SynBERC:MIT/Calendar/2007-8-8

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Anyone in the synthetic biology community is welcome to attend.

Wednesday August 8, 2007 at 12:30pm EST

32-D463, MIT

Topic of discussion

Post-seminar notes (please improve!)

Jason Kelly led a discussion on what we can do to make BioBricks more useful for academic research labs. This discussion also included generating a list of "wanted" BioBrick parts.

In a preliminary announcement, Randy Rettberg described the need for the Registry to upgrade parts documentation and the plan to have a new template for part entry online in time for this year's iGEM teams to use for entry of their parts. He also outlined the more structured process that will be used to screen parts for the Registry. (Add details...)

Wishlist of wanted parts

• BioBrick expression plasmids
  • T7 promoter - has patent issues, but it has been tried and tested.
    • "High demand to have the T7 expression system could move this into the BB plasmids."
    • A candidate is pET 28 -- a pre-prepped vector with T7 promoter and translation start site. (According to Tom Knight, pET vectors are in demand, but don't fit BioBrick format.)
  • T3
  • SP6
  • T7 RNA polymerase-containing strains
  • Also T3, SP6 What are the patent issues on these? (According to Tom Knight, T3 is unencumbered.)

• Popular plasmids
  • Any requests through the SynBERC BB plasmid project? (Colin)
  • Sauer lab would want typical ORI, non-ampicillin selection and T7 induction; otherwise they don't care. (The useful parts of pET vectors could be transferred into a BB-compatible plasmid.)
  • Xre protein, promoters (from Bacillus subtilis)
  • The pZE modular plasmid system - Stephanopoulous lab - should be covered by vector scaffold ... BBa_I51001
  • Gateway cloning system -- facilitates creation of fusion proteins.
  • SR6 (?) -- has increased host range.

• Yeast plasmids
  • Need to convert them to BB form (or get them from Pam Silver).

• Alternative antibiotic cassettes
  • Apramycin
  • Trimethoprim

• Transposase genes

• Plasmid origins
  • R6, P1, F, colE1

• Chromosomal integration fragments
  • Choice of locations on the chromosome

• Cell-killing enzymes
  • VSVG
  • SrcB

• Protein export tags
  • phoA
• Protein purification tags, antibody epitopes
  • FLAG
  • 6HIS - Sauer lab
  • Strep
  • GST
  • Chitin-binding
  • MBP
  • S tags (Berkeley Is this the same as strep?)

• Protein solubility
  • Maltose-binding protein

• Protein splicing domains (What's this?)

• Surface display proteins
  • LPP/OmpA
  • Neisseria IgA1

• New fluorescent proteins
  • EBFP2 blue
  • New red monomers

• Luminescent reporters
  • Renilla (sea pansy luciferase) [See BBa_J52008 (SCM)]
  • Photuris (firefly)
  • Bacterial (Photorhabdus)

• Promoters in reverse orientation

• Parts to enable custom insertions
  • In the middle of devices [e.g., a set of alternate restriction sites that are also kept out of BBs (and BB plasmids) when possible to support using them for editing] Reshma has designed some plasmids with sites that allow insertion of alternate origins.

• BioBrick every gene
  • From genome projects that have put all the coding sequences in separate plasmids.

• Add your wanted part here ...

Software tools

• Sequence analysis

• Strain organization/LIMS
  • Labs are interested in BB just to make compatible components across intra-lab projects
  • This feature is available in the software, just needs to be turned on.

• CAD tools
  • Which to build first?
  • Protein fusion
  • Gene expression with tags

• Construction planning/organization

• Convert existing parts to BB
  • e.g., VectorNTI->BBXML/import annotated sequence
  • Facilitate conversion to BB/transfer of things to Registry (Registry could accept non-BioBrick constructs and later BioBrick them on request)
  • BioBrick your vectors.

• Export to GenBank

Lab services

• Biobrick "domestication"
  • Site-directed mutagenesis to remove BB sites
  • Moving gene to BB vector via PCR
  • Sequence optimization and synthesis
• Archiving/Distribution (present activity) --> distribute strains

• Expert support
• "On-demand" BioBricking
  • Store part as info only until someone orders it, then BioBrick the physical DNA.
  • BB your favorite vector (as a service)
  • Automated assembly
  • Phone/email consultation on Registry issues (including the above points).

• Education
  • Get a document out on the statistics that show that the BB assembly done by hand works better than typical assembly approaches.

• Better distribution of strains

Other folks to talk to? Who is building big stuff?

• Metabolic engineering
  • multi-gene pathways (e.g. 5x Promoter.RBS.gene.term)
• Protein expression/purification
  • Varying 5' and 3' ends (purification tags, export sequences)
  • Multi-gene makes better use of BBs vs. say gateway system
  • Protein fusion - tom goingto start a sub-group working on this.
• Custom plasmid constuction
  • ORI, selectable markers, inducible promoters, etc
  • pZE ststem (lets you drop in ORIs: SC101, SC101*, p15A, colE1)
• Other suggestions?
  • Sri mentioned groups that get funding to move every gene from an organism into gateway system, we could raise money to do the same thing with BBs.

Live Notes

• Laub lab uses gateway.

Rocking the notetaking:

• Audience: Asking about creating a 5-gene operon, something to do with the PZE system, interested in using BB methods to build permutations of the transcription logic

• Audience: GST tags are key for surface plasmon resonance

Question: how do people keep track of plasmids and their constructs?

Problem: right now people do not have a way to import or export annotated sequence files from the registry.

Jason: one of the most useful functions of the Registry is documentation and archival of parts.

The Sauer lab just uses a notepad to keep track of plasmids.

We need better education about the ease of assembling pieces of DNA via BioBricks assembly. This should also involve statistics on assembly success rate.

The Registry could offer to BioBrick existing vectors that labs are using.

The rare Arginine codon in the BioBricks scar is a key issue when doing protein fusions.