Part:BBa_K822000

lldPRD operon promoter + RBS from E. coli

This is the promoter region for the lldPRD operon (previously named lct) of E. coli. The lldPRD operon encodes a lactate-dehydrogenase, and the lldPRD promoter has been shown to be induced by L-lactate.

The promoter is repressed/activated by the regulatory protein lldR encoded by the operon. In the absence of L-lactate, lldR binds to two operator sites O1 and O2 in the promoter region and inhibits expression. [1]

The activity of the promoter may be repressed by chloramphenicol and nutrient-rich growth environment. [2]

Characterization by ETH iGEM 2015

Check the characterization done by ETH Zurich 2015 iGEM team in the experience page.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 335
  

References

[1]: Dual role of lldR in regulation of the lldPRD operon, involved in L-lactate metabolism in E. coli. Aguilera et al. J. Bacteriol. 2008, 190(8):2997

[2]: Inducible Membrane-bound L-Lactate Dehydrogenase from Escherichia coli - purification and properties. Futai, M. and Kimura, H. Journal of Biological Chemistry. 1977, 252(16):5820-5827.

BBa_k2824006:T7-lldPRD operon promoter-GFP>/h2> <h2> Improve the Characterization of BBa_k82000

In the following figure, we compared the expression of lldPRD operon promoter-GFP and T7-lldPRD operon promoter-GFP. The T7 promoter could enhance gene expression when the concentration of lactic acid was less than 1 m mol/L. Considering that the lactate concentration in yogurt is generally no more than 1m mol/L, the conclusion can given that the T7 promoter can enhance the signal intensity for our experiments (Figure 1). <p> Figure 1:lldPRD operon promoter-GFP vs T7-lldPRD operon promoter-GFP

To see more details about the construction and result, click the hyperlink below: T7-lldPRD operon promoter-GFP composite:BBa_k2824006(https://parts.igem.org/Part:BBa_K2824006).

Improve the Characterization of BBa_k82000

<p> We can easily find that the Lldr-T7-lldPRD operon promoter-GFP part are in a higher level of expression comparing to the lldPRD operon promoter-GFP when the lactate concentration is lower than nearly 1.5 mM. Considering that the lactate concentration in yogurt is generally no more than 1 mM, the conclusion can give that the T7 promoter can enhance the signal intensity for our experiments (Figure 2).

Figure 1:Lldr-T7-lldPRD operon promoter-GFP

Figure 2:lldPRD operon promoter-GFP vs Lldr-T7-lldPRD operon promoter-GFP

To see more details about the construction and result, click the hyperlink below: Lldr-T7-lldPRD operon promoter-GFP composite: BBa_k2824008(https://parts.igem.org/Part:BBa_K2824008)

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Team Colourectal 2022 (Wageningen_UR)

Bas Raats

This contribution adds an alternative promoter that works similarly to lldPRD in different in the presence of glucose or the absence of oxygen.

The lldPRD promoter does not function in the presence of glucose or the absence of oxygen. Unfortunately, both conditions are found in the human colon, meaning that PlldPRD¬ cannot be used to sense lactate in a colonic environment (1). Zúñiga et al. developed a lactate sensitive promoter called ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia) (2). This promoter is sensitive to lactate in the presence of glucose and absence of oxygen, perfect for developing a biosensor to sense colon cancer.

To see morte details about this part please visit BBa_K4244000.

References

1. Schwerdtfeger LA, Nealon NJ, Ryan EP, Tobet SA. Human colon function ex vivo: Dependence on oxygen and sensitivity to antibiotic. PLoS One [Internet]. 2019 May 1 [cited 2022 Sep 30];14(5):e0217170. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217170 2. Zúñiga A, Camacho M, Chang HJ, Fristot E, Mayonove P, Hani EH, et al. Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments. ACS Synth Biol [Internet]. 2021;10(12):3527–36. Available from: https://doi.org/10.1021/acssynbio.1c00456

Additional Information by CUHK-HongKong-SBS 2024

In addition to encoding l-lactate dehydrogenase by the gene LldD, which enables Escherichia coli (E. coli) to utilize l-lactate as a carbon source, the lldPRD operon also encompasses the gene for the permease (LldP). Notably, the operon includes the gene for the transcriptional regulator (LldR), positioned between LldP and LldD (1). The expression of the lldPRD operon is regulated by LldR and ArcA (2).

It is particularly unusual that these three genes exhibit an overlapping arrangement wherein the stop codon of each upstream gene overlaps with the start codon of the downstream gene, resulting in three coding regions situated in three distinct reading frames (1). Subsequent research by Angel-Lerma et al. revealed that the protein levels of LldP, LldR, and LldD are not regulated at the transcriptional level. Instead, the modulation of protein dosage is primarily associated with RNase E-dependent mRNA processing events, which generate two distinct mRNA species from the lldPRD transcript. This processing occurs through endoribonucleolytic cleavage within the lldR mRNA, yielding intact lldP and lldD mRNAs (3).

Consequently, the availability of intact lldR mRNA for translation is significantly diminished, resulting in a marked reduction in the production of LldR. This mechanism ultimately leads to differential segmental stabilities among the cleavage products and variations in the translation efficiencies of the three cistrons encoding LldR, LldD, and LldP (3).

References

1. Dong, J. M., Taylor, J. S., Latour, D. J., Iuchi, S., & Lin, E. C. (1993). Three overlapping lct genes involved in L-lactate utilization by Escherichia coli. Journal of Bacteriology, 175(20), 6671–6678. https://doi.org/10.1128/jb.175.20.6671-6678.1993

2. Aguilera, L., Campos, E., Giménez, R., Badía, J., Aguilar, J., & Baldoma, L. (2008). Dual Role of LldR in Regulation of the lldPRD Operon, Involved in l-Lactate Metabolism in Escherichia coli. Journal of Bacteriology, 190(8), 2997–3005. https://doi.org/10.1128/JB.02013-07

3. Angel-Lerma, L. E., Merino, E., Kwon, O., Medina-Aparicio, L., Hernández-Lucas, I., Alvarez, A. F., & Dimitris Georgellis. (2020). Protein Dosage of the lldPRD Operon Is Correlated with RNase E-Dependent mRNA Processing. Journal of Bacteriology, 203(6). https://doi.org/10.1128/jb.00555-20