Part:BBa_K4229034

XiaI with SpyCatcher N-terminal and TnaB under the T7promotor and lacoperator

We had this BioBrick together with BBa_K4229033 on our Plasmid, which was transformed into BL21(DE3) E.Coli. We successfully produced Indigo and Indirubin with our transformed bacteria. The production of the compounds was shown visibly by the colour change of the culture[Fig.1]. After sample preparation, we could also measure the absorbance/fluorescence of both compounds at their respective absorption peaks (see [Fig.2] ) in all our production assays.

NMR and HPLC-followed mass spectrometry measurements of our samples additionally underlined that our synthesised products are Indigo and Indirubin.

The NMR proton-measurement of Indirubin from our bioproduction shows peaks at a shift of 7 – 7.6 ppm (see [Fig.3]), which roughly fits the expected spectrum of Indirubin from the literature [Ref.1]. However, the peaks are too weak to detect any further information. This might be due to too high contamination with other cell substances, as we did not have the equipment to purify them further. The most prominent peak at ~4 ppm is most likely from water, and the peak at ~2.5 ppm from DMSO. To prevent the massive water contamination, we freeze-dried the sample for Indigo before measuring. Another reason for the low signal of Indirubin could be that we had too little Indirubin in our sample.

After the HLPC separation of our Indirubin from our bioproduction, which fell out after ~16 min, the molecular mass could be detected via mass spectrometry ([Fig.4]). The result is as follows: ESI-MS, positive mode: m/z 263.1 (calculated mass for C16H10N2O2 [M + H]+ 263.2). It is likely to be Indirubin, with a literature value for its molecular weight of 262.26 [Ref.1].

[Fig.1]: A: Left Erlenmeyer flasks with 200 mL culture after Indigo-production, right Erlenmeyer flasks with 200 mL culture after Indirubin-production; B: Pellet of 1.5 mL bacterial culture after Indigo-production in 200 mL culture followed by centrifugation; C: Pellet of 1.5 mL bacterial culture after Indirubin-production in 200 mL culture followed by centrifugation. For Indirubin production, L-Cysteine was added to shift the reaction.

[Fig.3]: NMR proton-spectrum of Indirubin from own bioproduction using E. coli BL21 pCDFDuet-1 sTAF-sXTB. Indirubin-producing bacteria was spun down, the pellet was resuspended in KPI-buffer, sonicated, spun down again and resuspended in deuterated DMSO for measurement. B shows a zoomed-in section of A.

[Fig.2]: A: Indigo-concentration measured via fluorescence (excitation 612 nm, emission 670 nm), B: Indirubin-concentration measured via absorbance at 540 nm. Production with 200 mL cultures in 500 mL Erlenmeyer-flasks, at 25 °C after IPTG-induction at OD = 0.8, with L-Tryptophan-concentration of 5 mM. Photometric readout using a plate reader.

[Fig.4]: HPLC-followed mass spectrometry of Indirubin from own bioproduction using E. coli BL21 pCDFDuet-1 sTAF-sXTB. Indirubin-producing bacterial culture was spun down; the pellet was resuspended in KPI buffer, sonicated, spun down again and resuspended in deuterated DMSO for measurement. A: HPLC diagram, B: mass spectrometry diagram.

Through this western blots [Fig.5 and 6] we could successfully prove, that our target enzymes of the Indigo pathway are caught to the T1 protein of the wiffleball. We can see that the band for the T1 protein runs significantly lower if our indigo producing enzymes are not expressed. If mentioned enzymes are present however, they form a complex with the T1 proteins, as a result the band runs higher.

[Fig.5]

[Fig.6]

[Ref.1] PubChem, https://pubchem.ncbi.nlm.nih.gov/compound/10177,Computed by PubChem

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 965
  Illegal AgeI site found at 855
  
• 1000
  COMPATIBLE WITH RFC[1000]