Regulatory
Pbhr

Part:BBa_K2918000

Designed by: TUDelft2019   Group: iGEM19_TUDelft   (2019-09-24)
Revision as of 12:52, 17 October 2019 by Weronika77 (Talk | contribs) (→‎Strain Construction)

PBHR

DNA polymerase of the Φ29 bacteriophage

Sequence and Features

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]

The part has been confirmed by sequencing and there are no mutations.

Overview

The Φ29 replication mechanism is a unique protein-primed based replication of a linear plasmid. Protein primed replication, unlike the conventional DNA or RNA based primers, greatly simplifies the design of replication systems. The Φ29 replication begins at origins of replication (OriR and OriL) on two sides of a linear protein primed linear DNA (Nies et al, 2018).The Φ29 replication can be established by using four simple proteins: Φ29 DNA polymerase (DNAP/p2),terminal protein (TP/p3), single stranded binding protein (SSB/p5) and double stranded binding protein (DSB/p6). The replication process begins by binding of the Φ29 DNA polymerase and terminal protein complex at the origins of replication (Nies et al, 2018).The Φ29 DNA polymerase is a single subunit protein known to have high processivity, it has been shown to synthesize upto 70 kilobase pairs of DNA (Blanco et al, 1988). The double stranded DNA binding proteins (Nies et al, 2018).

Characterization

Strain Construction

The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible coding sequence overhangs. The part was then cloned in a level 0 MoClo backbone Modular Cloning

Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments. The MoClo system consists of Type IIS restriction enzymes that cleave DNA 4 to 8 base pairs away from the recognition sites. Cleavage outside of the recognition site allows for customization of the overhangs generated. The MoClo system is hierarchical. First, basic parts (promoters, UTRs, CDS and terminators) are assembled in level 0 plasmids in the kit. In a single reaction, the individual parts can be assembled into vectors containing transcriptional units (level 1). Furthermore, MoClo allows for directional assembly of multiple transcriptional units. Successful assembly of constructs using MoClo can be confirmed by visual readouts (blue/white or red/white screening). For the protocol, you can find it here.


Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2. The complete sequence of our parts including backbone can be found here.


Table 1: Overview of different level in MoClo

Level Basic/Composite Type Enzyme
Level 0 Basic Promoters, 5’ UTR, CDS and terminators BpiI
Level 1 Composite Transcriptional units BsaI
Level 2/M/P Composite Multiple transcriptional units BpiI

For synthesizing basic parts, the part of interest should be flanked by a BpiI site and its specific type overhang. These parts can then be cloned into the respective level 0 MoClo parts. For level 1, where individual transcriptional units are cloned, the overhangs come from the backbone you choose. The restriction sites for level 1 are BsaI. However, any type IIS restriction enzyme could be used.


Table 2: Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts

Basic Part Sequence 5' End Sequence 3' End Level 0 backbone
Promoter NNNN GAAGAC NN GGAG TACT NN GTCTTC NNNN pICH41233
5’ UTR NNNN GAAGAC NN TACT AATG NN GTCTTC NNNN pICH41246
CDS NNNN GAAGAC NN AATG GCTT NN GTCTTC NNNN pICH41308
Terminator NNNN GAAGAC NN GCTT CGCT NN GTCTTC NNNN pICH41276

References

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Categories
Parameters
None