Part:BBa_K5321000

Thrombin_HD22_29mer

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
  COMPATIBLE WITH RFC[1000]

Usage and Biology

In order to perform our proof of concept of our project, we choose thrombin as a model to mimic disease biomarkers, and its aptamers reported previously. Thrombin_HD22_29mer (DNA ligand 60-18[29]) is an aptamer originally characterized by Tasset et al. in 1997. It specifically targets the heparin-binding site of thrombin through hydrophobic interactions of the duplex.

Fig. 1 shows the interaction of Thrombin_HD22_29mer with human thrombin.

Figure 1 | The interaction of the Thrombin_HD22_29mer with human thrombin. (a) the site of crosslinking between thrombin_HD22_29mer and thrombin. The crosslinked tryptic peptide of thrombin is shown in boldface letters, and the arrow denotes the crosslink between T12 within the G-quadruplex of the DNA and Phe245 of thrombin. (b) the ribbon diagram of human thrombin, shown in purple, interacted with Thrombin_HD22_29mer in blue.

Characterization

We chose to use hybrid promoter + strong RBS + asd to characterize the strength of the promoter. This sequence was cloned on a low-copy spectinomycin-resistant plasmid pJUMP41-2A(sfGFP) (BBa_J428365) and tranformed into EcN △asd. Absence of the asd gene is lethal unless DAP is added exogenously. Our design ensures that the bacteria can only survive if the promoter initiates transcription. Thus, promoter strength was quantified as OD_600nm. Next are the specific characterization steps:

The overnight-cultured bacterial (EcN △asd) suspension, which transformed with the corresponding plasmid, was centrifuged at 9800g for 2 min. The supernatant was then discarded and the pellet was resuspended in sterile PBS. Repeat this operation twice to completely wash away the culture broth.

The last suspension was serially diluted in sterile PBS to reach OD_600nm = 0.3. Subsequently, it was inoculated into pre-prepared LB broth containing different sodium lactate (0, 0.1 mM, 1 mM, 10 mM) at a 1% inoculation rate. Spectinomycin (50 μg/ml) was added into the broth to maintain plasmid expression. Then all the culture grew separately under normoxic and anaerobic conditions for 8 - 16 h. Each group had 3 biological replicates. BBa_K1847008, a lactate-sensing promoter, was used for quality control. The result was shown as follows:

Figure 3 | Characterization results of lldRO1-PepT-lldRO2 (NS-WA). We defined the intensity (expressed as OD_600nm) under normoxic and lactic acid-free conditions as 1, and the relative values under other conditions were obtained by division. N = 3 biological replicates.

References

1. Aguilera, L. et al. Dual Role of LldR in Regulation of the lldPRD Operon, Involved in l-Lactate Metabolism in Escherichia coli. Journal of Bacteriology 190, 2997–3005 (2008).
2. Mengesha, A. et al. Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated salmonella. Cancer Biology & Therapy 5, 1120–1128 (2006).