Part:BBa_K1598008

J23101 promoter + RBS + Human FDXR + RBS + Human FDX1 + RBS + Human CYP11A1 + rrnb double terminator

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 3275
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 565
  Illegal XhoI site found at 1457
  Illegal XhoI site found at 3710
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1653
  Illegal AgeI site found at 1090
  
• 1000
  COMPATIBLE WITH RFC[1000]

Also called Cytochrome P450scc, Cholesterol Side Chain Cleavage Enzyme (P450scc) is a mitochondrial enzyme that converts cholesterol into pregnenolone, which is a neurosteroid as well as the precursor to a variety of steroid hormones and neurotransmitters. The gene that encodes it is CYP11A1. Furthermore, the catalysis involves 3 monooxygenase reactions, which requires two electron transport proteins: ferrodoxin (FDX1) and ferredoxin reductase (FDXR). Our biobrick consists of the iGEM prefix followed by the J23101 constitutive promoter, RBS, FDXR gene, RBS, FDX1 GENE, RBS, CYP11A1 gene, rrnb double terminator, and the iGEM suffix. This will be ligated onto a PSB1C3 backbone.

Emerging preclinical and clinical evidence suggests that pregnenolone may be a promising novel therapeutic candidate in schizophrenia. It has also been shown to reduce the side effects of benzodiazepines. Furthermore, cholesterol side chain cleavage is the 1st step in the biosynthesis of steroids such as testosterone, progesterone, cortisol, estrogen, etc. Therefore, this biobrick has a plethora of applications, and is in a way several biobricks in 1.

Usage and Biology

1. Antidepressant and Anxiolytic Side-Effects Reduction
2. Schizpohrenia Treatment
3. Memory, Cognition, and Neurotransmission
4. Steroid Biosynthesis

Usage and Biology

Relief of Side-effects from Depression/Anxiety drugs:

Pregnenolone and its derivates prevent the development of tolerance, and augment recovery from benzodiazepine withdrawal anxiety and hyperactivity in mice. Benzodiazepines, such as Valium, are one of the most popular antidepressant/anti-anxiety drugs in the world! Basically, this removes the need to constantly increase the dosage, and gets rid of many side effects and withdrawal symptoms, associated with treatments that act on the GABA receptor.[1]

Schizophrenia Treatment

Emerging preclinical and clinical evidence suggests that pregnenolone may be a promising novel therapeutic candidate in schizophrenia. Pregnenolone is a neurosteroid with pleiotropic actions in rodents that include the enhancement of learning and memory, neuritic outgrowth, and myelination. Further, pregnenolone administration results in elevations in downstream neurosteroids such as allopregnanolone, a molecule with neuroprotective effects that also increases neurogenesis, decreases apoptosis and inflammation, modulates the hypothalamic-pituitary-adrenal axis, and markedly increases GABA(A) receptor responses. In addition, pregnenolone administration elevates pregnenolone sulfate, a neurosteroid that positively modulates NMDA receptors. There are thus multiple mechanistic possibilities for pregnenolone as a potential therapeutic agent in schizophrenia, including the amelioration of NMDA receptor hypofunction (via metabolism to pregnenolone sulfate) and the mitigation of GABA dysregulation (via metabolism to allopregnanolone). [2]

Memory, Cognition, and Neurotransmission

Neurosteroids derived from Pregnenolone modulate several neurotransmitter systems such as gamma-aminobutyric acid type A (GABA(A)), N-methyl-D-aspartate (NMDA) and acetylcholine receptors. As physiologic consequences, they are involved in neuronal plasticity, learning and memory processes, aggression and epilepsy, and they modulate the responses to stress, anxiety and depression. There is evidence for a common mechanism of action between neurosteroids and sigma1-receptor ligands and focus on the potential therapeutic interests of such interaction in the physiopathology of learning and memory impairments, stress, depression and neuroprotection. [3]

Steroid Biosynthesis

Cloning

1. PCR Amplification

The concentration of each of the 6 parts was increased using PCR amplification.

2. Gel Extraction



3. Golden Gate Assembly



The insert was ligated into 3 different backbones. The backbones contain a sac B gene which prevented any false positives upon transformation.

4. Transformation

   Transformation was carried out according to the given protocol, with the exception of using competent E. Coli Top 10 cells.

5. Inoculation

   The cells were inoculated in 10mL of LB and left for 4 hours, after which ALA supplement was added to a final concentration of 0.064 mg/mL, and iron (ii) sulfate (FeSO4) was added to give a final concentration of 500 mM. After 20 hours, cholesterol was added to the final concentration 0.5 mM (193 mg/l) in a form of 100-fold concentrates of (i) 2-hydroxypropyl beta cyclodextrin and (ii) 97% DMSO.

6. Sample Preparation

   Liquid-Liquid Extraction using Ethyl Acetate was carried out according to standard protocols 4 hours after incubation with cholesterol.

Characterization

1. HPLC results
1. References were taken for different concentrations of pregnenelone

2. Standard Curve

3. Pregnenolone Results

2. Cell Growth
Photo: Pregnenolone Probiotic Prototype Cell Growth Curve