Composite

Part:BBa_K2918050

Designed by: TUDelft2019   Group: iGEM19_TUDelft   (2019-10-18)
Revision as of 10:59, 19 October 2019 by Weronika77 (Talk | contribs) (→‎Usage and Biology)

Cross-species based iFFL

Genetic implementation of an incoherent feed forward loop (iFFL) in which a stabilized broad host range (PBHR) promoter is controlling GFP expression.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 250
    Illegal XhoI site found at 3383
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
    Illegal AgeI site found at 1277
  • 1000
    COMPATIBLE WITH RFC[1000]

The two transcriptional units in this composite part are oriented outwards.

Usage and Biology

A Incoherent feed-forward loop (iFFL) is a unique control systems motif where the output signal is robust to changes in the input signal. This is achieved by the introduction of a repressor.

  • Figure 1: Overview of incoherent feed-forward loop

iFFL can be applied to genetic circuits to achieve expression independent from copy number, transcriptional and translational rates. To implement the iFFL in a genetic circuit, TALE proteins can be used. These proteins consist of repeats where 12th and 13th amino acids can vary, these are called the repeat variable diresidue (RVD). RVDs have been shown to bind to DNA in a simple one-to-one binding code (Doyle, Stoddard et al., 2013). The direct correspondence between amino acids allows scientists to engineer these repeat regions to target any sequence they want. In our system, we used the TALE protein as a repressor by engineering promoters to contain the binding site of this specific TALE protein (0.1 T7sp1 promoter, 0.5 T7sp1 promoter and PBHRsp1 promoter).
In our genetic circuit, a unrepressed promoter controls the expression of TALE protein while the promoters with the TALE binding sites drive expression of GFP.

insert image of TALE system.

When transcriptional units are placed in series, read through transcription due to low efficiency of terminators can occur. This could influence the behavior of the genetic circuit. Hence, the transcriptional units in the circuit are oriented to achieve insulation from influence of neighboring transcriptional unit.

An interesting application of the iFFL is to achieve controllable gene expression across different bacterial species. Gene expression in different bacterial contexts is influenced by changes in copy number, transcriptional and translational rates. iFFL based on broad host range promoters (PBHR promoter and PBHRsp1 promoter) has been demonstrated below to achieve controllable expression between E.coli and P.putida .

Strain Construction

The DNA sequence of the part was synthesized by IDT with flanking BsaI sites and AATG 3' overhang. The RBS was then cloned along with an altered ribozyme RiboJ in a level 0 MoClo backbone [http://www.addgene.org/47992/ pICH41246] and the sequence was confirmed by sequencing. The cloning protocol can be found in the modular cloning section below. Click here for the detailed protocol.

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

Characterization

References


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Parameters
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