Coding

Part:BBa_K2082106

Designed by: Carsten Hain   Group: iGEM16_Bielefeld-CeBiTec   (2016-10-13)


Error-prone polymerase I

Error-prone polymerase I - BBa_K2082106

BBa_K2082106 is a mutant of the E. coli polymerase I. Three point mutations (I709N, A759R and D424A) decreases the fidelity of the error prone polymerase I. Usage of the error prone polymerase I inside a polymerase I temperature sensitive strain at the non-permissive temperature leads to a mutation rate of 6×10-4 bp-1. (Camps, 2003)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 708
    Illegal XhoI site found at 1206
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 108
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1671


DNA polymerase I (Pol I) participates in lagging-strand replication of chromosomal DNA, DNA repair, and ColE1 plasmid replication. The error prone Pol I (EP Pol I) carries three point mutations (I709N, A759R and D424A), which decrease EP Pol I's fidelity dramatic (Camps, 2003).

EP Pol I mutations

The I709N mutation is located in motif A. This is a conserved sequence in the palm domain of the polymerase active site. The mutations lead to an enlargement of the substrate-binding pocket, which is a possible explanation for the increased error rate (Camps, 2003). The D424A mutation in the exonuclease domain leads to deactivation of the proofreading activity of the EP-Pol I. The amino acid replacements A759R is located in the O-helix, which is a conserved sequence (motif B) that is located close to the polymerase active site on dNTP binding. This may stabilize the enzyme with similar conformations, which leads to missed integration of nucleotides (Camps, 2003).

EP Pol I in in vivo library gernation

Because of EP Pol I's low fidelity the enzyme can be used in in vivo library generation by error prone DNA replication.
The main advantage of the EP Pol I to other unspecific mutators is, that the polymerase I replicates mainly plasmids from the ColE1 familiy (Camps, 2003) and only small fragments of the chromosome (okazaki processing). Therefore the mutation can be targeted to ones protein of interest onside ColE1 plasmids, like pSB1C3. Error prone polymerase I is therefore a fitting tool for further directed evolution approaches in future iGEM projects.

Characterization

We measured error prone polymerase I's mutation rate by reversion assays (Figure 1A), using a stop beta lactamase on a ColE1 plasmid as reporter as well as by high-throughput sequencing (Figure 1B).
A B

Figure 1: Mutation rate of error porne polymerase in comparison to wild type polymerase I (K2082107). Mutation rate was determined as revertant frequency by reversion assays (A) and mutations per bp by high-throughput sequencing
We also determined error prone polymerase I mutation spectrum (Figure 2A), finding a prevalence of transitions (Figure 2B).
A B

Figure 2: Mutation spectrum of error porne polymerase in comparison to wild type polymerase I (K2082107). The mutation spectrum was determined by high-throughput sequencing (A), showing a prevalence for transitions for the error prone polymerase I (B).

We determined the plasmid specifity of EP Pol I by comparing the mutation rate determined by our high-throughput sequencing experiment on different plasmids in the ColE1/pSC101 plasmid mixture.
Figure 3: Plasmid specifity of EP Pol I mutagenesis. The mutation rate of EP Pol I was determined on ColE1 and pSC101 plasmids by high-througput sequencing.

The mutation rate on ColE1 plasmids is higher when using EP Pol I (see figure 1). On pSC101 plasmids there is almost no difference in the mutation rate when using either EP Pol I or WT Pol I. Therefore the mutagenesis produced by EP Pol I is mostly specific for ColE1 plasmids and considerable lower on pSC101 plasmids. Because pSC101 plasmids use a replcation mechanism similiar to the chromosome it can be assumed, that the mutation rate inside the chromosome is only slightly increased when using EP Pol I. This confirms earlier results from Camps (Camps, 2003) who predicted a lower mutagenesis rate for the chromosome and pSC101 plasmids and measured this for the chromosome by rifampicin assays.

Application Protocol

  • Create competent cell of Pol I temperature sensitive strain (e.g. E. coli JS200, grow at 30°C) carrying BBa_K2082106 (it is strongly recommended to encode error prone polymerase I not on a ColE1 plasmid; use pSB4C5 instead)
  • For library generation of a single gene (Alexander, 2014)
    • Transform the gene of interest encoded on a ColE1 plasmid the preperated JS200
    • Grow cells at 37°C for ~24h
    • Isolate plasmids to obtain gene library
    • Repeat cycle multiple times for increased mutation rate
  • For mutagensis of a library (to circumvent transformation bottleneck of periodic retransformation)
    • Transform the gene of interest encoded on a ColE1 plasmid the preperated JS200
    • Grow cell at 37°C to saturation
    • Dilute cells 1:103-105
    • Repeat
    • Coupling with selection of improved proteins can be useful
    • This protocol has decreased mutation rate in comparison to periodic retransformation protocol, but it is especially useful when coupled with an in vivo selection system to sort out bad or defect clones

Characterization protocols

  • Reversion assay
    • Transform reporter plasmid into E. coli JS200
    • After regeneration inoculate prewarmed 10 mL LB with appropriate antibiotic with 100 μL
    • Grow for 24 h
    • Plate serial dilutions on LB agar plates with and without ampicillin
    • Determine total cell count and revertant count
    • Reversion frequency: number of Revertants/ number of viable cell count
  • NGS
    • Experimental setup as for reversion assays
    • Instead of plating isolate plasmids and use for Illumina MiSeq with the Illumina Nextera DNA Library Preparation Kit
    • Mapping of obtained read onto reference sequence, count coverage on non reference bases as number of mutations
    • Mutation rate: number of mutations/ number of sequenced bases
    • Divide mutations into groups based on refernce base and mutated base to obtain mutation spectrum
  • Alexander, David L.; Lilly, Joshua; Hernandez, Jaime; Romsdahl, Jillian; Troll, Christopher J.; Camps, Manel (2014): Random mutagenesis by error-prone pol plasmid replication in Escherichia coli. In: Methods in molecular biology (Clifton, N.J.) 1179, S. 31–44. DOI: 10.1007/978-1-4939-1053-3_3.
  • Camps, Manel; Naukkarinen, Jussi; Johnson, Ben P.; Loeb, Lawrence A. (2003): Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I. In: Proceedings of the National Academy of Sciences of the United States of America 100 (17), S. 9727–9732. DOI: 10.1073/pnas.1333928100.

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