Part:BBa_K2753018

TALE2 sp1

A member of the pTALE stabilized promoter family, characterized and sequenced by team GreatBay_China 2018.

An Introduction to TALE stabilised promoters

Genetic engineering requires elaborative design and sensible proportion of cellular components. For example, unbalance of synthesis enzymes in metabolic engineering would lead to not only reduced yield due to inefficient conversion, but also unnecessary waste of energy. Synthetic biologists can manipulate gene expression by methods like choosing promoters and RBS of suitable strength, and selecting vector with a desirable range of copy number. As a common engineering tool, plasmids are often assumed to have constant copy number in cells, which is untrue as plasmid copy number is actually subjected to huge variability. Altered growth conditions of the transgenic chassis like new culture medium composition or different temperature, and difference in the host growth phase would hugely affect plasmid copy number. Subsequently, this results in variable gene expression, leading to potential failure to perform the designated functions. Furthermore, even when placed in the genome, the genetic location of synthetic constructs also brings about uncertainty in gene expression: genes with closer proximity to the replication origin exists in larger number than those further away. In many situations a synthetic circuit previously placed on the plasmid needs to be integrated to the genome for preventing discard and improved stability, then a process of re-tuning is often required, which is not only time-consuming but also laborious.

pTALE promoters can achieve independence of expression level to copy number using an incoherent feedforward loop (iFFL) in which transcription-activator-like effectors (TALEs) function as a perfectly non-cooperative negative regulation. While copy number accretes gene expression, it also elevates the repression to the gene expression, thus has canceled out the effect of copy number on expression level. This design can also eliminate the impact of the location of genes (whether they are placed on plasmids or in the genome, and wherein the genome) on gene expression.

The copy number-free promoter family consists of seventeen members: fifteen promoters varying in strength, repressed by two different TALEs, and another two parental parts enabling future teams to build their own pTALE promoters by golden gate assembly as well as to use a “blue-green screening” method to easily distinguish successful constructs. （https://parts.igem.org/Parts:BBa_K2753013) (https://parts.igem.org/Parts:BBa_K2753014)

TALE1:

• pTALE1 sp1 (https://parts.igem.org/Parts:BBa_K2753030)
• pTALE1 sp2 (https://parts.igem.org/Parts:BBa_K2753024)
• pTALE1 sp3 (https://parts.igem.org/Parts:BBa_K2753025)
• pTALE1 sp4 (https://parts.igem.org/Parts:BBa_K2753026)
• pTALE1 sp5 (https://parts.igem.org/Parts:BBa_K2753027)
• pTALE1 sp6 (https://parts.igem.org/Parts:BBa_K2753029)

TALE2:

• pTALE2 sp1(https://parts.igem.org/Parts:BBa_K2753018)
• pTALE2 sp2(https://parts.igem.org/Parts:BBa_K2753019)
• pTALE2 sp3((https://parts.igem.org/Parts:BBa_K2753020)
• pTALE2 sp4((https://parts.igem.org/Parts:BBa_K2753021)
• pTALE2 sp5((https://parts.igem.org/Parts:BBa_K2753022)
• pTALE2 sp6((https://parts.igem.org/Parts:BBa_K2753023)

Biobrick Design

All promoters are placed on the standard biobrick assembly compatible backbone pSB1C3, with a RBS (BBa_B0034) reporter gene sfGFP (BBa_K1679038) downstream of the promoters in be tween the restriction sites of SpeI and PstI. (Figure. 3)

Characterization

In order to verify the stabilization effect of the pTALE promoter family, we assembled all promoter members onto three backbones:

• pUC20: ~500
• pR6K: ~15
• pSC101: ~1

An identical sfGFP is placed downstream of all promoters as a reporter gene. Three constitutive promoters: J23119, J23101, J23105, with the same sfGFP downstream were characterized along with the pTALE promoters as a reference of promoter strength. The green fluoresce was measured by flow cytometry. (see methods in our notebook).

The results indicate that pTALE promoters are able to buffer against the change in plasmid copy number and the location of genes in the organism, maintaining a reasonably constant fluoresce level.

To decide whether pTALE can perform as expected in metabolic pathways, maintaining the pre-set ratio of production enzymes for ensuring optimal flux to the product, we tested pTALE1 sp1 and pTALE2 sp1 by comparison to pTac, a frequently-used IPTG inducible promoter, using an geraniol synthesis operon (https://parts.igem.org/Part:BBa_K2753015) containing a GPPS (https://parts.igem.org/Part:BBa_K2753002) and a GES (https://parts.igem.org/Part:BBa_K2753003). Similar to the characterisation of green fluoresce characterization, three backbones: pUC20, pR6K, pSC101 are used. (Figure. 5)

With pTac promoters, geraniol yield increased with the copy number of the vector, showing positively correlated relation. However, when TALE stabilized promoter (TALEsp) was used, higher the copy number of the vector, lower the production of geraniol, being the opposite of pTac. And the stronger TALEsp promoter, TALE1sp1 gave generally reduced yield compared to its weaker counterpart TALE2sp1. Interestingly, the graphs of the two TALEsp promoters appeared to be seemingly parallel to each other.

We surmised that the yield of geraniol was affected by two factors: the expression level of enzymes and the cellular burden. As for enzyme expression, there exists an optimal gene expression level that produces just enough enzyme to metabolize all the substrate. The yield would be the greatest at this level. But if lower than this level, production would increase with enzyme expression since there is a surplus of substrates. And if the enzyme expression is higher, the more enzymes now becomes a cellular burden as it no longer contributes to more product.

In the case of pTac, it fits into the scenario when the gene expression is lower than the optimum: higher copy produced more enzymes to catalyze geraniol synthesis reaction. As for TALE stabilized promoters, the expression level of enzymes would remain unchanged regardless of copy number due to the stabilization nature of pTALE (LINK more about pTALE). So in low copy vector where the cellular burden isn’t significant, the product yield is only related to the strength of the promoter, explaining why pTALE sp2 has better performance than pTALE sp1 as it’s closer to the optimum. But when the copy number increases, the expression level is unaffected while the cellular burden rises sharply because much more TALE would be made to stabilize expression, leaving less energy available for geraniol synthesis. And it in turns answers why a negative trend of production is shown when regulated by pTALE.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1513
  Illegal NheI site found at 1921
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 113
  Illegal XhoI site found at 2152
  Illegal XhoI site found at 2692
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 747
  Illegal AgeI site found at 849
  Illegal AgeI site found at 1767
  Illegal AgeI site found at 2277
  
• 1000
  COMPATIBLE WITH RFC[1000]