This page will provide a complete overview of the 50 parts we have designed and/or used for the project.
1. Basic components
In this project,we used basic components like CRISPR-MAD7 nuclease,RecE/T recombinase,some different promoters,plasmid vectors.
We constructed a single-plasmid system for CRISPR-MAD7 nuclease editing by inserting CRISPR-MAD7 nuclease sequences into different vector skeletons to edit genes in Corynebacterium glutamicum. In addition, we guided CRISPR-MAD7 nuclease to recognize different PAM sites by altering gRNA to improve the editing efficiency of the CRISPR-MAD7(Cas12a) gene editing system in valley rod.
The designed promoter sequence was ligated with CRISPR-MAD7 nuclease to construct an expression vector. The expression vector was transformed into E. coli to amplify the plasmid and verified to be correct by sequencing, and then transformed into Corynebacterium glutamicum as a means of observing the expression effect of the CRISPR-MAD7(Cas12a) system. It was observed whether the promoter could enhance the expression of CRISPR-MAD7(Cas12a) system in Corynebacterium glutamicum.
The recE/T system is a genetic engineering tool used for homologous recombination in bacteria.Its primary role is to facilitate precise genetic modifications by promoting homologous recombination between a target bacterial genome and introduced DNA fragments. To this end, we used the recE/T system to improve the efficiency of gene editing for Corynebacterium glutamicum.
The plasmid vector is a self-replicating DNA molecule that transfers DNA fragments (target genes) to a recipient cell under a genetically engineered recombinant DNA technology.Plasmid vectors introduced the CRISPR components into the cells to ensure that they function properly within the cell. By selecting different plasmid vectors for experiments, the most suitable ones were selected to improve the efficiency of gene editing.
See the following table for specific basic components.
| Graph of parts | Name | Type | Description | Length |
|---|---|---|---|---|
|
BBa_K4687000 | Coding | We used CRISPR-MAD7 nuclease to build our biobricks. | 3792 |
|
BBa_K4687001 | Coding | The recE/T system is a genetic engineering tool used for homologous recombination in bacteria. | 4 |
|
BBa_K4687002 | Regulatory | The designed PlacM promoter sequence was ligated with CRISPR-MAD7 nuclease to construct an expression vector. | 58 |
|
BBa_K4687003 | Regulatory | We ligated the designed Ptrc promoter sequence with CRISPR-MAD7 nuclease to construct an expression vector. | 17 |
|
BBa_K4687004 | Regulatory | We ligated the designed Ptac promoter sequence with CRISPR-MAD7 nuclease to construct an expression vector. | 16 |
|
BBa_K4687005 | Regulatory | We ligated the designed Ptuf promoter sequence with CRISPR-MAD7 nuclease to construct an expression vector. | 21 |
|
BBa_K4687006 | Plasmid | E.coli-Corynebacterium glutamicum shuttle expression vectors based on the medium-copy-no plasmid pGA1. | 4803 |
|
BBa_K4687007 | Plasmid | Pragenuclear plasmid in E. coli. As the cloning vector, which originated from the phage subsp. | 2961 |
|
BBa_K4687008 | Plasmid | E. coli-C. glutamicum shuttle expression vector. | 5758 |
|
BBa_K4687009 | Plasmid | Constitutive expression of FnCpf1 and recT in C. glutamicum. | 10947 |
|
BBa_K4687010 | RNA | gRNA (TTTA(+)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (TTTA(+)) is constructed. | 21 |
|
BBa_K4687011 | RNA | gRNA (TTTC(-)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (TTTC(-)) is constructed. | 21 |
|
BBa_K4687012 | RNA | gRNA (TTTT(-)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (TTTT(-)) is constructed. | 21 |
|
BBa_K4687013 | RNA | gRNA (TTTG(-)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (TTTG(-)) is constructed. | 21 |
|
BBa_K4687014 | RNA | gRNA (TTTC(+)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (TTTC(+)) is constructed. | 21 |
|
BBa_K4687015 | RNA | gRNA (TTTA(-)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (TTTA(-)) is constructed. | 21 |
|
BBa_K4687016 | RNA | gRNA (CTTC(+)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (CTTC(+)) is constructed. | 21 |
|
BBa_K4687017 | RNA | gRNA (CTTA(+)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (CTTA(+)) is constructed. | 21 |
|
BBa_K4687018 | RNA | gRNA (CTTG(-)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (CTTG(-)) is constructed. | 21 |
|
BBa_K4687019 | RNA | gRNA (CTTT(+)) sequences are integrated into expression vectors using primers,then,a plasmid with a specific gRNA (CTTT(+)) is constructed. | 21 |
|
BBa_K4687020 | DNA | We designed donor DNA to interact with CRISPR-MAD7(Cas12a) gene editing systems to achieve targeted gene knockout. | 2000 |
|
BBa_K4687021 | DNA | We designed donor DNA to interact with CRISPR-MAD7(Cas12a) gene editing systems to achieve targeted gene knockout. | 2000 |
|
BBa_K4687022 | DNA | We designed donor DNA to interact with CRISPR-MAD7(Cas12a) gene editing systems to achieve targeted gene knockout. | 1983 |
|
BBa_K4687023 | DNA | We designed donor DNA to interact with CRISPR-MAD7(Cas12a) gene editing systems to achieve targeted gene knockout. | 3983 |
|
BBa_K4687046 | RNA | sgRNA1 sequences are integrated into expression vectors using primers,then,a plasmid with a specific sgRNA is constructed. | 21 |
|
BBa_K4687047 | RNA | sgRNA2 sequences are integrated into expression vectors using primers,then,a plasmid with a specific sgRNA is constructed. | 21 |
|
BBa_K4687048 | Regulatory | We ligated the designed promoter sequence with CRISPR-MAD7 nuclease to construct an expression vector. | 35 |
|
BBa_K4687049 | Terminator | The donor DNA can be expressed correctly so that the gene can be accurately repaired by homologous recombination after being cut during gene editing in Corynebacterium glutamicum, thus surviving. | 86 |
|
BBa_K4687050 | Terminator | In this experiment T2 terminator mainly regulates the expression of CRISPR-MAD7 nuclease. So that CRISPR-MAD7 nuclease can be expressed correctly for gene editing in Corynebacterium glutamicum. | 28 |
2. Composite components
On the premise of basic components,we make composite parts by combining basic parts,in order to simplify the editing process, improve marginal efficiency.After the experiment, according to the experimental results, the best composite parts are selected.Then we used selected composite components to knock out four specific genes in Corynebacterium glutamicum crtI、crtR、crtEb and crtEb/crtR.To increase the yield of lycopene.
See the following table for specific composite components.
| Name | Type | Description | Length | |
|---|---|---|---|---|
|
BBa_K4687024 | Composite | A composite genome editing system like recE/T and CRISPR-MAD7(Cas12a) leverages the strengths of both systems to achieve highly controlled and efficient genetic modifications. | 7221 |
|
BBa_K4687025 | Composite | A recombinant plasmid with pEC-XK99E as the vector backbone, Ptrc as the promoter of CRISPR-MAD7(Cas12a) and recE/T recombination system was constructed by using PCR and one-step cloning method. | 8612 |
|
BBa_K4687026 | Composite | A recombinant plasmid with pEC-XK99E as the vector backbone, PlacM as the promoter of CRISPR-MAD7(Cas12a) and recE/T recombination system was constructed by using PCR and one-step cloning method. | 8667 |
|
BBa_K4687027 | Composite | A recombinant plasmid with pEC-XK99E as the vector backbone, Ptac as the promoter of CRISPR-MAD7(Cas12a) and recE/T recombination system was constructed by using PCR and one-step cloning method. | 8625 |
|
BBa_K4687028 | Composite | A recombinant plasmid with pEC-XK99E as the vector backbone, Ptuf as the promoter of CRISPR-MAD7(Cas12a) and recE/T recombination system was constructed by using PCR and one-step cloning method. | 8630 |
|
BBa_K4687029 | Composite | We combined the recE/T and CRISPR-MAD7(Cas12a) systems.Using this system, we optimized the CRISPR-MAD7 promoter sequence.And we will change the plasmid vector to pBluescript obtain the recombinant plasmid. | 6825 |
|
BBa_K4687030 | Composite | We combined the recE/T and CRISPR-MAD7(Cas12a) systems.Using this system, we optimized the CRISPR-MAD7 promoter sequence.And we will change the plasmid vector to pXMJ19 obtain the recombinant plasmid. | 9622 |
|
BBa_K4687031 | Composite | We combined the recE/T and CRISPR-MAD7(Cas12a) systems.Using this system, we optimized the CRISPR-MAD7 promoter sequence.And we will change the plasmid vector to pJYS1 obtain the recombinant plasmid. | 14811 |
|
BBa_K4687032 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687033 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687034 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687035 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687036 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687037 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687038 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687039 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687040 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687041 | Composite | A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. | 14840 |
|
BBa_K4687042 | Composite | We need to knock out crtEb/crtR, a downstream gene of lycopene in the terpenoid biosynthesis pathway. After cleaving the DNA gap by CRISPR-MAD7 nuclease, the donor DNA is repaired by homologous recombination to achieve the knockout of the targeted gene. | 16848 |
|
BBa_K4687043 | Composite | We need to knock out crtEb/crtR, a downstream gene of lycopene in the terpenoid biosynthesis pathway. After cleaving the DNA gap by CRISPR-MAD7 nuclease, the donor DNA is repaired by homologous recombination to achieve the knockout of the targeted gene. | 16848 |
|
BBa_K4687044 | Composite | We need to knock out crtEb/crtR, a downstream gene of lycopene in the terpenoid biosynthesis pathway. After cleaving the DNA gap by CRISPR-MAD7 nuclease, the donor DNA is repaired by homologous recombination to achieve the knockout of the targeted gene. | 16831 |
|
BBa_K4687045 | Composite | We need to knock out crtEb/crtR, a downstream gene of lycopene in the terpenoid biosynthesis pathway. After cleaving the DNA gap by CRISPR-MAD7 nuclease, the donor DNA is repaired by homologous recombination to achieve the knockout of the targeted gene. | 22260 |
