Genetic circuits for directed evolution of bioluminescent proteins

Bioluminescent technologies are amongst the most commonly used tools for quantifying and visualising biological processes, and novel functions are being engineered for their application inside and outside the lab. Current methods to engineer bioluminescence-based tools rely on random or semi-random mutagenesis approaches which require intensive genotype screening, and are typically performed on expensive robotic workstations. In this project, we aimed to develop an in vivo platform to automate the screening of bioluminescent protein libraries using engineered bacteria. We repurposed a light-sensing circuit that allows individual bacteria to transduce the bioluminescence emission of an intracellular luciferase variant into expression of antibiotic resistance genes. This synthetic ability should allow bacteria expressing brightest luciferase variants to outcompete the rest of the population and become automatically selected by simply growing them under the appropriate antibiotic pressure. The current version of the genetic circuit confers modest but detectable growth and survival advantages to glowing cells; however, it requires further optimisation for robust performance.Open Acces

Estudi de la importància de la proteïna SIRT3 sobre el mitocondri en la línia cel·lular de càncer de còlon SW620

[cat] La SIRT3 és una proteïna desacetilasa de localització mitocondrial, que té una important repercusió en la majoria de processos relacionats amb aquest orgànul. De fet, és indispensable per a una correcta funció metabòlica i determinant per a evitar el dany oxidatiu i la reprogramació metabòlica mediada per ROS, que actuen conjuntament en la carcinogènesi. No obstant, en les cèl·lules cancerígenes, SIRT3 atorga una elevada resistència a l’estrès i a la manca de nutrients, promovent la proficiència mitocondrial –juntament amb PGC-1– necessària pel creixement tumoral i la metàstasi, pel que es contempla com un regulador instrumental en la biologia del càncer i, presumiblement, una excel·lent diana terapèutica. Malgrat ser un dels càncers més incidents a nivell mundial –i de cada dia més proper, per l’evolució del nostre estil de vida–, es troben pocs estudis relacionats amb la biogènesi mitocondrial i la fosforilació oxidativa en el càncer de còlon; i, que nosaltres coneguem, cap que hagi estudiat la SIRT3 en aquest context. Per a aquest motiu, es va realitzar un silenciament estable del gen SIRT3 en la línia de càncer colorectal SW620 i s’estudiaren els nivells proteïcs dels cinc complexes OXPHOS, PGC-1α, TFAM i la mateixa SIRT3; a més dels nivells de mRNA dels darrers tres factors. Els resultats d’aquest estudi mostren una disminució en la majoria de complexes de la CTE, probablement, degut a la incapacitat per a mantenir els nivells de TFAM necessaris per a l’expressió de les seves subunitats mitocondrials, que ocorre per una co-inducció insuficient de PGC-1α i SIRT3. Conjuntament amb aquests resultats, hem pogut detectar l’activació d’algun tipus de mecanisme compensatori per part d’aquestes cèl·lules per a mantenir els nivells proteics de PGC-1α, que malgrat això, no ha resultat efectiu per a fer front al silenciament, ja que l’activitat d’aquest co-activador s’ha vist compromesa per algun motiu que encara desconeixem.[eng] SIRT3 is a mitochondrial deacetylase protein related with the oxidative and replicative state of this organelle. Although it regulates the majority of its functions, SIRT3 has a determing role in avoiding oxidative damage and metabolic reprograming induced by ROS signalling in carcinogenesis. However, in cancer cells, this protein confers a great resistance to several kinds of stress –such as nutrient scantiness or chemotherapy– by promoting mitochondrial proficiency (together with PGC-1), which is at the same time, a required condition for distant metastasis and invasive properties. For this reason, SIRT3 is considered as an instrumental regulator of cancer biology and, presumably, an excellent therapeutic target too. Despite being the most common cancer type worldwide, regardless of sex, with the second highest mortality rate and an each day closer illness –due to the evolution of our life style–, there are few studies related to colorectal cancer and mitochondrial biogenesis and, to our knowledge, none regarding SIRT3 in this context. Therefore, we performed a stable silencing of this gene in the SW620 human colorectal cancer cell line and then, protein levels of all OXPHOS complexes, PGC-1α, TFAM and SIRT3, and mRNA levels of the last three factors were determined. The results of this study show that when silencing SIRT3 expression, a significant decrease in most of the ETC complexes occurs, apparently, due to the unsatisfactory co-induction of PGC-1α and SIRT3, which disables the maintenance of the necessary levels of TFAM to express their mitochondrial-encoded subunits. Together with these results, we were able to detect the activation of some type of compensatory mechanism to mantain PGC-1α protein levels, which, in spite of everything, was not effective facing the silencing, because its activity seemed to be impaired for some reason we still do not know

Bioluminescence-Driven Optogenetics

Bioluminescence-based technologies are among the most commonly used methods to quantify and visualise physiology at the cellular and organismal levels. However, the potential of bioluminescence beyond reporter technologies remains largely unexplored. Here, we provide an overview of the emerging approaches employing bioluminescence as a biological light source that triggers physiological events and controls cell behaviour and discuss its possible future application in synthetic biology

Detection and Quantification of Antibody to SARS-CoV-2 Receptor Binding Domain Provides Enhanced Sensitivity, Specificity and Utility

Background: Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity.Methods: The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA) provides the most sensitive format. It has been exploited in a novel hybrid manner employing an S1 solid-phase preferentially presenting RBD once solid-phase bound, coupled with a labelled RBD conjugate, used in a two-step sequential assay.Findings: This assay showed a specificity of 100% on 825 pre COVID-19 samples and a potential sensitivity of 99.6% on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralisation and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine. The early response at presentation with illness, elevated responsiveness with disease severity, detection of asymptomatic seroconversion and persistence after the loss of antibody to the nucleoprotein (anti-NP) are all documented.Trial Registration: The ISARIC WHO CCP-UK study was registered at https://www.isrctn.com/ISRCTN66726260 and designated an Urgent Public Health Research Study by NIHR.Interpretation: The hybrid DABA displays the attributes necessary for an antibody test to be used in both clinical and reference serology. It allows the neutralising antibody response to be inferred early in infection and potentially in vaccine recipients. It is also of sufficient sensitivity to be used to provide serological confirmation of prior infection and provides a more secure measure for seroprevalence studies in the population generally than does anti-NP based on the Architect platform.Funding: This work is variously supported by grants from: the National Institute for Health Research (NIHR; award CO-CIN-01), the Medical Research Council (MRC; grant MC_PC_19059 and MC_PC_19078), MRC NIHR (grant CV220-111) and by the NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford (award 200907), NIHR HPRU in Respiratory Infections at Imperial College London with PHE (award 200927), Wellcome Trust and Department for International Development (DID; 215091/Z/18/Z), the Bill and Melinda Gates Foundation (OPP1209135), Liverpool Experimental Cancer Medicine Centre (grant reference C18616/A25153), NIHR Biomedical Research Centre at Imperial College London (IS-BRC-1215-20013), EU Platform for European Preparedness Against (Re-)emerging Epidemics (PREPARE; FP7 project 602525), and NIHR Clinical Research Network for providing infrastructure support for this research.Declaration of Interests: RST, MOM and PC report patent pending (Patent Application No. 2011047.4 for “SARS-CoV-2 antibody detection assay). All other authors declare no competing interests.Ethics Approval Statement: The use of tissues was approved by the CDRTB Steering Committee in accordance with the responsibility delegated by the National Research Ethics Service (South Central Ethics Committee – C, NRES reference 15/SC/0089).Written informed consent was obtained from all patients. Ethical approval was given by the South Central–Oxford C Research Ethics Committee in England (reference: 13/SC/0149), Scotland A Research Ethics Committee (reference: 20/SS/0028) and World Health Organization Ethics Review Committee (RPC571 and RPC572l; 25 April 2013