Part:BBa_K1598008:Experience

We like to consider this biobrick our little miracle, and have numerous people to thank for it. The idea to express Pregnenolone using E Coli. came to us in the last week of August, a few weeks before wiki freeze. It was a divine commandment that came straight from the most important people in the world, those suffering from conditions like anxiety, schizophrenia, and depression, who were the reason we were doing this project in the first place. In all the interviews we did for the Human Practices aspect of the project, there was one recurring theme- debilitating side-effects.

Whenever we asked any mentally distressed people if they'd consume our psychobiotics instead of the conventional drugs they've been on, they always had one answer. People were willing to take something as novel and as controversial as genetically modified bacteria that make neuroactive compounds only, and only if it was a significant improvement to their current medication. The antidepressants and anxiolytics of today are riddled with problems. The consumer develops a tolerance to them over time, so the dosage has to be constantly increased, resulting in extravagant costs. Withdrawal anxiety worse than any hangover is experienced when the treatment is discontinued, and finally they have crippling side effects which include weight gain, memory loss, sedation, and hyperactivity.

After reading about side-effects for a couple of days, we discovered Pregnenolone, a neurosteroid that reduced he side-effects of various anti-depressants [1]. And thus, on the Monday 3 weeks before wiki freeze began our quest for Pregnenolone. We were fortunate to have the expertise of UCL's very own Dr. Fiona Truscott, who specialized in Cytochrome P450s, such as P450scc which converts cholesterol into Pregnenolone. With her help we designed the pregnenolone synthesis biobrick, but there were some problems. Firstly, for pregnenolone to be synthesized by our probiotic in the gut, our bacteria needed to synthesize its 2 electron transport partners, Ferredoxin and Ferredoxin Reductase, as well. The made our insert a whopping 3910 base pairs long. Putting such a biobrick together seemed like a daunting task for a team that had been struggling with 3A assembly for over 2 months. Moreover, characterizing this biobrick isn't easy. It involves the use of a technique that is dreaded by most of our department: HPLC.

So, it was completely natural for our whole team and its advisors to think that this was a bad idea, which probably wouldn't work in the 3 weeks we had left. But, I was hooked. The more I read about it, the more I was drawn to this part, and the plethora of possibilities. Recent pre-clinical and clinical evidence shows that Pregnenolone could also be used as an adjunctive therapy for schizophrenia- another debilitating mental health condition [2]. Moreover, Pregnenolone is the pre-cursor to all steroids; therefore, this biobrick has innumerable applications and endless potential for further development [3]. So, it was imperative to find a way to put this biobrick together in the limited time we had left.

Just as we were losing hope, the project found an unlikely saviour- Synthace. The synthetic biology start-up believed in our cause and agreed to work with us! Game on. Using Synthace's highly optimized Golden Gate assembly process, which had been developed using Design of Experiments, Automation and the best that synthetic biology can offer, gave us a real shit at this. And, Boom! The whole team was in. However, just as a plan was forming, we realized that we'd run out of all of our free DNA synthesis from IDT. Shattered. Although the team was finally united and believed in the project, our other sponsors didn't, and we can't blame them since it's only sensible. We were told, 'With the best will in the world, it will not be possible for synthetic DNA to be double-checked, ordered, synthesized and delivered to UCL by a service provider, assembled by Synthace and then transformed into E. coli cells whose properties must then be assayed... all in the 10 working days we have left in the competition. In point of fact the DNA synthesis will take longer than 10 working days.'

We were on our own. But, we were the UCL iGEM 2015. We never give up, give in, or back down. In our desperation, we contacted everyone we knew and had collaborated with, looking for someone to synthesize the five parts we needed. We asked all the iGEM teams we knew if they had any free IDT DNA synthesis they could spare us, and found 2 extremely generous donors in the Birkbeck iGEM team and the iGEM team from Manchester-Graz. It was the Friday 2 weeks before wiki-freeze, and 3 parts had been ordered, but we still needed 2 more, and as a last resort, we contacted every iGEM Software team, hoping that they didn't need to synthesize as much as other teams. A few moments before the end of the day, we got the response we were waiting. From half-way across the world, University of Michigan had come to our rescue and we'd finally ordered all the needed DNA! The primers were ordered by a very kind UCL professor, who used his own research grant to help us. But, the struggle had only begun.

A whole week passed, and there was no sign of the of the parts we needed. It was Monday. Yes, the Monday before wiki freeze, when the first 2 of the parts arrived. And just as the whole plan was falling apart, everything fell into place on the next day, thanks to IDT. In those 4 days, we carried out PCR amplification, gel extraction, Golden Gate assembly, cloning, and HPLC in one fell swoop. Thanks to innumerable people, we were able to grab the one chance we had to make and characterize this biobrick, and we did it. The drama, the stress, the 8 hours spent on the HPLC machine on deadline day, the skipped meals- it was all worth it. We'd created something that could really change the lives of people that suffered from mental health conditions.

What makes this part so special is that it truly embodies the spirit of iGEM and is a testament to everything this competition stands for. A bunch of silly, naive, overly ambitious kids managed to pull of an extremely intricate collaboration to make a biobrick that expresses 3 different genes, and is one of the biggest parts in iGEM this year. This was done against all the odds in 4 days. The parts were synthesized by 4 different universities on 2 different continents, assembled with the help of a start-up, and characterized under the supervision of numerous researchers. It captures the essence of iGEM and shows that if the scientific community is united by a cause, it can achieve anything.- Yash Mishra, UCL iGEM 2015

Applications of BBa_K1598008

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)

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.

HPLC reference results

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.

UNIQf92d2e5c5cf90ba1-partinfo-00000006-QINU UNIQf92d2e5c5cf90ba1-partinfo-00000007-QINU

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