purple caps), Top 10 (Tom) (big clear tubes), BL21 (Invitrogen) (greenish brown caps)
- Used 2μL of plasmid/DNA.
- Heat shocked the BL21(DL3) cells
- 42 c for 50 seconds
- Used 500μL 2xYT media for DH5&alpha cells
- Used 500μL S.O.C. media for other 3 cells
- horizontal shaking for 60 minutes
- Plated 200μL cells
- Cell Types Transformed:
Notes
- Invitrogen Top 10 BSMT tube fell. It was put on the microfuge for a second to get the cells off the side of the tube.
- For TOP10 cells use this transformation protocol, which is similar but slightly different from the what we did previously
Updates on Research in Biosynthetic Pathways for Acid Production
- I emailed out the two referenced papers on this pathway: CA-->BA-->SA
- Unfortunately, it seems that the gene's sequence encoding the enzyme responsible (benzoic acid 2-hydroxylase) for catalyzing the BA-->SA reaction is not in Pubmed. It seems that the last paper written on the enzyme was in 2000 and did not seem to have any information on sequence information.
- Here is a piece from a paper that seems to explan the CA-->BA reaction. "Our results revealed the metabolic pathway that leads from l-phenylalanine to the major aryl metabolites produced by B. adusta. The presence of 13C-labelled trans-cinnamic acid together with PAL activity shows that trans-cinnamic acid is a key pathway intermediate. In addition, trans-cinnamic acid is an efficient precursor of benzoic acid, benzyl alcohol, and benzaldehyde. trans-Cinnamic acid can be subsequently hydroxylated to β-hydroxyphenylpropionic acid (Fig. 2, pathway 2), which in turn can be converted via a β-oxidation step to benzoic acid. This was confirmed by the presence of acetophenone as a degradation product of β-hydroxyphenylpropionic acid. Furthermore, trans-cinnamic acid is the precursor that is most efficiently converted to benzoic acid among the putative precursors which we tested. This confirms that there is a β-oxidation process in B. adusta and that benzoic acid is the major product of this process. β-Oxidation has been found previously in several other fungi (13), although apparently not in B. adusta. To our knowledge, this is the first time that β-oxidation of this nature has been found in a white rot fungus." http://www.pubmedcentral.gov/articlerender.fcgi?artid=92016
- http://www.biology.lsa.umich.edu/research/labs/pichersky/references/pub16.pdf
