From 2010.igem.org
- The notebook is organized into weeks. Click on the Saturday of each week to view that week's notebook entry
Weekly Lab Notebook
June 6 - June 12
Colin - This week, I realized that the website needed a lot of help, so i spent a lot of time brainstorming. However, it seemed that I couldn't figure out what exactly i should do to make it look nice. I'm looking at some other web sites, and I feel like I am close to a design.
Jeremy - Implementing mapping from reactions to enzymes to genes from Entrez
Beginning work on plasmid display via BioPython
Started hooking up Biobrick entries/database to plasmid design,
including plasmid backbone, promoter, rbs, and terminator elements
Creating new reaction network with RPAIR changes
Anna - We looked over Colin’s summary. Discussed the plasmid designer, particularly some more of our
concerns with it. The main problem we worry about now is the plasmid size limit. What we can do to
alleviate this issue would be to create cis and trans regulation, or fusion proteins. If we want the two
genes, have both driven by the same promoter, which will then construct an RNA sequence with
ribosome binding sites and stop codons, etc. The fusion will simply be the transcription and translation
sequence when designing the plasmid. This would require us to eliminate the ribosome binding sites
between genes and add more amino acids as a linker to create one single protein as opposed to two
separate proteins. Generally, we would probably want the two separate proteins, but sometimes it could
be ideal, such as in the cases for green fluorescent proteins. Also, a fusion protein would be a better
chance of increasing the reaction rate. Perhaps a better option would be to provide the user an option to
design a fusion protein as well. We learned a lot about plasmid design. Particularly, we discussed
restriction enzyme sites where we can insert a gene of interest. Another thing we have to remember to
include in the plasmid design would be a resistance marker. The resistance marker is important in
determining if the cell took up the plasmid of interest. The ORI is the segment that hooks into the
bacterial system or where the DNA polymer sits to replicate and keep it within the cell. IMPTools again
helps with plasmid design in giving us the actual genes we want to insert into the segment, it does not
tell use what promoters, stop codons, or plasmids are most ideal . Will need to determine the best
pathway from IMPTools and utilize that specific pathway in the plasmid design. When designing the
plasmid, we have to incorporate binding sites, promoters and restriction sites. It is important that there
are no restriction sites within the genes, especially not within the resistance markers. We also discussed
the option of designing two plasmids for pathways that are particularly large. We discussed how cloning
sites affect copy number. Multiple cloning sites will yield high copy number and vice versa. Now, to
figure out the best promoters, we must decide between inducible or constitutive promoters. Promoters
are not in the KEGG database, but are in the biobricks database! Finally, we focused on which organisms
specifically to use in the plasmid design and how different organisms will affect the plasmid design. We
again decided to focus on E. coli.
Bobak - I spent this week upgrading the Imptools website so that it couldn't be hacked using Cross Site Request Forgery. Django has tools to make this easy, so I spent friday experimenting with the KEGG PATHWAY database. As it turns out, KEGG PATHWAY is available chiefly in image format, and was therefore impractical to implement. In addition the well-traversed metabolic synthesis pathways were of little interest to us.
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