University of Washington 2010 iGEM Team Accomplishments

Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device

• We synthesized the CapD protein coding sequence optimized for E. Coli expression and in accordance with BioBrick standards, <partinfo>BBa_K314011</partinfo>, and determined its catalytic constants, as shown here: Find it on our wiki

• We BioBricked the Tse2/Tsi2 <partinfo>BBa_K314203</partinfo>, toxin-antitoxin locus with F2620, an HSL inducible promoter, showed expression of the toxin in response to HSL, and presented evidence that both the toxin and antitoxin are working as expected. Find it on our wiki

Characterize the operation of at least one new BioBrick Part or Device and document it in the registry

• We re-engineered CapD into CapD_CP by circularly permuting it. CapD_CP can be found in the parts registry under <partinfo>BBa_K314012</partinfo>. As a result of this reengineering, CapD_CP no longer needs to autocleave itself to become active, resulting in substantially higher yields of active protein after purification. We expressed and characterized the enzyme as can be found here: Find it on our wiki

• We introduced and characterized a commonly used f1 phage replication origin, <partinfo>BBa_K314110</partinfo>. By incorporating the f1 origin into current BioBrick plasmids new plasmids can replicate as double stranded DNA in bacteria or as single stranded DNA by M13 helper phage. The part was characterized as shown here: Find it on our wiki

Characterize or improve an existing BioBrick Part or Device and enter this information back on the Registry

• We made a set of protein expression cassette's, allow future teams to make generators in a single round of cloning. To do this we used some new parts (f1 ori, lacI, T7 Promoter) as well as several existing parts (<partinfo>BBa_B0034</partinfo>, High Const# <partinfo>BBa_J23100</partinfo>, Low Const# <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_R0011</partinfo>). We then characterized each cassette's ability to express GFP (<partinfo>BBa_E0040</partinfo>). Information and characterization of the cassette's in different base plasmids (<partinfo>pSB1C3</partinfo>, <partinfo>pSB1A3</partinfo>, <partinfo>pSB3K3</partinfo>, <partinfo>pSB4A5</partinfo>) can be found in our wiki here: Find it on our wiki
  • <partinfo>BBa_K314100</partinfo> High Constitutive expression cassette
  • <partinfo>BBa_K314101</partinfo> Low Constitutive expression cassette
  • <partinfo>BBa_K314103</partinfo> Lac Induced expression cassette
  • <partinfo>BBa_K314104</partinfo> T7 Induced expression cassette

Develop and document a new technical standard that supports the sharing of BioBrick Parts or Devices, either via physical DNA or as information via the internet

• We developed a new standard, described in [http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_68:_Standard_for_the_Electronic_Distribution_of_SBOLv_Diagrams BBF RFC 68], for the electronic distribution of diagrams based on [http://bbf.openwetware.org/RFC.html#BBF_RFC_16:_Synthetic_Biology_Open_Language_Visual_.28SBOLv.29_Specification SBOLv] symbols.
• We developed a new software tool, WikiDust, that allows users to quickly create standards-compliant diagrams and upload them to the iGEM wiki or other websites.

Gain real-world synthetic biology experience

• Our team was comprised of senior high school students who had never pipetted before to graduating seniors with several years of lab experience. By the end of the summer we had learned many skills in synthetic biology, from cloning genes, to developing enzyme assays, to writing code!

Contributions

• Please see our Team Page for Who Did What.

University of Washington 2010 iGEM Parts Submitted

Gram(-) Parts Submitted

Tse2	BBa_K314200-A toxic protein originating from Pseudomonas aeruginosa that has been shown to arrest growth in both prokaryotic and eukaryotic cells when expressed intracelluarly.
Tsi2	BBa_K314201-Protein originating from Pseudomonas aeruginosa that confers immunity to the toxic protein Tse2.
Tse2/Tsi2	BBa_K314202-Tse2/Tsi2: toxin antitoxin locus.
F2620-Tse2/Tsi2	BBa_K314203-HSL inducible Tse2/Tsi2 generator.

Gram(+) Parts Submitted

CapD (K314011)	A protein native to Bacillus anthracis that favors transpeptidation. Find it on our wiki
CapD_CP (K314012)	Circular permuted CapD with a Foldit-designed linker Find it on our wiki
CapD_CP_F24H (K314015)	Mutated CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG. Find it on our wiki
CapD_CP_F24H_L40R_T59N_M61S (K314017)	Mutated CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG. Find it on our wiki
High Constitutive Expression Cassette (K314100)	High constitutive protein expression insert includes f1 origin k314110, a high expression promoter J23100, and the Elowitz standard RBS B0034.
Low Constitutive Expression Cassette (K314101)	Low constitutive protein expression insert includes f1 origin K314110, a medium expression promoter J23114, and the Elowitz standard RBS B0034.
Lac Induced Expression Cassette (K314103)	Lac induced protein expression insert includes f1 origin K314110, Lac I generator K314111, a lac I repressed promoter R0011, and the Elowitz standard RBS B0034.
T7 Expression Cassette (K314104)	Lac induced protein expression insert includes f1 origin K314110, Lac I generator K314111, a T7 promoter k314112, and the Elowitz standard RBS B0034.
f1 origin	Phage origin recognized by M13. Used to make SS DNA when infected with M13k07 helper phage.
Lac I	Lac I gene with promoter and RBS
T7 Promoter	T7 promoter with RBS B0034