Part:BBa_K2839012

TALEsp1 Pupsp1 promoter with translational control

1.Short Description

This part consists of a TAL Effector (TALEsp1), a TALEsp1-stabilized promoter (pupsp1) , 12.1 riboswitch and a fusion fluorescent marker (sfGFP+ 99 nucleotides from luciferace).

This part was designed and submitted by iGEM Thessaloniki, is RFC[10] compatible and can be used to achieve stabilised expression of the sfGFP marker, decoupled from gene/plasmid copy number. It, also, achieves on the fly inducibility of the sfGFP production without the need for promoter and/or RBS replacement. sfGFP is produced only when theophylline is present.

2. Biology and Functionality

Transcription Activator- Like Effectors (TAL Effectors or TALEs) are DNA binding proteins that are naturally expressed by members of the Xanthomonas genus when infecting plants. They contain a central repeat domain which defines their binding to specific promoter sequences in the plant genome, activating transcription and facilitating the bacterial infection.

Because of their modular architecture and their ability to recognise specific promoter sequences, TALEs are widely used in genome editing when precise targeting is required. They can be designed to tightly bind to target sites and either activate or repress transcription.(1)

Aptamers are oligonucleotides or peptide molecules that change their conformation when bound to the target molecule. They can be used for the regulation of gene expression, which becomes dependent of the inducer ’s concentration. We use a Theophylline aptamer which achieves translational control of protein production. Gene expression is off when Theophylline is absent, because the RBS and start codon are hidden inside the aptamer’ s loop. When theophylline is added, the aptamer’s conformation changes, the RBS is revealed and the ribosomes can initiate translation.

3.Usage in our project

• Promoter Stabilization:

Here, we utilize the ability of the TAL effectors to bind to specific promoter sequences and act as repressors by preventing the recruitment of the RNA polymerase in order to create a type I incoherent Feedforward Loop (iFFL) network motif that renders the expression of the downstream sequences independent of the plasmid’s copy number. A promoter under this effect is called a Stabilized Promoter.

Copy number positively affects GOI expression, while its repressor, being under the same positive influence, compensates for this change. If the hill coefficient that characterizes the repression is 1, and thus the repression is perfectly non-cooperative, it results in the disassociation of the GOI’s expression from the plasmid’s copy number.

Each stabilized promoter is characterized by its Strength, the Gene of Interest expression level and Error, the relative change in the Gene of Interest expression as the Copy number increases from the lowest to the highest Copy Number measured. As the expression level of the Repressor increases, the stabilization Error and the promoter’s Strength decrease, leading to weaker but more stable expression across different copy numbers.

This Tale - promoter pair exhibit orthogonal behaviour and the tale repression is characterized by a hill coefficient of 1. Furthermore, TALEsp1 protein does not interfere with the host's native genetic circuitry. It tightly binds to it’s operator and leads to a stable expression of the downstream fluorescent marker decoupled from plasmid copy number.

• Inducibility:

In order to achieve on the fly inducibility of the system, we introduced a theophylline riboswitch, 12.1 Part: BBa_K2839017. We chose the theophylline riboswitch, since it is completely orthogonal and thus provides greater predictability in the outcome of the induction and shows high dynamic range, which is essential in order to allow for more precise control of the expression level.Gene expression is off when Theophylline is absent, because the RBS and start codon are hidden inside the aptamer’ s loop. When theophylline is added, the aptamer’s conformation changes, the RBS is revealed and the ribosomes can initiate translation.Theophylline is a small molecule which is permeable to bacterial cell membranes and has negligible growth inhibition at the concentrations used for our experiments. Furthermore, it is not a component of the widely used liquid mediums and it is neither a precursor in metabolic pathways of bacteria nor a cofactor of enzymes.

4.RFC[10] Compatibility, Illegal Sites Removal

This part is RFC[10] compatible since the sequence was optimized to eliminate illegal restriction sites.

5.Design

Wanting to expand on the TAL Effector stabilized promoters system, we designed a tool that would allow, on top of the stabilization of a promoter,inducible expression depending on the theophylline concentration in the culture. The post and pre-aptamer regions are important for the riboswitch’s functionality and changing these sequences might have varying effects on the behaviour of the riboswitch due to possible changes in the secondary structure of the aptamer. Since we decided to use sfGFP as our fluorescent marker and in order to avoid RNA interactions between the marker and the riboswitch that could deteriorate it’s functionality, we fused the N-terminus of sfGFP with the first 33 amino-acids of luciferase marker. We selected sfGFP over other available marker since its maturation and fluorescence is unaffected by fusion partner misfolding [2].

6. Cloning Strategy

• PCR amplification of Ribo12.1-1c3 plasmid with In1,In2 primers.These primers amplify the riboswitch characterization device and incorporate BsaI sites at the external regions of the amplified device.
• PCR amplification of pTHSSe_59 plasmid with Ri1,Ri2 primers.

These primers amplify the TALEsp1 cassette together with the plasmid backbone and incorporate BsaI restriction sites flanking the amplified sequence.Restriction digestion with BsaI restriction enzyme of the previously amplified sequences, creates sticky complementary ends and thus they can be assembled together.

• Golden Gate assembly reaction between the amplified products from step1 and step2.

This reaction forms the Talesp1-Pupsp1-RIbo12.1-1c3 plasmid containing the theophylline inducible TALE stabilized promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 2734
  Illegal XhoI site found at 3763
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1707
  Illegal AgeI site found at 3181
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 23