Vibriosis is a prevalent aquatic disease caused by Vibrio species and has led to massive loss of brown-marbled grouper, Epinephelus fuscoguttatus. The complexity of molecular mechanisms associated with immune defence can be studied through transcriptomics analysis. High quality and quantity of total RNAs are crucial for the veracity of RNA sequencing and gene expression analysis. A low quality RNA will compromise downstream analysis, resulting in loss of time and revenue to re-acquire the data again. Thus, a reliable and an efficient RNA isolation method is the first and most important step to obtain high quality RNA for gene expression studies. There are many aspects need to be considered when deciding an extraction method, such as the cost-effectiveness of the protocol, the duration of chemical exposure, the duration required for a complete extraction and the number of sample-transferring. A good RNA extraction protocol must be able to produce high yield and purity of RNA free from enzyme inhibitors, such as nucleases (RNase), phenols, alcohols or other chemicals carryover, apart from protein and genomic DNA contamination, to maintain isolated RNA integrity in storage condition. In this study, TransZolTM Up produced clean and pure RNA samples from control gills only but not from the infected gill and whole-body tissues. Modified conventional CTAB (conventional hexadecyltrimethylammonium bromide) method was then used as an alternative method to isolate RNA from gill and whole-body tissues of Vibrio-infected E. fuscoguttatus. Modified CTAB method produced intact RNA on gel electrophoresis with higher RIN number (>6.5) for infected gill and whole-body tissues, suggesting that this method could also be used to isolate high quality RNA from fish samples. Therefore, this method is potentially suitable to be used to extract RNA from other fish species especially those that have been infected.
Flavonoid is an industrially-important compound due to its high pharmaceutical and cosmeceutical values. However,
conventional methods in extracting and synthesizing flavonoids are costly, laborious and not sustainable due to small
amount of natural flavonoids, large amounts of chemicals and space used. Biotechnological production of flavonoids
represents a viable and sustainable route especially through the use of metabolic engineering strategies in microbial
production hosts. In this review, we will highlight recent strategies for the improving the production of flavonoids
using synthetic biology approaches in particular the innovative strategies of genetically-encoded biosensors for in
vivo metabolite analysis and high-throughput screening methods using fluorescence-activated cell sorting (FACS).
Implementation of transcription factor based-biosensor for microbial flavonoid production and integration of systems
and synthetic biology approaches for natural product development will also be discussed.
Microbial production of natural products using metabolic engineering and synthetic biology approaches often involves
the assembly of multiple gene fragments including regulatory elements, especially when using eukaryotes as hosts.
Traditional cloning strategy using restriction enzyme digestion and ligation are laborious and inflexible owing to the
high number of sequential cloning steps, limited cutting sites and generation of undesired ‘scar’ sequences. In this study,
a homology-based isothermal DNA assembly method was carried out for one-step simultaneous assembly of multiple DNA
fragments to engineer plant phenylpropanoid biosynthesis in Saccharomyces cerevisiae. Rapid construction of yeast
plasmid harboring dual gene expression cassettes was achieved via isothermal assembly of four DNA fragments designed
with 20 bp overlapping sequences. The rate-limiting enzyme of phenylpropanoid pathway, cinnamate 4-hydroxylase
encoded by C4H gene from Polygonum minus was cloned in tandem with yeast promoter and terminator elements of S.
cerevisiae for efficient construction of phenylpropanoid biosynthetic pathway in recombinant yeast. The assembled pAGCAT (C4H-ADH1t-TEF1p) shuttle plasmid and transformation of S. cerevisiae with the plant C4H gene were confirmed
via PCR analysis. Based on these findings, the yeast shuttle plasmid harboring P. minus phenylpropanoid biosynthesis
gene was efficiently constructed to be the starting platform for the production of plant natural products in geneticallyengineered S. cerevisiae.
The giant freshwater prawn holds a significant position as a valuable crustacean species cultivated in the aquaculture industry, particularly well-known and demanded among the Southeast Asian countries. Aquaculture production of this species has been impacted by Macrobrachium rosenbergii nodavirus (MrNV) infection, which particularly affects the larvae and post-larvae stages of the prawn. The infection has been recorded to cause mortality rates of up to 100% among the affected prawns. A simple, fast, and easy to deploy on-site detection or diagnostic method is crucial for early detection of MrNV to control the disease outbreak. In the present study, novel single-stranded DNA aptamers targeting the MrNV capsid protein were identified using the systematic evolution of ligands by exponential enrichment (SELEX) approach. The aptamer was then conjugated with the citrate-capped gold nanoparticles (AuNPs), and the sensitivity of this AuNP-based aptasensor for the detection of MrNV capsid protein was evaluated. Findings revealed that the aptamer candidate, APT-MrNV-CP-1 was enriched throughout the SELEX cycle 4, 9, and 12 with the sequence percentage of 1.76%, 9.09%, and 12.42%, respectively. The conjugation of APT-MrNV-CP-1 with citrate-capped AuNPs exhibited the highest sensitivity in detecting the MrNV capsid protein, where the presence of 62.5 nM of the viral capsid protein led to a significant agglomeration of the AuNPs. This study demonstrated the practicality of an AuNP-based aptasensor for disease diagnosis, particularly for detecting MrNV infection in giant freshwater prawns.