Part:BBa_K4179019

XimD + XimE under T7 promoters + LacI + Lac operator

This composite part comprises of flavin-dependent monooxygenase - XimD (BBa_K4179016) and SnoaL-like cyclase - XimE (BBa_K4179018), under the T7 promoter (BBa_J64997). XimD has (BBa_K2906017) as an RBS while XimE has (BBa_K3875029). XimE is constituvely expressed. Between the promoter and RBS of XimD there is a Lac operator (BBa_K1624002) that makes the expression of XimD to be under the regulation of LacI (BBa_C0012). LacI is cloned under the native pLacI promoter (BBa_K3842018).

Usage and Biology

The purpose of the composite part is to introduce XimD and XimE into E. coli to allow the formation of decursinol (a pyran) from 7-methyldesuberosin. To ensure more of the pyran product (decursinol) in comparison with the furan product (marmesin), the team constrained a surplus of XimE via genetic manipulations.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1772
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1772
  Illegal NheI site found at 1502
  Illegal NheI site found at 1995
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1772
  Illegal BglII site found at 3282
  Illegal BamHI site found at 1457
  Illegal XhoI site found at 1189
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1772
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1772
  Illegal NgoMIV site found at 178
  Illegal NgoMIV site found at 1237
  Illegal AgeI site found at 1751
  
• 1000
  COMPATIBLE WITH RFC[1000]

Quantitative PCR

Following the successful XimD+XimE design cloning process, the final plasmid was transformed into E.coli BL21 DE3 strain. The next step was conducting a qPCR assay for two main purposes:

1) evaluation of XimD and XimE transcription.

2) assessment of our LacI-based regulatory system effect.

Figure 1: (A-C) Amplification curves of XimD, XimE and housekeeping gene transcripts. Five ml of E. coli BL21 cells were allowed to grow over night in the presence (induced) or absence (non-induced) of one mM IPTG, before RNA extraction. RNA was then subjected to one h of DNase (Invitrogen, #AM2238) and converted to cDNA (Invitrogen, #4311235). Finally, samples underwent quantitative PCR with gene-specific primers. IPTG presence resulted in the induction of XimD (A) by 14-fold (D), while XimE transcription remains relatively unchanged (B, D). rssA was used as a housekeeping gene. The threshold for analysis was set at 0.3. Dashed line: Ct value for each sample. NTC: non-template control. The XimD-XimE plasmid was used as a positive control. D. Fold-change of XimD and XimE transcription. The Ct values acquired correspond to the initial total RNA value levels. Hence, the values were normalized to the Ct values of endogenous housekeeping gene, rssA, a known housekeeping gene [1]. This figure was created with the assistance of user-created functions [2,3].

Referring to each one of our set purposes:

1) Our assay confirmed both genes were transcribed.

2) The XimD gene was cloned under LacI regulation. Thus, when introducing IPTG to the system we expected transcription levels to rise. Induction level was revealed to be 14-fold after six h of incubation with a one mM of IPTG (figure 1D). Interestingly, XimE seemed to have also been affected in the IPTG-induced culture, marking a 3-fold increase in transcription (figure 1D). A possible explanation is the proximity of the XimD and XimE genes in our construct. When we introduce IPTG to the system, the Lac repressor detach from the Lac operator regulating the XimD gene [4]. This in turn, permits the recruitment of T7 RNA polymerase to the XimD T7 promoter. Since the XimE promoter is compatible with T7 RNA polymerase as well, the IPTG induction might have caused a spatial proximity of T7 RNA polymerase and the XimE promoter, which increased its transcription.

References

1. Peng, S., Stephan, R., Hummerjohann, J., & Tasara, T. (2014). Evaluation of three reference genes of Escherichia coli for mRNA expression level normalization in view of salt and organic acid stress exposure in food. FEMS microbiology letters, 355(1), 78-82.‏

2. Víctor Martínez-Cagigal (2022). Shaded area error bar plot (https://www.mathworks.com/matlabcentral/fileexchange/58262-shaded-area-error-bar-plot), MATLAB Central File Exchange. Retrieved October 8, 2022.

3. Douglas Schwarz (2022). Fast and Robust Curve Intersections (https://www.mathworks.com/matlabcentral/fileexchange/11837-fast-and-robust-curve-intersections), MATLAB Central File Exchange. Retrieved October 8, 2022.