Part:BBa_K4380007

ep-PCR reverse for sequence mutations

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

Vilnius-Lithuania Igem 2022 project NanoFind used this part as a way mutate proteins on the bacterial surface. The team was working to create an easily accessible nanoplastic detection tool, using peptides, whose interaction with nanoplastic particles would lead to an easily interpretable response. The system itself focused on smaller protein molecules, peptides, which are modified to acquire the ability to connect to the surface of synthetic polymers – plastics. The detection system works when peptides and nanoplastic particles combine and form a sandwich complex - one nanoplastic particle is surrounded by two peptides, attached to their respective protein. The sandwich complex consisted of two main parts – one is a peptide bound to a fluorescent protein, another peptide is immobilized on a cellulose membrane by a cellulose binding domain. A bacterial cell surface display system was used as a novel way to expose the same peptides onto the bacterial membrane, allowing to try to amplify the signal and to find novel peptides by using a peptide evolution protocol. This primer was used as a reverse primer for peptide evolution protocol.

Direct evolution of peptides

Vilnius Lithuania iGEM 2022 team has successfully put two plastic binding peptides - LCI and TA2 - through error-prone PCR (epPCR) reactions and were able to get peptide libraries for specific peptide evolution approaches. (Figure 1)

Figure 1: Peptide libraries, generated by epPCR (Parts:TA2 system:BBa_K4380020 and LCI system: BBa_K4380019).

Two different error-prone PCR strategies were chosen in order to find peptides with higher specificity (Table 1, Figure 2).

• epPCR using modified oligonucleotides 8-Oxo-dGTP and dPTP.
• epPCR utilizing low fidelity of Taq polymerase (with commercial kit and manual substrate selection).

Table 1. epPCR results for LCI peptide.

Method	Mutations
epPCR using modified oligonucleotides	45 bp/kb
PickMutant™ Error-Prone PCR Kit (Cat. No. MT040)	27 bp/kb
epPCR using low fidelity of recombinant Taq polymerase	42 bp/kb

Table 1. epPCR results for TA2 peptide.

Method	Mutations
epPCR using modified oligonucleotides	72 bp/kb
PickMutant™ Error-Prone PCR Kit (Cat. No. MT040)	9 bp/kb
epPCR using low fidelity of recombinant Taq polymerase	27 bp/kb