Team:Berkeley/Methods

Purpose:

When a particular protein sequence is known, but the codon usage per amino acid is not, designing primers that account for the codon degeneracy allows for PCR extraction of the given gene from a DNA template. This technique is especially useful when searching for a gene that has highly conserved regions in the amino acid sequence, such as actin, but may have different codon usage across the array of species in which the protein is found.

In iGEM 2013 Berkeley’s project, identifying a B-glucosyltransferase that acts on indoxyl was key, as a sequence for an indoxyl-specific B-glucosyltransferase does not exist in literature. Furthermore, none of the genomes of the indigo producing plant species have been sequenced, making our task even more difficult. By taking advantage of conservation patterns among B-glucosyltransferases from sequenced plant species related to indigo producing plants, we were able to design degenerate primers aimed to enrich cDNA libraries of the indigo plants for B-glucosyltransferases.

Materials:

• 1. DNA sequence editor (ApE)
• 2. Protein database (UniProt, NCBI)
• 3. BLASTX, PBLAST
• 4. Access to an Oligo synthesis facility
• 5. Sequence alignment software (Clustal Omega)
• 6. JalView 2.0 or other multiple sequences alignment viewer
• 7. Primer design software to ensure stability of designed primers

Methods:

• In our case, we did not have access to the sequence of our protein of interest. So we relied on the information available for the general family of B-glucosyltransferases into which our desired enzyme falls.

Purpose:

Materials:

• Purified Enzyme: FMO or GLU
• Substrate:
  • FMO: Indole, NADPH (co-factor)
  • GLU: Indican
• TECAN Plate Reader and TECAN Plate
• Solvents: DMSO, Water

Methods:

Purpose:

The Berkeley iGEM team 2013 has determined the enzyme kinetics of FMO and GLU (data for: FMO and GLU) based on the Michaelis-Menten kinetics model. Our purpose of determining kinetics was to give a quantitative data of how fast these enzymes can turnover their respective substrates, which is very useful when considering scaling up the process and for future iGEM teams when they use our enzymes.

Materials:

• Purified Enzyme: FMO or GLU
• Substrate:
  • FMO: Indole, NADPH (co-factor)
  • GLU: Indican
• TECAN Plate Reader and TECAN Plate
• Solvents: DMSO, Water

Methods:

Note: We followed the protocol from Bio-Rad’s Bradford Assay to determine the concentration of purified FMO and GLU.

We determined the concentration of our purified enzyme to be 4.2 mg/L for FMO and 3.5 mg/L for GLU.

Note: We assumed that the oxidation of indoxyl to indigo is much faster than that of the turnover rate of indole to indoxyl by FMO.

1. 1. In each well of the TECAN plate, we added 0.3 mM of NADPH, purified FMO and various indole concentrations (0, 0.10, 0.19, 0.39, 0.78, 1.55, 3.10, 6.20, 12.40 mM) at 5% DMSO.

Note: The 5% DMSO originates from the fact that indole had to be dissolved in DMSO. We recommend minimal DMSO because FMO is sensitive to DMSO.

2. 2. Using a TECAN plate reader, we determined the absorbance at 620 nm (peak wavelength at which indigo absorbs) every minute for 50 minutes.
3. 3. The absorbance data was converted into concentration of indigo based on the indigo standard we have determined previously.
4. 4. We wrote the following MATLAB code to automate analysis and generated the Michaelis-Menten Km.