Difference between revisions of "Team:UCL/Sensors"

Background

In order to target mental health issues such as depression and anxiety in a responsive manner it is necessary to design sensors that respond only when triggered by specific biomarkers. This means that mood sensing probiotics designed by our team will only release neuroactive compounds when a patient actually goes through clinical depression or a panic attack instead of being constitutively active. This is achieved by transforming bacteria with plasmids with signal sensing promoters upstream of the effector genes.

Infamous Insidious Inflammation

Clinical depression is likely caused by a chronic low grade-response to inflammation [Link] M. Berk et al. So depression is an inflammatory disease, but where does the inflammation come from? in European Journal for Medical Research, 2013 [1]. Although the pathway from inflammation to depression is complex and not fully understood it has been shown that the immune response is often accompanied by symptoms such as oxidative and nitrosative stress in the gut. [Link] Leonard B. and Maes M. Mechanistic explanations how cell-mediated immune activation, inflammation and oxidative and nitrosative stress pathways and their sequels and concomitants play a role in the pathophysiology of unipolar depression in Neuroscience & Biobehavioral Reviews Volume 36, Issue 2, February 2012, Pages 764–785 [2].

Therefore, one of the promoters we have used is the PyeaR promoter, which is sensitive to nitric oxide in the cell and was inserted upstream of different effectors. This promoter uses the E. coli native enzyme NsrR which inhibits the expression of the PyeaR promoting sequence. [Link] HJ Beaumont et al. Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor in Molecular Microbiology Volume 54, Issue 1, October 2004, Pages 148-58[3] Nitric oxide in the cell inactivates this enzyme, thus increasing the rate of transcription of any downstream genes.

BBa_K381005 Testing The PyeaR promoter (BBa_K216005) from the 2009 Edinburgh iGEM team was the part we wanted for our Nitric Oxide sensing device. To test if this promoter works, we used the BBa_K381001 (BBa_K216005 + BBa_E0840) part made by 2011 BCCS-Bristol iGEM In order to test the promoter, we transformed the DH5-α with BBa_K381001 and measured the fluorescence with a plate reader after being induced with varying concentrations of potassium nitrate (0 mM - 40 mM) at OD = 0.4.

Figure 1. Fluorescence of cultures when PyeaR promoter was induced at varying concentrations of potassium nitrate.

Fig. 1 shows that at after around OD = 0.4, fluorescence becomes higher in the cultures containing potassium nitrate compared to the culture with no nitrate. On another note, no large increase in fluorescence was seen from increasing the concentration of potassium nitrate beyond 5 mM, suggesting that 5 mM potassium nitrate is enought to induce the promoter to maximum strength. Surprisingly, the cultures with 5 mM and 10 mM potassium nitrate had a generally stronger induction compared to the 25 mM and 40 mM cultures.

Accelerative Antagonistic Anxiety

There are several different kinds of anxiety and panic disorders. One common feature of these diseases is the occurrence of panic attacks which are caused by a sudden surge in epinephrine. It has also been shown that patients with panic disorder have an elevated level of arterial epinephrine even at rest. [Link]Wilkinson D. et al. Sympathetic Activity in Patients With Panic Disorder at Rest, Under Laboratory Mental Stress, and During Panic Attacks in Archives of general Psychiatry, 1998;55(6):511-520 [4]

As the gut is strongly innervated by the sympathetic nervous system and plays an important role in inflammation. [Link]Straub R. H. The role of the sympathetic nervous system in intestinal inflammation in Gut. 2006 Nov; 55(11): 1640–1649[5] and because many E. coli strains have an inbuilt system for response to epinephrine we designed a construct with an epinephrine sensitive promoter, flhDC. This promoter is controlled by the transcription factor QseB which is activated by the epinephrine sensing membrane bound protein QseC. [Link]Clarke M. and Sperandio V. Transcriptional regulation of flhDC by QseBC and sigma (FliA) in enterohaemorrhagic Escherichia coli. in Mol Microbiol. 2005 Nov;58(4):1203[6]

In a study with rats researchers have already tested whether QseC could sense epinephrine. QseC-loaded liposomes were treated with the physiologically intestinal-relevant concentration of 5–50 μM epinephrine. [Link] M Clarke et al. The QseC sensor kinase: A bacterial adrenergic receptor. in Proc Natl Acad Sci U S A. 2006 Jul 5; 103(27): 10420–10425[7]

The flhDC promoter can therefore be useful for immediate response in the case of a sudden surge in epinephrine during a panic attack.

BBa_K554001 Testing

The flhDC promoter BBa_K554001 from the 2011 UNICAMP-EMSE Brazil iGEM team was what we needed.

In order to test the promoter, we ligated the GFP reporter part, BBa_I13504, downstream of the promoter, transformed it in DH5-α and measured the fluorescence on a plate reader after being induced with varying concentrations of epinephrine at OD=0.1.

Figure 2. Fluorescence of DH5-α cultures when flhDC promoter was induced at varying concentrations of (-)-epinephrine.

Fig. 2 shows that at after around OD = 1, fluorescence becomes higher in the cultures containing epinephrine compared to the culture with no epinephrine. On another note, no large increase in fluorescence was seen from increasing the concentration of epinephrine beyond 5 μM, showing that 5 μM epinephrine is enough to induce the promoter to maximum strength.

Overpowering Obstructive Osmotic Stress

BBa_R0082 Testing

The OmpR promoter ( BBa_R0082) from 2004 Antiquity was the part we wanted for our osmotic stress sensing device. To test if this promoter works, we ligated BBa_R0082 with GFP reporter part, BBa_I13504, downstream of the promoter , transformed it in DH5-α, and measured the fluorescence with a plate reader after being grown in LB containing varying concentrations of NaCl.

Figure 3. Fluorescence of OmpR promoter-GFP DH5-α cultures when grown at varying concentrations of NaCl.

Fig. 3 shows that at after around OD = 0.2, fluorescence becomes higher in the cultures containing NaCl compared to the culture with no epinephrine with the exceptions of 0.2% and 0.4% NaCl. However at OD=0.5, the culture with 0.2% NaCl started to fluoresce more than the 0% culture. Overall, the cultures containing 0.05% and 0.1% had the highest amount of fluorescence, suggesting that at around 0.05% - 0.1% or perhaps a lower NaCl concentration, the OmpR promoter is induced at its optimal strength.