Difference between revisions of "Part:BBa K5321000"
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'''Figure 1 | The theoretical model of hybrid promoter lldRO1-PepT-lldRO2.''' When lactate is low, lldR acts as an inhibitory protein and inhibits promoter expression by binding to the O1 and O2 sites. When the lactate concentration rises, lactic acid molecules trigger an allosteric reaction by binding to the lldR protein, making the inhibitory effect disappear. pPepT is a characteristic hypoxia-induced promoter in ''E. coli'' Nissle 1917 (EcN), which is regulated by the Fumarate and Nitrate Reduction regulator (FNR). See BBa_K4713111 for more information about pPepT.<br>
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'''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.<br>
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Before constructing the hybrid promoter, we imagined two failure situations ('''Fig. 2a'''): first, the lldR protein binding at the O1 site can cover up the FNR binding site of PepT, so that the FNR protein cannot bind to the FNR-box to initiate downstream transcription due to steric hindrance (condition 1); second, as the distance between promoters O1 and O2 lengthens (pPepT is 19 bp longer than the original promoter), the active lldR protein bound to the O1 site cannot activate transcription (condition 2).
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Therefore, we constructed 6 versions of the plldR operon through shortening or lengthening the distance between O1 and O2. Combined with 4 variants of pPepT ('''BBa_K4713111 to BBa_K4713114'''), a total of 24 candidates was constructed ('''Fig. 2b'''). 14 candidates have been successfully characterized, uploaded as '''BBa_K4713001 to BBa_K4713014'''. <b><font color=#FF0000>Among them, we recommended '''BBa_K4713008''' as the prior part to be engineered.</font></b> We wish future teams could recharacterize our parts and share their experiences.
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'''Figure 2 | Rational design of hybrid promoters.''' Our hybrid promoter containing two promoters. (a) According to the principle of two promoters, we may encounter two problems when combining. The first one is that FNR protein cannot bind normally due to steric hindrance effect. The second is that the distance between the promoter and O1 will affect the intensity of induced activation. (b) Based on this, we designed a total of 24 versions of promoters. <br>
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You may visit Parts '''BBa_K4713111 to BBa_K4713114''' to learn about the different sequences of mutated pPepT, our engineered hypoxia promoter.
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lldRO1-PepT-lldRO2 (NS-WA) is Hypoxia-lactate-induced hybrid Promoter Type 1. This sequence is constructed based on variant wild-type pPepT(BBa_K4713111). '''There are 128 binary pairs between two LldR binding sites and there are 54 binary pairs between O1 and pPepT.'''
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===Characterization===
 
===Characterization===

Revision as of 12:19, 20 September 2024

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 OD600nm. 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 OD600nm = 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 OD600nm) 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).