Part:BBa_K4182012

RK2 Ori

This is an improvements on the previous pGPP synthesis circuit (BBa_K3052010) by the replicon optimization from p15Aori (5-10 copies) with the lower copy RK2 ori (1-3 copies), trying to reduce the metabolic stress and enhance the protein expression level.

RK2 ori is a 2.22-kb replication origin of the broad-host-range IncPa plasmid formed by the vegetative origin (oriV) and the replication protein trfA. The standardized ori segment is formed by oriV followed by the gene that encodes the replication protein TrfA, which is expressed under its native BHR promoter. This origin of replication is among the least restrained and it keeps its copy number per cell very low (1-3 copies).

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 399
  Illegal NgoMIV site found at 523
  
• 1000
  COMPATIBLE WITH RFC[1000]

Profile

Base Pairs

532

Source

E.coli

Usage&Biology

This is an improvements on the previous pGPP synthesis circuit (BBa_K3052010) by the replicon optimization from p15Aori (5-10 copies) with the lower copy RK2 ori (1-3 copies), trying to reduce the metabolic stress and enhance the protein expression level.

1. Plasmid map before and after replicon optimization

FIG.1 p15Aori-pGPP (2019XJTU-China parts BBa-K3052010)

FIG.2 Replicon optimized plasmid 2 (421ori-pGPP) (2022-XJTU-China parts BBa_K4182012)

2. Introduction of Plasmid 2 (precursor pGPP synthesis circuit)

1.Mevalonate (MVA) pathway

Fig 3 MVA pathway and constructed gene cluster

The MVA pathway mainly exists in eukaryotes. It starts from acetyl-CoA to form IPP and DMAPP via mevalonate, with the help of several enzymes including AtoB, HMGS, HMGR, MK, PMK, MPD and Idi. Then IPP and DMAPP are further condensed to form geranyl pyrophosphate (GPP) catalyzed by geranyl pyrophosphate synthase (trGPPS). GPP is a very common precursor of fragrance molecules, terpenes and our herbicide aspterric acid (AA).

2. Promoter optimization to RK2 ori (oriV+trfA)

FIG 4 Structure of RK2 ori

RK2 is a 2.22-kb replication origin of the broad-host-range IncPa plasmid formed by the vegetative origin (oriV) and the replication protein trfA. The standardized ori segment is formed by oriV followed by the gene that encodes the replication protein TrfA, which is expressed under its native BHR promoter. This origin of replication is among the least restrained and it keeps its copy number per cell very low (1-3 copies).

Our plasmid 2 derivate from RK2 ori, and its low copy number is regulated by iterons mechanism. TrfA protein is a replication regulator belonging to RepA protein family, which can regulate plasmid replication by binding to the repeat sequence (iterons) near the replication start site. TrfA will initiate plasmid DNA replication once binding to the origin. However, with the increase of plasmid copy, the concentration of the iterons and TrfA protein increases, leading to the TrfA-TrfA protein interaction and lock the two plasmid DNA like a handcuff, then replication terminates and the copy-number of the plasmid is regulated.

Since the MVA pathway involves many genes (the p15Aori-pGPP plasmid is 15kb in length) and the previous data showed that the expression level of each enzyme in the cell was not high (See the results of team 2019-XJTU-China). So we tried to replace the replicon p15Aori (5-10 copies) with the lower copy RK2 ori (1-3 copies), to reduce the pressure of cell growth and metabolism as well as protein expression by reducing the copy number of plasmid.

3.Construction process and verification of plasmid 2

Because of the large size of the plasmid and the difficulty of gene manipulation, the Gibson Assembly method was used in our study: oriV-trfA fragment was obtained by PCR, while the linear pGPP fragment (~12 kb) was obtained by enzyme digestion of Sac I and Bln I. For the recovery of linear large pGPP fragment, a variety of strategies were employed and a new direct recovery method by the silica-gel particle was found to be efficient, mainly because the silica-gel particles were uniformly dispersed in buffer, which can avoid the DNA fragment break caused by excessive shear force. So centrifugation was conducted at 8000 rpm instead of 12000 rpm, and be gentle during the purification. Finally we obtained pure pGPP fragment with a concentration of 4.1 μg/μL.

FIG.5: Fragment used for construction of plasmid 2 (1: M, DL10000, 2: oriV+TrfA 3: pGPP linear fragment)

After Gibson assembly, we obtained only 6 colonies on LB medium with low cloning efficiency, and the colony PCR of clone 1-4 showed positive results. Then plasmids were extracted and sent to the company for sequencing. Sequencing results showed No 4 was correct.

FIG.6 Colony PCR verification of plasmid 2. M, DL5000

The strain harboring our plasmid 2 was cultured to OD600= 0.6-0.8, and IPTG with a final concentration of 1mM was added for 6 hours. RT-qPCR was performed after induction. The results showed that the transcription level of key enzyme GPPS was significantly increased after the replacement of lower copy number replicon, which proved the effectiveness of the strategy. When the metabolic pressure was reduced, cell growth and metabolism were enhanced, leading to the increased protein expression. It will also facilitate the synthesis of GPP and AA, our final product.

FIG.7 RT-qPCR results of GPPS in three strains

References

[1]Rafael Silva-Rocha, Esteban Martínez-García, Belén Calles, Max Chavarría, Alejandro Arce-Rodríguez, Aitor de las Heras, A. David Páez-Espino, Gonzalo Durante-Rodríguez, Juhyun Kim, Pablo I. Nikel, Raúl Platero, Víctor de Lorenzo, The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes, Nucleic Acids Research, Volume 41, Issue D1, 1 January 2013, Pages D666–D675, https://doi.org/10.1093/nar/gks1119

[2]Jorge Alonso-Gutierrez, Rossana Chan, Tanveer S. Batth, Paul D. Adams, Jay D. Keasling, Christopher J. Petzold, Taek Soon Lee, Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production, Metabolic Engineering, Volume 19,2013, Pages 33-41, ISSN 1096-7176,https://doi.org/10.1016/j.ymben.2013.05.004.

[3]Figurski, D.H. and Helinski, D.R. (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl Acad. Sci. USA, 76, 1648–1652.

[4]Santos,P.M., Di Bartolo,I., Blatny,J.M., Zennaro,E. and Valla,S.(2001) New broad-host-range promoter probe vectors based on the plasmid RK2 replicon. FEMS Microbiol. Lett., 195, 91–96.