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. Coil expression and in accordance to 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, and showed expression of the toxin in response to HSL. 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 autoclave 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 this into current BioBrick plasmids the plasmids can be replicated as double stranded DNA by 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, lacR, T7 Promoter) as well as several existing parts (BBa_B0034, High Const# J23100, Low Const# J23114, R0011). We then characterized each cassette's ability to express GFP (##). The results of each expression cassette in pSB1C3 are listed here. Further information and characterization of the cassette's in different base plasmids (1A3, 3K3, 4A5) can be found in our wiki here: Find it on our wiki
  • <partinfo>BBa_K314100</partinfo>High Const
  • <partinfo>BBa_K314101</partinfo>Med Const
  • <partinfo>BBa_K314103</partinfo>Lac Induc
  • <partinfo>BBa_K314104</partinfo>T7 Induc

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

• We developed a new software tool that allows users to make interactive images that link to the desired part?
• We developed a new software tool that allows users to submit many BioBricks with the single click?

Gained 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 gene's, to developing enzyme assays, to writing code!

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

<partinfo>K314011 DeepComponents</partinfo> Find it on our wiki

<partinfo>K314012 DeepComponents</partinfo>A circularly permuted CapD with a Foldit-designed linker. Find it on our wiki

<partinfo>K314015 DeepComponents</partinfo>A mutated CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG. Find it on our wiki

<partinfo>K314017 DeepComponents</partinfo> A mutated CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation. Find it on our wiki <partinfo>K314100 DeepComponents</partinfo> <partinfo>K314101 DeepComponents</partinfo> <partinfo>K314103 DeepComponents</partinfo> <partinfo>K314104 DeepComponents</partinfo>

CapD	A protein native to Bacillus anthracis that favors transpeptidation.
CapD_CP	Circular permuted CapD with a Foldit-designed linker
CapD_CP_F24H	Mutated CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG.
CapD_CP_F24H_L40R_T59N_M61S>	Mutated CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG.
High Constitutive Expression Cassette>	High constitutive protein expression insert includes f1 origin k314110, a high expression promoter J23100, and the Elowitz standard RBS B0034.
Medium Constitutive Expression Cassette	Medium constitutive protein expression insert includes f1 origin K314110, a medium expression promoter J23114, and the Elowitz standard RBS B0034.
Lac Induced Expression Cassette	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	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 Promotor	T7 promoter with RBS B0034