Difference between revisions of "Part:BBa K1598008"

Latest revision as of 17:54, 25 September 2015

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

Sequence and Features

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

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 reduces a plethora of side-effects, such as sedation, memory loss, etc., of various antidepressants through its interactions with receptors in the brain such as GABA (A), and the production of other neuro-active compounds.[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

Pregnenolone is the precursor to all steroids, and the conversion of cholesterol into Pregnenolone is the rate limiting step of the biosynthesis of all steroids (http://www.sciencedirect.com/science/article/pii/S0143416006000856). Hence, this biobrick unlocks steroidogenesis, and paves the way for a innumerable applications in the fields of hormone replacement, fitness, birth control, immunorepression, etc.

(Source:https://en.wikiversity.org/wiki/Wikiversity_Journal_of_Medicine/Diagram_of_the_pathways_of_human_steroidogenesis)

Cloning

PCR Amplification

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

Gel Extraction

After PCR amplification, gel extraction was carried out to ensure that the parts were correct, and to separate them.

Golden Gate Assembly

Golden Gate assembly was carried out using a highly optimized protocol designed by Synthace. SapI as the enzyme used, and the insert was ligated into 4 different backbones: pSB1C3, a custom chloramphenicol backbone optimized by Synthace, pSEVA 251, and pSEVA 441. All the backbones except for pSB1C3 contain a sac B gene which prevented any false positives upon transformation when sugar was added to the agar plates during transformation.

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

Transformation

Transformation was carried out according to standard protocol using competent E. Coli Top 10 cells.

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. The ALA supplement contains heme protein needed by the cells to make CYP11A1, along with iron. Both of these compound are readily available in the gut. No inducer was need as the promoter used was constitutive.

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.

Sample Preparation

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

Characterization

High Performance Liquid Chromatography was used to charachterize the biobrick. This was done by using the HPLC system Series 1200 (Agilent, USA) at 50˚C and a flow rate of 1 ml/min. The stationary phase used was a Symmetry column (Waters, USA) 250 mm × 4.6 mm (with a guard column 20 mm × 3.9 mm) packed with reverse phase ODS (5 µm). The liquid phase used was a solution of 52% acetonitrile, 48% H2O and 0.01% acetic acid. A buffer of 0.01% acetic acid was used for elution.

Peak detection was carried out by UV absorbance at 200 nm. Identification of the peaks and the quantification of pregnenolone was carried out using standard technique.

Results As shown by the image below, pregenolone is seen as a clear peak at around 11mins.
A standard curve of area under the peak vs. concentration of pregnenolone in standard solution was plotted based on the peaks, as shown below.

References were taken for different concentrations of pregnenelone

Results

Therefore, a maximum pregnenolone production of 0.007 mg/ml within 4 hours of substrate addition was observed! This was approximately 17.5 times greater than the amount quantified in past research [4]. Furthermore, this is possibly the first time Human FDX1 gene, Human FDXR gene, and Human CYP11A1 were expressed together in E Coli., as no literature on this particular combination of genes being expressed by E. Coli. was found.

Cell Growth

As illustrated by the graphs above the cell growth rate of the E. Coli with the part in 3 different backbones was studied using OD600 measurements taken every 10 minutes, overnight. This was used to get a better estimate of the best backbone for the part, and as seen in the graphs above, there is no significant difference in cell growth caused by a different backbone.

References

• [1] Reddy DS and Kulkami SK Neurosteroid coadministration prevents development of tolerance and augments recovery from benzodiazepine withdrawal anxiety and hyperactivity in mice. in Methods Find Exp Clin Pharmacol. 1997 Jul-Aug;19(6):395-405
• [2] Marx CE et al. Pregnenolone as a novel therapeutic candidate in schizophrenia: emerging preclinical and clinical evidence. in Neuroscience. 2011 Sep 15;191:78-90. doi: 10.1016/j.neuroscience.2011.06.076. Epub 2011 Jul 1.
• [3] Maurice T et al. Neuroactive neurosteroids as endogenous effectors for the sigma1 (sigma1) receptor: pharmacological evidence and therapeutic opportunities. in Jpn J Pharmacol. 1999 Oct;81(2):125-55.
• [4] Desislava S. Makeeva Functional reconstruction of bovine P450scc steroidogenic system in Escherichia coli in American Journal of Molecular Biology, 2013, 3, 173-182