Difference between revisions of "Part:BBa K4182012"
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<partinfo>BBa_K4182012 short</partinfo>
 
<partinfo>BBa_K4182012 short</partinfo>
  
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Optimize and improve the replicon ori of 2019XJTU-iGEM parts BBa-K3052010 to reduce the metabolic pressure of cells and improve the protein expression level
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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).
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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==Usage&Biology==
 
==Usage&Biology==
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===Optimize and improve the replicon ori of 2019XJTU-iGEM parts BBa-K3052010 to reduce the metabolic pressure of cells and improve the protein expression level===
+
 
 +
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====
 
====1. Plasmid map before and after replicon optimization====
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[[File:XJTU-p2-1.png|400px]]
 
[[File:XJTU-p2-1.png|400px]]
  
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FIG.1 p15Aori-pGPP 2019XJTU-Igem parts BBa-K3052010
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FIG.1 p15Aori-pGPP (2019XJTU-China parts BBa-K3052010)
  
 
[[File:XJTU-p2-2.png|400px]]
 
[[File:XJTU-p2-2.png|400px]]
  
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FIG.2 Optimized plasmid 2 (i.e. 421ori-pGPP)
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FIG.2 Replicon optimized plasmid 2 (421ori-pGPP)  
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2022-XJTU-China parts BBa_K4182012
  
  
  
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====2. Principle and process of plasmid 2 construction====  
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=====2.1 mevalonate (MVA) pathway=====
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====Introduction of Plasmid 2 (precursor pGPP synthesis circuit)====  
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1.Mevalonate (MVA) pathway
  
 
[[File:XJTU-p2-3.png|400px]]
 
[[File:XJTU-p2-3.png|400px]]
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[[File:XJTU-p2-4.png|400px]]
 
[[File:XJTU-p2-4.png|400px]]
  
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The MVA pathway mainly exists in eukaryotes. It uses acetyl-CoA as raw material to form IPP and DMAPP through the precursor mevalonate. Then, under the action of polyisoprenyl diphosphate synthase, IPP and DMAPP are further condensed to form geranyl pyrophosphate (GPP). The reaction is also called the cytoplasmic pathway because it occurs in the cytoplasm.
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Fig 3 MVA pathway and constructed gene cluster
  
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According to works of literature, carbon sources like glucose are first metabolized to acetyl-CoA which is then converted into IPP and DMAPP, the two universal precursors of all terpenes. Then IPP and DMAPP will be further converted to geranyl pyrophosphate (GPP) by a geranyl pyrophosphate synthase (GPPS) which is a common precursor of fragrance molecules.  
+
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).  
  
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acetyl-CoA is catalyzed by acetyl-CoA acetyltransferase to produce acetyl-CoA, which is catalyzed by 3-hydroxy-3-methylglutaryl synthase hydroxymethylglutaryl-CoA (HMG-CoA) is formed under the catalysis of coenzyme A synthase. HMG-CoA is then reduced by hydroxymethylglutaryl-CoA reductase (HMGR) to form mevalonate (MVA). mevalonate kinase (MK), phosphomevalonate kinase (MVA), MVA, MVA, HMGR, MVA, MVA, MVA, MVA, MVA, MVA. PMK, mevalonate diphosphate decarboxylase, MPD, and isopentenyl diphosphate isomerase (Idi) catalyze the formation of IPP and DMAPP. Finally, they are further condensed to form geranyl pyrophosphate (GPP).
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2. Promoter optimization to RK2 ori (oriV+trfA)
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atoB（acetyl-CoA acetyltransferase）；
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HMGS（3-hydroxy-3-methylglutaryl coenzyme A synthase）；
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HMGR（3-hydroxy-3-methylglutaryl-coenzyme A reductase）；
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MK（mevalonate kinase）:
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PMK（phosphomevalonate kinase）；
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MPD（mevalonate diphosphate decarboxylase）；
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=====2.2 Promoter optimization oriV+trfA=====
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[[File:XJTU-p2-5.png|400px]]
 
[[File:XJTU-p2-5.png|400px]]
  
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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 from its native BHR promoter. The immediate source of this RK2 origin was pJB785TT and the relevant sequence of this origin was used for formatting the corresponding module. This origin of replication is among the least restrained and it keeps its copy number per cell very low.
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FIG 4 Structure of RK2 ori (换图)
 +
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.
  
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The 421-oripGPP vector is pSEVA421, and the replication start site is from RK2, which belongs to the repeat plasmid. trfA on the vector sequence belongs to the repA gene, and its expressed protein RepA protein can regulate plasmid replication by binding to the repeat sequence near the replication start site. According to the steric hindrance model, the regulation of plasmid copy by RepA depends on the concentration of duplicates. The function of RepA is essentially DNA polymerase, and the first effect of RePA binding to repeats is to initiate plasmid DNA replication. With the replication of the plasmid, the concentration of the repeats increases. The RepA bound to the duplicator interacts with the RepA bound to the other duplicator, coupling two segments of plasmid DNA through the RePA-RePA interaction, forming steric hindrance between the two duplicators and terminating replication initiation.
 
  
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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, we tried to replace the replicating P15Aori (5-10 copies) with the lower copy oriV+trfA (1-3 copies). To reduce the pressure of cell growth and metabolism by reducing the copy number, to improve the protein expression.
+
3. Construction process and verification of plasmid 2
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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.  
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====3. Construction process and verification of plasmid 2====
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Because of the large size of the plasmid and the difficulty of gene manipulation, the Gibson ligation method was used: oriV-trfA PCR was used to obtain the linear pGPP fragment, while the linear PGPP fragment was obtained by enzyme digestion. Restriction enzymes Sac I and Bln I were used to digesting the linearized 12-kb GPP fragment. For the recovery of linear large fragments, we optimized a variety of protocols and tried a new direct purification method using silica gel particles. Compared with the silica gel column, the silica gel particles are uniformly dispersed in the sol buffer system, which can avoid the breakage of DNA fragments caused by excessive shear force on the DNA adsorbed to the silica gel particles. Compared with the conventional silica gel column, the centrifugal speed involved in the operation is also reduced from 12000 rpm to 8000 rpm. Be careful and gentle during operation to avoid damaging the integrity of DNA. GPP fragments were eventually recovered to a lower concentration (4.1 μg/μl) and visualized on agarose gels (Figs. 3-9, GPP).
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[[File:XJTU-p2-6.png|400px]]
 
[[File:XJTU-p2-6.png|400px]]
  
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FIG.3: M, DL10000, 2: oriV+TrfA 3: pGPP linear fragment
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FIG.5: Fragment used for construction of plasmid 2
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The high molecular weight of plasmids was associated with low ligation efficiency and only 6 colonies on two solid media. Monoclonal colonies were selected for colony PCR (Figure 3-11) and 1-4 showed positive results. Four positive Wells were selected and transferred to liquid culture, bacteria were stored, and plasmids were extracted and sent to the company for sequencing. Sequencing results showed that fragments 1 and 4 had been correctly ligated, and strain 4 was stored.
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(1: M, DL10000, 2: oriV+TrfA 3: pGPP linear fragment)
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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.
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[[File:XJTU-p2-7.png|400px]]
 
[[File:XJTU-p2-7.png|400px]]
  
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FIG.4 pGPP plasmid colony PCR. M GL Marker for 5000
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FIG.6 Colony PCR verification of plasmid 2. M, DL5000
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The positive strain 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 replicons, which proved the effectiveness of the strategy. When the metabolic pressure of cells was reduced, cell growth and metabolism were enhanced, and protein expression was also increased. This also facilitates the synthesis of GPP and AA, our final product.
+
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.
  
 
[[File:XJTU-p2-8.png|400px]]
 
[[File:XJTU-p2-8.png|400px]]
  
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FIG.5 RT-qPCR results of GPPS in three strains
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FIG.7 RT-qPCR results of GPPS in three strains
  
 
==References==
 
==References==

Revision as of 03:27, 14 October 2022


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

XJTU-p2-1.png

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

XJTU-p2-2.png

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



Introduction of Plasmid 2 (precursor pGPP synthesis circuit)

1.Mevalonate (MVA) pathway

XJTU-p2-3.png

XJTU-p2-4.png

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)

XJTU-p2-5.png

FIG 4 Structure of RK2 ori (换图) 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.


XJTU-p2-6.png

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.


XJTU-p2-7.png

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.

XJTU-p2-8.png

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.