Team:TUDelft/Part Collection

PHOCUS

Parts

To construct our parts, we used different approaches. Some were synthesised by IDT, which allowed us to obtain sequences codon-optimized for Escherichia coli (our host strain). Others were obtained by PCR amplification from the genomes of Bacillus subtilis or Escherichia coli BL21 (DE3) and assembled in the desired backbone from the BglBrick collection [1] by Gibson Assembly. Additionally, some were ordered from Twist Bioscience already cloned in a vector ready for expression.

Basic parts


Favourite
part 		Name Type Description
BBa_K3407000 Coding Reversible nonoxidative vanillate / 4-hydroxybenzoate decarboxylase from Bacillus subtilis (bsdBCD)
BBa_K3407001 Coding Cry7Ca1 toxin from Bacillus thuringiensis strain BTH-13
BBa_K3407002 Coding Mini-endoribonuclease 3 (Mini-3) from Bacillus subtilis
BBa_K3407003 Coding YmdB from Escherichia coli BL21 (DE3)
BBa_K3407004 Coding Fox-1 RNA Binding domain (RBD)
BBa_K3407005 Coding Fox-1 RNA Binding domain (RBD) with mutations F126A and F160A
BBa_K3407006 Coding shRNA targeting eGFP(del6)
BBa_K3407007 Coding shRNA targeting eGFP(del6), mutated
BBa_K3407008 DNA sgRNA targeting T7 gene 4.3
BBa_K3407013 DNA T7 truncated promoter

Favourite basic part

Our favourite basic part is BBa_K3407004, which encodes for the Fox-1 RNA Binding domain (RBD). This part leads to a functional expressed product that successfully binds to a specific sequence in the loop of our designed shRNA and can therefore be used to transport it to its target location. Fox-1 RBD has been designed to be used for in vitro assays by fusing a His-Tag 6x to its N-terminal, facilitating purification with simple and economic Nickel-IMAC (Immobilised Metal Affinity Chromatography) or other Ni-related techniques such as beads. We believe this part is a very versatile BioBrick, as any future projects seeking a strong and specific interaction between protein and ssRNA can potentially use it. Future iGEM teams working with RNA can make use of it to create fusion proteins involved in RNA modification, detection, translation, recognition or delivery, where a strong and specific interaction is needed. As seen in our results, this protein domain is soluble, not leading to inclusion bodies even when expressed under strong T7 transcription. Furthermore, it is small, requiring little resources per active unit, which might reduce problems related to cell burden. Its reduced size and increased solubility further supports its application as parts of other phusion proteins, as longer amino acid sequences are thought to increase the chances of misfolding.


Composite parts


Favourite
part 		Name Type Description
BBa_K3407016 Composite T7p - RBD - bsdBCD - T7t
BBa_K3407017 Composite T7p - RBD - Cry7Ca1 toxin (from Bacillus thuringiensis strain BTH-13) - T7t
BBa_K3407018 Composite araBAD - RBD - Mini-endoribonuclease 3 (Mini-3) from Bacillus subtilis - T1t
BBa_K3407019 Composite tetA/tetR - RBD - YmdB from Escherichia coli BL21 (DE3) - T1t
BBa_K3407020 Composite T7p - Fox-1 RNA Binding domain (RBD) - T1t
BBa_K3407022 Composite T7p - shRNA targeting eGFP(del6)
BBa_K3407023 Composite T7p - targeting eGFP(del6), mutated
BBa_K3407024 Composite T7p - RBD - Fox-1 RNA Binding domain (RBD) with mutations F126A and F160A - T1t
BBa_K3407025 Composite sgRNA targetting T7 gene 4.3

Favourite composite part

Our favourite composite part is BBa_K3407022, as it allows the expression of a designed shRNA under a viral T7 promoter. It opens the possibility for future iGEM teams to easily build any short hairpins for RNAi-related assays, rendering it a BioBrick of simple expression with high versatility. The loop allows processing by Dicer-2 from D. melanogaster while giving a novel functionality allowing protein-RNA interaction (Fox-1 RBD - loop interaction).


Part collection

Our part collection has been designed to allow the successful in vivo production and delivery of short-hairpin RNAs (shRNAs) to induce gene silencing by the RNA interference (RNAi). RNAi is a biological response against double-stranded RNA (dsRNA) that is triggered when dsRNA is present in the cytoplasm of most eukaryotic organisms, leading to gene silencing [2].

shRNAs are single RNA molecules with a strong, self-assembling secondary structure consisting of a double-stranded RNA (dsRNA) region and a single-stranded RNA (ssRNA) loop. They represent a dsRNA precursor that will be processed by Dicer in the first step of the RNAi pathway, and their expression allows transient or stable gene silencing. shRNAs are simple to transcribe, self-assemble and they are stable [3]. Furthermore, shRNAs are more specific than longer dsRNA sequences, as the expected cleavage products are predictable thus providing control in minimizing off-target effects.

In this collection, future iGEM teams can find the components that are needed to express shRNAs both in vitro and in vivo. It also includes proteins that can additionally be expressed to transport these shRNAs to their target and protect them from degradation by RNAses, thus enhancing their silencing effect. Furthermore, our design could allow expression of multiple shRNAs from a single transcript, which opens the possibility to target specifically one gene by using different shRNAs, or even targeting multiple genes at the same time. The components of our collection are the following:


Short-hairpin RNAs (shRNAs) and Fox-1 RNA Binding Domain (RBD)

shRNAs consist of two palindromic sequences containing the mRNA target sequence, that self-assemble into double-stranded RNA (dsRNA) upon transcription. We have designed a shRNA that contains 27 nucleotides of dsRNA as well as a single-strand RNA (ssRNA) loop, which contains a specific sequence that is recognized by Fox-1 RNA binding domain (RBD). Binding of Fox-1 RBD to the shRNA can allow its transport to the target cell, by for example fusing this Fox-1 RBD to another protein such as a cell-penetrating peptide (CPP). This will facilitate the entrance of the designed shRNA to the target cell where the RNAi response will take place.


YmdB, an RNAse inhibitor for the expression of shRNA in vivo

shRNA is susceptible to be degraded by the bacteria RNAses when produced in vivo. In order to circumvent this problem, the RNAse activity can be reduced by expression of RNAse inhibitors. An example is YmdB, an RNAseIII inhibitor from E. coli [4]. Therefore, this inhibitor would be co-expressed together with the dsRNA molecule.


Mini-3, a specific endoribonuclease for production of multiple shRNAs from a single transcript

Production of multiple shRNAs could be achieved by the production of a tandem shRNA (tshRNA), consisting of multiple shRNAs produced in a single transcript. Mini-3, is an endoribonuclease that would cleave the shRNAs out of the transcript recognizing “ACCU'' sequence at the base of each hairpin. After cleavage, Mini-3 produces a GG 3’ overhang, which is used by Dicer to cut the shRNA into small interfering RNAs (siRNA). This overhang has been also added in our designed shRNAs. These siRNAs will target and subsequently cleave the complementary mRNAs, causing gene silencing [5].


Favourite
part 		Name Type Description
BBa_K3407002 Coding Mini-endoribonuclease 3 (Mini-3) from Bacillus subtilis
BBa_K3407003 Coding YmdB from Escherichia coli BL21 (DE3)
BBa_K3407004 Coding Fox-1 RNA Binding domain (RBD)
BBa_K3407005 Coding Fox-1 RNA Binding domain (RBD) with mutations F126A and F160A
BBa_K3407006 Coding shRNA targeting eGFP(del6)
BBa_K3407007 Coding shRNA targeting eGFP(del6), mutated
BBa_K3407018 Composite araBAD - RBD - Mini3 - T1t
BBa_K3407019 Composite tetA/tetR - RBD - YmdB - T1t
BBa_K3407020 Composite T7p - RBD - Fox-1 RBD - T1t
BBa_K3407022 Composite T7p - shRNA* - eGFP
BBa_K3407023 Composite T7p - RBD - Fox-1 RBD* - T1t
BBa_K3407024 Composite pKDsgRNA_4.3