Part:BBa_K2839000

TALEsp1-Pupsp1 stabilized promoter

1.Short description:

This part consists of a TAL Effector (TALEsp1), a TALEsp1-stabilized promoter (pupsp1) and a fluorescent marker (sfGFP). This part was originally designed by Thomas Hale Segall-Shapiro (Shapiro et al) and iGEM Thessaloniki modified it to exclude 2 PstI recognition sites, thus making it RFC[10] compatible .It can be used to achieve stabilised expression of the sfGFP marker, decoupled from gene/plasmid copy number.

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 programmed/designed to tightly bind to target sites operating as transcription factors by either activating or repressing transcription initiation or elongation. and either activate or repress transcription.(1)

• DNA binding proteins
• Modular Architecture
• Precise Binding with negligible off target effects

3.Usage in our Project:

As a chassis for hosting the system’s devices we use DH5a E.coli cells. 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.

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.

When compared to Part:BBa_K2839014 , Part:BBa_K2839000 has a higher relative promoter strength but due to the strength - error tradeoff has a greater Error.

4.RFC[10] Compatibility, Illegal Sites Removal:

The original sequence by Shapiro et al. contains 2 PstI illegal sites, which we removed in order to make the part RCF[10] compatible. We achieved this by using 2 sets of primers with BsaI site overhangs, each with the function of removing one PstI recognition site.

5.Cloning Strategy:

We cloned the Talesp1 pupsp1 stabilized cassette in plasmid backbones with different ORIs in order to test if the device’s output remains stable when the copy number changes. Specifically, we initially used two sets of standardized primers in order to amplify the Talesp1 pupsp1 stabilized cassette from pTHSSe_59 plasmid and the pSB1c3 backbone. These primers belong to the Metabrick platform and incorporate BsaI restriction sites flanking the amplified products. The amplified products after restriction digestion with BsaI, acquire complementary sticky ends and can be ligated together using Golden Gate Assembly reaction forming the talesp1-1c3 plasmid. Since the assembled device is RFC10 compatible, it can be easily cloned in different backbones.

6.Results and 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.

• Interlaboratory Collaboration Measurement Study:

This part was used as the Test Device 3 in our http://2018.igem.org/Team:Thessaloniki/Collaborations/ Interlaboratory variation Collaboration.

References

[1]Brophy, J. A. N., & Voigt, C. A. (2014). Principles of genetic circuit design. Nature Methods. https://doi.org/10.1038/nmeth.2926

[2]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

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 3677
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

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

  Illegal BsaI site found at 23