Engineering

Design-Build-Test-Learn Cycles

Three Wet-lab-based DBTL cycles

pET-CH1
pET-F4
SDS-PAGE
Design
Build
Learn
Test
DBTL

Design

To express peach chitinase at a high level and inducible, we chose the pET-28a(+) vector, which was driven by the T7 promoter.

Build

  1. Nucleotide sequence of chitinase Prunus persica (peach), named CH1 in this study was downloaded from KEGG.
  2. PpCH1 gene fragments was synthesized free of charge by GenScript China.
  3. Amplification of pET-28a(+) plasmid DNA by DH5α, which was linearized for subsequent cloning applications.
  4. A recombinant pET-CH1 plasmid was constructed and confirmed by sequencing.

Learn

We tried twice, but failed to determine which band corresponds to our target protein. The total protein extraction process should be optimized.

Test

A recombinant pET-CH1 plasmid was used for protein expression analysis.

Design
Build
Learn
Test
DBTL

Design

To express specific dsRNA: F4 fragment for the purpose of knocking down sGFP expression.

  1. The nucleotide sequence of superfold GFP (sGFP) was provided by PLD Technology.
  2. Oligowalk from Mathews group was used for siRNA design.
  3. A double-stranded RNA (dsRNA) fragment targeting sGFP (F4 fragment) was designed with restriction sites.

Build

  1. F4 fragment was synthesized by GenScript.
  2. Double restriction digestion of F4 fragment and pET-28a(+) vector.
  3. Ligation with T4 DNA ligase and transformation into E. coli DH5α.
  4. Plasmid extraction and confirmation by sequencing.

Learn

The short F4 fragment and secondary structure interference with cloning are issues that require testing of alternative cloning strategies.

Test

Sanger sequencing results indicated incomplete insertion of F4 fragment into the vector.

Design
Build
Learn
Test
DBTL

Design

To observe protein by performing SDS-PAGE safely in a high school laboratory with precast PAGE Gels.

Build

Total protein extraction from E. Coli.

Perform SDS-PAGE.

Learn

Although we did not obtain the expected clear bands for robust expression, the established experimental workflow demonstrates the feasibility of our cloning strategy, confirming that our system can support protein expression with further refinement.

Faint bands also indicated partial expression, and further optimization of the protein extraction protocol, induction time, and IPTG concentration is needed to improve yield.

Test

We failed to determine which band corresponds to our target protein.

Engineering Success

Key Achievements:

  • An IPTG-inducible expression system for the predicted chitinase from Prunus persica (peach) (the CDS part: BBa_25CSQLVI) was successfully constructed using the T7-driven pET-28a vector (BBa_25YSLUTM).
  • Two methods were employed for plasmid construction: homologous recombination and double restriction digestion with T4 DNA ligase ligation.
  • The process of performing SDS-PAGE safely in a high school laboratory was initially explored.