Part:BBa_K4059037:Design

PsiD, PsiK, and PsiM, E. coli K12 codon optimized, with LacI

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 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 1709
  Illegal BsaI.rc site found at 3372
  Illegal SapI site found at 3978
  

Design Notes

After successful transformation of pSB1K3-LacI-T7lac-PsiDKM into E. Coli ER2566, the transformant was grown to OD600 = 0,3 in LB-media with kanamycin (5 0μg/mL). At OD600 = 0,3 the culture was induced with both 0,1mM and 1mM IPTG. Each hour 5mL culture was harvested. The experiment was continued for 3 hours. By inducing with IPTG we were able to test the enzymatic activity of PsiDKM using real-time qPCR.

Prior to using real-time qPCR, the RNA was purified. The RNA purification was confirmed on a 1% agarose gel.

After successful cDNA synthesis real-time qPCR were used to analyze the activity of PsiDKM after inducing with IPTG.

[1] Link to our results, go to https://2021.igem.org/Team:SDU-Denmark/Results

qPCR analysis of PsiDKM: Gene expression of the enzymes PsiD, PsiK and PsiM and the T7 promoter. After inducing with 1mM IPTG it is confirmed that the enzymes and T7 promoter generally increase in gene expression, confirming the function of the inducible system. However, it is seen that the gene expression for all enzymes and T7 decreases after 3 hours, which contrasts with what is expected. An explanation for the decrease is that the cells might be stressed after induction of proteins, which could affect the gene expression. The optimal gene expression is therefore seen after either 1- or 2 hours.

Detection for psilocybin

Colour reaction:To test the initial production experiments with only PsiDKM, we performed a colour reaction using the Ehrlich reagent. This is a compound known for its ability to bind indoles, which is a functional group that characterizes psilocybin. Binding to different indoles results in different colours.1 However, every single intermediate of the pathway including the precursor 4-hydroxyindole consist of an indole. In addition, indoles are found in many naturally occurring indoles such as amino acids, hereunder tryptophan. As the first samples were not divided up between cell and media, a huge uncertainty is related to using this method. However, when conducting this test, we observed a distinct variation between our transformants and the control (normal ER2566). Both groups had undergone the exact same treatment (addition of 4-hydroxyindole, methionine, serine and induction with IPTG), which is why we found the outcome intriguing.

[2] Link to our results, go to https://2021.igem.org/Team:SDU-Denmark/Results

LC/MS:To test whether the PsiDKM construct was able to produce psilocybin or not, 4-hydroxyindol was added to cultures containing our plasmid with PsiDKM. These cultures were separated into media and cells. These samples were freeze dried and resuspended in a solution containing 50% acetonitrile and 1% formic acid. This was the analyzed via Triple Quad Liquid Chromatography-Mass Spectrometry. Fragment ions as well as standard curve used for quantification are described in detail in the LC/MS section within PsiH (indsæt link her til PsiH).

Both fragment ions are present in all three PsiDKM samples, this is visualized on the figure below, indicating that the system can produce psilocybin when 4-hydroxy indole is added. Two out of three can be quantified using the standard curve, these calculations are shown below. These indicate that PsiDKM system #1 can produce 0,65ng/mL psilocybin and PsiDKM system #2 is able to produce 2,4ng/mL psilocybin

[3]

LC/MS of PsiDKM: Arbitrary areas of three different PsiDKM samples of fragtment ions m/z=204 and m/z=160.

To read more about the results, go to https://2021.igem.org/Team:SDU-Denmark/Results

Source

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