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.

7.Results, Characterization:

We measured the fluorescence intensity of the constructs in different plasmid backbones with different copy numbers to investigate their performance in promoter stabilization and its correspondence with expected results.

For the sfGFP fluorescence intensity measurements flow cytometry was our primary measuring method, while a plate reader was also used. All measurements were performed in biological replicates (n=3) and cells were maintained at mid-log phase, unless other stated. Sample preparation, technical details and the raw data can be found in our [http://2018.igem.org/Team:Thessaloniki/Experiments/ protocol] and [http://2018.igem.org/Team:Thessaloniki/Results/ results ] pages, respectively.

• Different Copy number Measurements:

Fluorescence intensity measurements of this construct were conducted in DH5α E. coli cells to determine the functionality of promoter stabilization over different copy numbers.

The construct was inserted in plasmid backbones with different ORIs (psc101,p15A, pUC19-derived pMB1) and transformed into DH5a cells. After that, colony PCR was performed in order to identify the colonies with the correct insert. Single colonies were picked and prepared to be measured.

• Flow cytometry measurements:

Sample preparation

In order to prepare the cultures for flow cytometry measurements we followed the protocol created by Adam Mayer et al [1]. In particular the correct colonies were inoculated into 1 ml Lb + antibiotics and grown overnight at 37 °C in a shaking incubator adjusted to 250 rpm.The overnight growths were diluted 1:200 into 1 ml LB + antibiotics and grown at 37 °C into shaking incubator .After 2 hours the growths were diluted 1:500 into prewarmed LB + antibiotics + inducer where necessary and grown at 37 °C, 250 rpm for 5 hours.After growth, 20 μl of culture sample was diluted into 180 μl PBS + 200 μg/ml kanamycin to inhibit translation. The samples were stored at 4°C for 1 hour and then measurements were performed using the CyFlow Cube8 Sysmex Partec Flow Cytometer.

Flow Cytometry Results

Fig1: TALE1sp1 Pupsp1, TALEsp2 Pupsp2, non stabilized constitutive pT7A1w1 promoter flowcytometry fluorescence measurements at three different copy numbers. Error bars represent standard deviation from three biological replicates.

• Plate Reader Measurements

For the plate reader measurements and sample preparation we followed the the iGEM 2018 InterLab Study Protocol.

Fig2: TALE1sp1 Pupsp1, TALEsp2 Pupsp2, non stabilized constitutive pT7A1w1 promoter Plate Reader fluorescence measurements at three different copy numbers. Error bars represent standard deviation from three biological replicates.

The results demonstrate that sfGFP expression level, under the control of TALEsp1-Pupsp1 stabilized promoter (BBa_K2839000) and TALEsp2-Pupsp2 stabilized promoter (BBa_K2839014), does not significantly change when expressed from vectors with different copy number. Whereas, sfGFP expression driven from a non stabilized constitutive promoter changes when different copy number plasmids are used for its expression.

We measured the fluorescence intensity of the constructs in pSB1C3 backbone to investigate the riboswitch performance and its correspondence with expected results.

For the sfGFP fluorescence intensity measurements flow cytometry was our measuring method. All measurements were performed in biological replicates (n=3) and cells were maintained at mid-log phase, unless other stated. Sample preparation, technical details and the raw data can be found in our protocol and results pages, respectively

• Flow Cytometry Results

Fig 1: sfGFP expression from TALEsp1 Pupsp1 promoter, induced by different theophylline concentrations. Error bars represent standard deviation from three biological replicates.

8.References:

[1]Segall-Shapiro, T. H., Sontag, E. D., & Voigt, C. A. (2018). Engineered promoters enable constant gene expression at any copy number in bacteria. Nature Biotechnology. https://doi.org/10.1038/nbt.4111

[2] Pédelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology. https://doi.org/10.1038/nbt1172

[3]A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Research, 37(1), 184–192. https://doi.org/10.1093/nar/gkn924

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