Part:BBa_K2839000

TALEsp1-Pupsp1 stabilized promoter

Contents:

1. One Short Description
2. Two Biology and Functionality
3. Tree Usage in our Project
4. Four Illegal Sites Removal
5. Five Cloning Strategy
6. Six Results, Characterization
7. Seven Potential Applications
8. Eight References

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 BBa_K2839014 , 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.

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 protocol and results pages, respectively.

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