Part:BBa_K2918029

OriL-GFP-Kan-OriR

Linear plasmid for replication by Φ29 replication machinery.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 664
  Illegal EcoRI site found at 2056
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 664
  Illegal EcoRI site found at 2056
  Illegal NheI site found at 956
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 664
  Illegal EcoRI site found at 2056
  Illegal BamHI site found at 873
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 664
  Illegal EcoRI site found at 2056
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 664
  Illegal EcoRI site found at 2056
  
• 1000
  COMPATIBLE WITH RFC[1000]

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

Usage and Biology

The Φ29 replication mechanism involves replication of a protein-primed based replication linear DNA. Protein primed replication, unlike the conventional DNA or RNA primed mechanism, does not depend on specific sequences of DNA/RNA which simplifies the design of replication systems. 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 (OriL and OriR), which flank the protein-primed linear plasmid (Salas et al., 1994). The double stranded DNA binding proteins aid in the process of replication and bind more intensely at the origins of replication (OriL and OriR), destabilizing the region and facilitating strand displacement. Single stranded binding proteins bind to the displaced DNA strand preventing strand switching of the DNA polymerase and protecting the linear plasmid from host nucleases (Salas et al., 1994). The replication mechanism is depicted in Figure 1. If you want to read more about this mechanism, you can take a look at our Design page!

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  Figure 1: Overview of phi29 replication mechanism

The Φ29 replication system is promising in many ways:

• The Φ29 DNA polymerase has the highest processivity of all known single subunit DNA polymerases (Blanco et al., 1989), and can be used for whole genome amplification.
• The Φ29 machinery along with cell-free expression systems can be used to establish the three dogmas of biology in vitro. Setting the basis for artificial cell development.
• The existing DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide sequences.
• We envision that the unique configuration of the double-stranded, protein-capped linear replicon will be a basis for many new engineered protein-DNA complexes.
• Orthogonal replication enables not only replication independent from the host, but also the ability to engineer the orthogonal DNA polymerase’s fidelity without introducing mutations in the cell’s genome which makes in vivo directed evolution a possibility.

Characterization

To characterize our linear construct we wanted to demonstrate in vitro replication of our linear construct (OriL-GFP-Kan-OriR, link to page) and show that our construct can be propagated by the phi29 replication machinery. We used an in-vitro transcription-translation system (PUREfrex®2.0) supplemented with dNTPs and purified auxiliary proteins: single stranded binding protein (SSB) and double stranded binding protein (SBD), needed for an efficient DNA replication. We used 1nM as the starting DNA concentration of our OriL-GFP-Kan-OriR with a twofold excess of plasmid DNA encoding for both DNAP and TP. After a 4 hour incubation at 30०C, we purified the DNA from the reaction as described in the complete protocol. The size of our construct was determined by DNA gel electrophoresis. As a control, we used samples with and without dNTPs. On our gel, we observed that our linear construct (~2,3 kb) was successfully amplified, as shown in Figure 1. These results show that the phi29 replication machinery can be used for orthogonal DNA replication in-vitro. We demonstrated that our origin-flanked linear construct (OriL-GFP-Kan-OriR) can be replicated in vitro by the phi29 minimal replication machinery.

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  Figure 1: Purified OriL-GFP-Kan-OriR from a coupled DNA transcription-translation and replication reaction with (+) and without (-) dNTPs, amplified in vitro using the PUREfrex 2.0 system An intense band in the expected size (~2.3 kb) is observed from the +dNTPs reaction, demonstrating the OriL-GFP-Kan-OriR in-vitro replication.

Successful replication of our linear construct has been shown on the agarose gel (figure 1). A very intense band is visible at a size of 2.3 kb, corresponding to the size of OriL-GFP-Kan-OriR construct. This indicates that there has been successful in vitro amplification of our construct.

Strain Construction

The construct was assembled by golden gate assembly based modular cloning system. First, the individual transcriptional units were cloned into level 1 destination vectors [http://www.addgene.org/47998 pICH47732] and [http://www.addgene.org/47998/ pICH47742] by BpiI based golden gate assembly. The multi-transcriptional unit construct was assembled by a BsaI based golden gate. The assembly was a one-pot restriction-ligation reaction where the individual level 1 constructs were added along with the destination vector [http://www.addgene.org/47998/ pICH8031] and the construct was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.

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

• Blanco, L., Lázaro, J. M., De Vega, M., Bonnin, A., & Salas, M. (1994). Terminal protein-primed DNA amplification.Proceedings of the National Academy of Sciences of the United States of America.
• Blanco, L., Bernads, A., Lharo, J. M., Martins, G., & Garmendia, C. (1989). Highly Efficient DNA Synthesis by the Phage 429 DNA Polymerase.