Team:Lethbridge HS/Parts

Project

Parts

• Description

• What is nuclease? What is dextranase?

• What we are doing differently

• Extracellular Polymeric Substance Matrix

Parts

All constructs are in vector pSB1C3.
For more info on our parts please visit the Parts Registry.

Our Petal Death Protein (PDP) part (BBa_K179200) was design to produce a modest amount of Oxalic acid. The PDP gene was obtained from Dianthus caryophyllus (Carnation) (Clove pink). PDP catalyzes the production of Oxalic acid from Oxaloacetate: Oxaloacetate + H2O = oxalic acid + acetate. The first 3 amino acids represent a propeptide and are not included in the PDP sequence. The PDP gene is under control of the constitutively active medium transcription promoter (J23107) and a strong RBS (B0034). This combination was chosen to allow us to tune the amount of oxalic acid produced through control of PDP production. Mutation of the transcription promoter to strong or weak activity will result in more or less PDP production and subsequent changes in oxalic acid production. The same principle can be applied to the RBS from strong to medium to weak. We tuned the oxalic acid production to achieve mite death at the lowest possible concentration while preventing any possible harm to bee health.

Petal Death Protein Composite construct
BBa_K1792000
Petal Death Protein Basic construct
BBa_K1792001

All constructs are in vector pSB1C3.
For more info on our parts please visit the Parts Registry.

The Nuclease is from Staphylococcus aureus. This part is the same as BBa_K729004 (UCL 2012) with the addition of a C-terminal his-tag. The Nuclease is an enzyme that catalyzes the hydrolysis of both DNA and RNA at the 5' position of the phosphodiester bond. The α-Dextranase gene is from Chaetomium gracile (Fungi). It performs hydrolysis of 1,6-alpha-D-glucosidic linkages in dextran. This is a very large gene (1,818 nucleotides, native sequence). Both the nuclease part (BBa_K1792004) and dextranase part (BBa_K1792002) include a T7 promoter, which is IPTG inducible to get maximum yield of protein. The promoters and RBS are also selected for greatest yield of protein when induced. These proteins contain a N-terminal bacterial secretion tag. Which allows the proteins to be exported from cells and easily separated by centrifugation. The proteins also contain a 6X histidine tag for possible downstream purification and concentration via affinity chromatography (if necessary). Stop codons take care of the protein termination for both the nuclease and dextranase. There is a double terminator on the nuclease construct to stop mRNA production after the gene of interest is translated. The dextranase construct was very large, so to minimize part size a terminator element was not included. As a result, dextranase mRNA synthesis will continue until the transcription complex falls off.

Nuclease Composite construct
BBa_K1792004

Nuclease Basic construct
BBa_K1792005

Dextranase Composite construct
BBa_K1792002

Dextranase Basic construct
BBa_K1792003