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Plasmid Tightly Regulated Copy-Control System (PTRCCS)

1．Usage
1.1 Briefing on PTRCCS
PTRCCS, namely the Plasmid Tightly Regulated Copy-Control System in pGF (plasmid Genome Fast) Vector [1], can help the artificially synthesized genomes achieve stable replication in E. coli, and tightly control the copy number conversion of the synthetic genome in the E. coli - EPI300 strain, converting the single copy into the copy number of up to 100. It has been reported that a variety of artificially synthesized genomes, such as Saccharomyces cerevisiae genome [2] and plant virus genome [3], have been successfully synthesized using vectors containing this system.

1.2 The significance of PTRCCS to our project
This year, we successfully employed this system to convert the assembled mitochondrial genomes of S. cerevisiae into E. coli for stable cloning.
In the course of our experiments, there has been a very serious problem, which was stable genomic clones could not be obtained in E. coli and random mutations and deletions occurred after the assembly of mitochondrial genomes in Saccharomyces cerevisiae. Later, with reference to the design of pGF Vector, we added this system to our vector and successfully obtained the artificial genome synthesized by stable cloning in E. coli.

1.3 Providing reference for the teams to participate
Based on our researches, it is safe to say that we are the first team in all iGEM teams to manually design and synthesize vital genomes. With consideration of the importance of PTRCCS to the successful competition of our project this year, we strongly recommend that PTRCCS be used by all teams working on related subject. Good news is that there are already commercial products based on this system and hopefully the complete vector DNA containing the system will be soon available. If necessary, please feel free to contact us and we are more than glad to provide the complete vector containing PTRCCS we used this year.

2．Biology
PTRCCS consists of two parts: (1) ParA-ParB-ParC plasmid partition system; (2) oviS/oviV copy-control system.

Figure 1.Gene map of Plasmid Tightly Regulated Copy-Control System (PTRCCS)

2.1 ParA-ParB-ParC plasmid partition system：
The ParA-ParB-ParC from the F plasmid in E. coli consists of three elements that are essential for plasmid partition: Protein SopA, Protein SopB and Cis-acting region sopC. The system ensures the proper distribution of newly-replicated plasmids to daughter cells during cell division, when these proteins mutually impact. [4]

2.2 oriS/oriV Copy-Control system：
The oriS/oriV Copy-Control system is comprised of the oriS(ori2)-repE-incC system that controls a single copy of the plasmid and the oriV/(TrfA) system that implements a strictly controllable multicopy. [5] The oriS(ori2)-repE-incC system derives from F plasmid in E. coli, composed of replicon oriS(ori2), protein repE and incompatibility region incC. In the single copy mode, plasmid replication initiates at oriS (ori2), which consists of (1) four directly repeated sequences of 19 bp (iterons), (2) an AT-rich region, and (3) binding sites for the host DnaA protein. The RepE protein (251 residues, 29 kD), when in the monomeric form, mediates the assembly of a replication complex at oriS. The dimeric form of RepE binds to the inverted repeats of the repE operator exerting autogenous repression. [6]
The oriV/(TrfA) system derives from RK2 Vector. The oriV origin of replication consists of eight 17- bp direct repeats (iterons) that bind a monomeric form of the initiation protein TrfA [7]. DNA replication oriV is completely inactive in the commonly used hosts, because they do not produce the TrfA replication protein upon which replication at oriV depends. To supply the TrfA protein, Jadwiga Wild and his partner constructed special hosts, in which the synthesis of copy-up TrfA mutant protein is very tightly controlled by the ParaBAD (PBAD) promoter and AraC protein. [8]

3.Characterization
3.1 PTRCCS verified to be useful
We have not been able to convert the assembled mitochondrial genome of S. cerevisiae into E. coli for stable cloning even after numerous attempts before applying this system. Nonetheless, hardly had we equipped the vectors with this system when we successfully realized the cloning of the assembled mitochondrial genome in Saccharomyces cerevisiae which was later transferred into E. coli for stable cloning.

Figure 2. Gel electrophoresis of H2 after PCR,M is GeneRuler High Range DNA Ladder(Thermo Scientific);L1 is H2(Half of the genomic DNA).

3.2 Characterization Purpose：
Jadwiga Wild and his partner pointed out in their work that vectors containing this system were capable of conversion from single copy mode to multiple copy mode only after induction and the copy number was determined by the length of the sequences inserted in the vectors. [8]
Therefore, we measured the copy number of the vector containing the system in the case of inserting the minimal S. cerevisiae genome sequence we designed this year.
We adopted the QPCR method to measure the E. coli plasmid copy number, which was ever carried out by Lee C, Kim J, Shin S G, et al. [9]

The follows are needed to characterize the part:

3.3 Protocol of Copy Number Determination qPCR
3.3.1 Lysate standard sample qPCR
1. Inoculate a single colony into 5 mL of liquid LB medium with corresponding antibiotic and incubate in the shaker at 37 °C;
2. After 14-16h of growth, transfer 100 μL of suspended cells to 5 mL of fresh liquid LB medium with corresponding antibiotic and incubate at 37 °C until the OD600 reaches 0.7-0.8;
3. Spin down a suspended 1 mL of cells of 0.7 OD600 at 8.0g for 15 min
(Growth conditions are specified at the end of the protocol);
4. Remove the medium and resuspend the cell pellet in 1 mL of PBS;
5. Spin down the suspended of cells at 8.0 g for 15 min;
6. Repeat steps 2 and 3;
7. Completely remove PBS from the cell pellet;
8. Incubate cells at 95 °C for 10 min;
9. Store cells at -20 °C for 10 min;
10. Completely resuspend dry cell pellet in 100 μL of water by pipetting. Then vortex for 30s and spin down;
11. Make an initial dilution by transferring 10 μL of resuspended cell to 40 μL of water. Pipet carefully vortex for 30s and spin down;
12. Make a second dilution by transferring 10 μL of to 90 μL of water. Pipet carefully vortex for 30s and spin down;
13. For X reactions, make two different mixes using chromosome gene and plasmid gene primers:
X*6 μL of water
X*1 μL Forward primer 20 uM
X*1 μL Reverse primer 20 uM
X*10 μL of SYBR Green
14. First, transfer 18 μL of mix with chromosome primers to first X tubes, then transfer 18 μL of plasmid primers mix to other X tubes (X*2 tubes);
15. Add 2 μL of each diluted sample to the tubes;
16. Tenderly close the caps;
17. Run the reaction.

For steps 13-15, increase the volumes by a factor of desired technical replicate numbers.

3.3.2 Data Analysis
(1) By using the equation from standard curve that relates plasmid Ct value to real plasmid number calculate the plasmid number in the sample.
(2) By using the equation that relates chromosome Ct to real chromosome number calculate the number of chromosomes in the sample.
(3) Chromosome number = cell number. Therefore, by dividing the obtained plasmid number by chromosome number we can find the plasmid per cell number.

3.4 Result

Figure 3. PTRCCS-Vector-Blank qPCR Result

Table 1. PTRCCS-Vector-Blank qPCR Result and plasmid copy number

Figure 4. PTRCCS-Vector-MitoCRAFT qPCR Result

Table 2. PTRCCS-Vector-MitoCRAFT qPCR Result and plasmid copy number

4. Reference

[1]. Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector. Virologica Sinica (2016), 31 (2), 160-167.
[2]. “Perfect” designer chromosome V and behavior of a ring derivative. Science 355, eaaf4704 (2017).
[3]. Construction and Rescue of a Functional Synthetic Baculovirus. ACS Synthetic Biology 2017 6 (7), 1393-1402.
[4], [5]. DNA sequence requirements for interaction of the RK2 replication initiation protein with plasmid origin repeats. J Biol Chem. 1993 Feb 15; 268(5):3662-9.
[6]. Crystal structure of a prokaryotic replication initiator protein bound to DNA at 2.6 A resolution.EMBO J. 1999 Sep 1; 18(17):4597-607.
[7]. The plasmid RK2 initiation protein binds to the origin of replication as a monomer. J Biol Chem. 1996 Mar 22; 271(12):7072-8.
[8]. Conditionally Amplifiable BACs: Switching From Single-Copy to High-Copy Vectors and Genomic Clones. Genome Research. 2002;12(9):1434-1444.
[9]. Lee C, Kim J, Shin S G, et al. Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli[J]. Journal of biotechnology, 2006, 123(3): 273-280.

5. Others
5.1 The use of this system requires the presence of an E. coli EPI300 strain containing an inducible expression of the TrfA protein.
5.2 We were accorded with the plasmid DNA containing this system by Dai Junbiao, researcher of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.