From 2010.igem.org

Latest revision as of 03:07, 28 October 2010

	Home Team Colin Hoffman Jeremy Kemball Anna Kropornicka Bobak Hadidi Acknowledgements Team Profile Project BioMORTAR Website IMPtools Website IMPtools v1.0 Plasmid Designer Modeling Next Version Cooperation Press Newsletter Notebook Bootcamp Safety

• • • 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.