Team:BUCT-China/Basic Part
According to the requirements and regulations of the basic parts, I will split our glyoxylic acid operon and fatty acid operon(composite parts) into 11 basic parts (BBa_K2782000~BBa_K2782011). Below I will introduce these basic accessories one by one:
This sequence is responsible for binding to the repressor protein, preventing the slip of the RNA polymerase, thereby preventing transcription, and is a composite part (the core part of BBa_K27820011).
Source:
- Escherichia coli strain C600 chromosome, complete genome.
- Sequence ID: CP031214.1
Design Notes ：
- Add a restriction site at the necessary position.
This sequence is responsible for the synthesis of a repressor protein specific to the glyoxylate operon after transcription and translation, thereby binding to a specific site, thereby preventing the relevant transcription from proceeding, and is a composite part (part of BBa_K2782011).
Source:
- Escherichia coli strain C600 chromosome, complete genome.
- Sequence ID: CP031214.1
Design Notes ：
- Add a restriction site at the necessary position.
This sequence is a glyoxylate operon sigma factor binding site sequence, a promoter core sequence, and an RNA polymerase binding site, which is responsible for binding to the sigma factor at the time of transcription, thereby starting transcription, and is also an RNA polymerase binding site, responsible for Binding to RNA polymerase begins transcription.
Source:
- Escherichia coli strain C600 chromosome, complete genome
- Sequence ID: CP031214.1
Design Notes ：
- Add a restriction site at the necessary position.
This sequence is the ribosome binding site on the glyoxylate operon and is responsible for binding to the ribosome to initiate transcription.
Source:
- Escherichia coli strain Es_ST2350_SE1_NDM_03_2018 chromosome, complete genome
- Sequence ID: CP031321.1
Design Notes ：
- Add a restriction site at the necessary position.
Responsible for recording and showing whether transcription is carried out to monitor whether the glyoxylate operon works
Source:
- Synthetic construct clone pEY-sRFP-A, complete sequence
- Sequence ID: KX682231.1
Design Notes ：
- Add a restriction site at the necessary position.
- BBa_K2782000:Ribosome binding site preceding the repressor protein sequence of the fatty acid operon
As a ribosome binding site, after the transcription is completed, the 16SrRNA gene can be accurately recognized by the base complementation, thereby starting the subsequent translation work. This ribosome binding site serves as a ribosome binding site prior to the repressor sequence of the fatty acid operon.
Source：
- Integration expression vector pSe1Bb1s-gfp, complete sequence
- Sequence ID: KJ814971.1
Design Notes ：
- Note the distance between the control RBS and the start codon ATG.
- Retain the consensus sequence of the ribosome binding site - AGGAGG-
The sequence-expressing repressor recognizes a specific operator (the operon is the repressor binding site), and when the manipulation sequence binds to the repressor, it blocks the binding of the RNA polymerase to the promoter sequence, preventing RNA polymerase from following the DNA moves forward, preventing transcription mediated negative regulation. When a certain amount of fatty acid is present, it can bind to the repressor protein, and the binding of the repressor protein to the operon fails, and the RNA polymerase binds to the promoter sequence and moves forward along the DNA, after which the gene begins to express.
Source：
- Escherichia coli strain C600 chromosome, complete genome
- Select seq CP031214.1
Design Notes ：
- The base sequence is edited based on the degeneracy of the protein.
- Amino acids associated with binding sites and activities cannot be altered.
We are proposing a newly designed fatty acid opperon acts as a molecular switch that signals fatty acids to regulate the expression of subsequent genes. It can act as a molecular switch with fatty acids as a signal to regulate the expression of subsequent genes. In combination with a repressor protein, RNA polymerase cannot Bind to the promoter sequence, or RNA polymerase cannot move forward along the DNA, repress transcrip .An appropriate amount of fatty acid is added to block the binding of the protein to the fatty acid opperon. The gene can be expressed after the repressor protein leave fatty acid opperon .
Source：
- Escherichia coli strain C600 chromosome, complete genome
- Select seq CP031214.1
Design Notes ：
- The base sequence is edited based on the degeneracy of the protein.
- Amino acids associated with binding sites and activities cannot be altered.
As a ribosome binding site, after transcription is completed, the 16SrRNA gene can be accurately recognized by base complementation, and the subsequent fluorescent protein translation work begins.
Source：
- PREDICTED: Apteryx rowi REV3 like, DNA directed polymerase zeta catalytic subunit (REV3L), transcript variant X1, mRNA S
- equence ID: XM_026065528.1
Design Notes ：
- The base sequence is edited based on the degeneracy of the protein.
- Amino acids associated with binding sites and activities cannot be altered.
The fluorescent protein family is a homologous protein with a relative molecular mass of 20,000 to 30,000 found in the genus and corals. Red fluorescent protein (RFP) is a photoprotein (drFP583 or DsRed) isolated from coral (Discosoma sp.) that emits bright red fluorescence. These commonly used reporter genes can also be used in combination to detect the expression of two or even three genes. The choice of reporter gene depends on its sensitivity, reliability, and dynamic range of monitoring. The well-stabilized reporter gene is suitable for gene transcriptional kinetics research and high-throughput screening, especially for qualitative research of gene transfer.
Source：
- Synthetic construct clone pEY-sRFP-A, complete sequence
- Sequence ID: KX682231.1
Design Notes ：
- The base sequence is edited based on the degeneracy of the protein，and the primary structure of the protein is not changed as much as possible.
- Amino acids associated with binding sites and activities cannot be altered
A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription, and it contains a conserved sequence required for RNA polymerase specific binding and transcription initiation, and the promoter itself is not transcribed. The promoter is typically located upstream of the transcription start site. The promoter part we submitted is after the fatty acid operon. When the signaling molecule fatty acid appears, it binds to the repressor protein, causing the repressor to detach from the operon. The sigma factor and RNA polymerase can recognize the position on the promoter. Point to start transcription.
Source：
- Cloning vector pTHSSe_71, complete sequence
- Sequence ID: MH029788.1
Design Notes ：
- Information on the promoter of a homologous gene can be used as a reference
