Part:BBa_K2918006
Medium T7 promoter
A T7 promoter with a strength that should give 50% of normal expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The T7 promoter variant was obtained by performing biased randomization of the wild type T7 promoter. The promoter variant contains a two nucleotide substitutions compared to the wild type promoter.
Strain Construction
Aim: To clone the promoter in a level 0 MoClo backbone (pICH41233).
Procedure: The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible promoter overhangs. The cloning protocol can be found in the MoClo section below.
Modular Cloning
Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments. The MoClo system consists of Type IIS restriction enzymes that cleave DNA 4 to 8 base pairs away from the recognition sites. Cleavage outside of the recognition site allows for customization of the overhangs generated. The MoClo system is hierarchical. First, basic parts (promoters, UTRs, CDS and terminators) are assembled in level 0 plasmids in the kit. In a single reaction, the individual parts can be assembled into vectors containing transcriptional units (level 1). Furthermore, MoClo allows for directional assembly of multiple transcriptional units. Successful assembly of constructs using MoClo can be confirmed by visual readouts (blue/white or red/white screening). For the protocol, you can find it here.
Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2. The complete sequence of our parts including backbone can be found here.
Table 1: Overview of different level in MoClo
Level | Basic/Composite | Type | Enzyme |
---|---|---|---|
Level 0 | Basic | Promoters, 5’ UTR, CDS and terminators | BpiI |
Level 1 | Composite | Transcriptional units | BsaI |
Level 2/M/P | Composite | Multiple transcriptional units | BpiI |
For synthesizing basic parts, the part of interest should be flanked by a BpiI site and its specific type overhang. These parts can then be cloned into the respective level 0 MoClo parts. For level 1, where individual transcriptional units are cloned, the overhangs come from the backbone you choose. The restriction sites for level 1 are BsaI. However, any type IIS restriction enzyme could be used.
Table 2: Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts
Basic Part | Sequence 5' End | Sequence 3' End | Level 0 backbone |
---|---|---|---|
Promoter | NNNN GAAGAC NN GGAG | TACT NN GTCTTC NNNN | pICH41233 |
5’ UTR | NNNN GAAGAC NN TACT | AATG NN GTCTTC NNNN | pICH41246 |
CDS | NNNN GAAGAC NN AATG | GCTT NN GTCTTC NNNN | pICH41308 |
Terminator | NNNN GAAGAC NN GCTT | CGCT NN GTCTTC NNNN | pICH41276 |
None |