Vector Architecture Research Articles

Co-designing ab initio electronic structure methods on a RISC-V vector architecture

Ab initio electronic structure applications are among the most widely used in High-Performance Computing (HPC), and the eigenvalue problem is often their main computational bottleneck. This article presents our initial efforts in porting these codes to a RISC-V prototype platform leveraging a wide Vector Processing Unit (VPU). Our software tester is based on a mini-app extracted from the ELPA eigensolver library. The user-space emulator Vehave and a RISC-V vector architecture implemented on an FPGA were tested. Metrics from both systems and different vectorisation strategies were extracted, ranging from the simplest and most portable one (using autovectorisation and assisting this by fusing loops in the code) to the more complex one (using intrinsics). We observed a progressive reduction in the number of vectorised instructions, executed instructions and computing cycles with the different methodologies, which will lead to a substantial speed-up in the calculations. The obtained outcomes are crucial in advancing the porting of computational materials and molecular science codes to (post)-exascale architectures using RISC-V-based technologies fully developed within the EU. Our evaluation also provides valuable feedback for hardware designers, engineers and compiler developers, making this use case pivotal for co-design efforts.

Hardware Acceleration for High-Volume Operations of CRYSTALS-Kyber and CRYSTALS-Dilithium

Many high-demand digital services need to perform several cryptographic operations, such as key exchange or security credentialing, in a concise amount of time. In turn, the security of some of these cryptographic schemes is threatened by advances in quantum computing, as quantum computer could break their security in the near future. Post-quantum cryptography (PQC) is an emerging field that studies cryptographic algorithms that resist such attacks. The National Institute of Standards and Technology (NIST) has selected the CRYSTALS-Kyber Key Encapsulation Mechanism and the CRYSTALS-Dilithium Digital Signature algorithm as primary PQC standards. In this article, we present field-programmable gate array (FPGA)-based hardware accelerators for high-volume operations of both schemes. We apply high-level synthesis (HLS) for hardware optimization, leveraging a batch processing approach to maximize the memory throughput and applying custom HLS logic to specific algorithmic components. Using reconfigurable FPGAs, we show that our hardware accelerators achieve speedups between 3 \(\times\) and 9 \(\times\) over software baseline implementations, even over ones leveraging CPU vector architectures. Furthermore, the methods used in this study can also be extended to the new CRYSTALS-based NIST FIPS drafts, ML-KEM and ML-DSA, with similar acceleration results.

Co-designing ab initio electronic structure methods on a RISC-V vector architecture.

Ab initio electronic structure applications are among the most widely used in High-Performance Computing (HPC), and the eigenvalue problem is often their main computational bottleneck. This article presents our initial efforts in porting these codes to a RISC-V prototype platform leveraging a wide Vector Processing Unit (VPU). Our software tester is based on a mini-app extracted from the ELPA eigensolver library. The user-space Vehave and a RISC-V vector architecture implemented on an FPGA were tested. Metrics from both systems and different vectorisation strategies were extracted, ranging from the most simple and portable one (using autovectorisation and assisting this by fusing loops in the code) to the more complex one (using intrinsics). We observed a progressive reduction in the number of vectorial instructions, executed instructions and computing cycles with the different methodologies, which will lead to a substantial speed-up in the calculations. The obtained outcomes are crucial in advancing the porting of computational materials and molecular science codes to (post)-exascale architectures using RISC-V-based technologies fully developed within the EU. Our evaluation also provides valuable feedback for hardware designers, engineers and compiler developers, making this use case pivotal for co-design efforts.

Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling

BackgroundBacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work.ResultsIn the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743.ConclusionsThe results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.

Extension VM: Interleaved Data Layout in Vector Memory

While vector architecture is widely employed in processors for neural networks, signal processing, and high-performance computing; however, its performance is limited by inefficient column-major memory access. The column-major access limitation originates from the unsuitable mapping of multidimensional data structures to two-dimensional vector memory spaces. In addition, the traditional data layout mapping method creates an irreconcilable conflict between row- and column-major accesses. Ideally, both row- and column-major accesses can take advantage of the bank parallelism of vector memory. To this end, we propose the Interleaved Data Layout (IDL) method in vector memory, which can distribute vector elements into different banks regardless of whether they are in the row- or column-major category, so that any vector memory access can benefit from bank parallelism. Additionally, we propose an Extension Vector Memory (EVM) architecture to achieve IDL in vector memory. EVM can support two data layout methods and vector memory access modes simultaneously. The key idea is to continuously distribute the data that needs to be accessed from the main memory to different banks during the loading period. Thus, EVM can provide a larger spatial locality level through careful programming and the extension ISA support. The experimental results showed a 1.43-fold improvement of state-of-the-art vector processors by the proposed architecture, with an area cost of only 1.73%. Furthermore, the energy consumption was reduced by 50.1%.

Trehalose-polyamine/DNA nanocomplexes: impact of vector architecture on cell and organ transfection selectivity.

A novel family of precision-engineered gene vectors with well-defined structures built on trehalose and trehalose-based macrocycles (cyclotrehalans) comprising linear or cyclic polyamine heads have been synthesized through procedures that exploit click chemistry reactions. The strategy was conceived to enable systematic structural variations and, at the same time, ensuring that enantiomerically pure vectors are obtained. Notably, changes in the molecular architecture translated into topological differences at the nanoscale upon co-assembly with plasmid DNA, especially regarding the presence of regions with short- or long-range internal order as observed by TEM. In vitro and in vivo experiments further evidenced a significant impact on cell and organ transfection selectivity. Altogether, the results highlight the potential of trehalose-polyamine/pDNA nanocomplex monoformulations to achieve targeting transfection without the need for any additional cell- or organ-sorting component.

A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing

ABSTRACT The development of CRISPR-Cas-based engineering technologies has revolutionized the microbial biotechnology field. Over the years, the Class II Type II CRISPR-Cas9 system has become the gold standard for genome editing in many bacterial hosts. However, the Cas9 system does not allow efficient genomic integration in Pseudomonas putida , an emerging Synthetic Biology host, without the assistance of lambda-Red recombineering. In this work, we utilize the alternative Class I Type I-C CRISPR-Cas3 system from Pseudomonas aeruginosa as a highly efficient genome editing tool for P. putida and P. aeruginosa . This system consists of two vectors, one encoding the Cas genes, CRISPR array and targeting spacer, and a second Standard European Vector Architecture vector, containing the homologous repair template. Both vectors are Golden Gate compatible for rapid cloning and are available with multiple antibiotic markers, for application in various Gram-negative hosts and different designs. By employing this Cas3 system, we successfully integrated an 820-bp cassette in the genome of P. putida and performed several genomic deletions in P. aeruginosa within a week, with an efficiency of >83% for both hosts. Moreover, by introducing a universal self-targeting spacer, the Cas3 system rapidly cures all helper vectors, including itself, from the host strain in a matter of days. As such, this system constitutes a valuable engineering tool for Pseudomonas , to complement the existing range of Cas9-based editing methods, and facilitates genomic engineering efforts of this important genus. IMPORTANCE The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.

Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria.

Modular cloning has become a benchmark technology in synthetic biology. However, a notable disparity exists between its remarkable development and the need for standardization to facilitate seamless interoperability among systems. The field is thus impeded by an overwhelming proliferation of organism-specific systems that frequently lack compatibility. To overcome these issues, we present Golden Standard (GS), a Type IIS assembly method underpinned by the Standard European Vector Architecture. GS unlocks modular cloning applications for most bacteria, and delivers combinatorial multi-part assembly to create genetic circuits of up to twenty transcription units (TUs). Reliance on MoClo syntax renders GS fully compatible with many existing tools and it sets the path towards efficient reusability of available part libraries and assembled TUs. GS was validated in terms of DNA assembly, portability, interoperability and phenotype engineering in α-, β-, γ- and δ-proteobacteria. Furthermore, we provide a computational pipeline for parts characterization that was used to assess the performance of GS parts. To promote community-driven development of GS, we provide a dedicated web-portal including a repository of parts, vectors, and Wizard and Setup tools that guide users in designing constructs. Overall, GS establishes an open, standardized framework propelling the progress of synthetic biology as a whole.

Masked Language Modeling for Resource Constrained Biological Natural Language Processing.

Recent advances in Natural Language Processing (NLP) have produced state of the art results on several sequence to sequence (seq2seq) tasks. Enhancements in embedders and their training methodologies have shown significant improvement on downstream tasks. Word vector models like Word2Vec, FastText & Glove were widely used over one-hot encoded vectors for years until the advent of deep contextualized embedders. Protein sequences consist of 20 naturally occurring amino acids that can be treated as the language of nature. These amino acids in combinations with each other makeup the biological functions. The choice of vector representation and architecture design for a biological task is highly dependent upon the nature of the task. We utilize unlabelled protein sequences to train a Convolution and Gated Recurrent Network (CGRN) embedder using Masked Language Modeling (MLM) technique that shows significant performance boost under resource constraint setting on two downstream tasks i.e., F1-score(Q8) of 73.1% on Secondary Structure Prediction (SSP) & F1-score of 84% on Intrinsically Disordered Region Prediction (IDRP). We also compare different architectures on downstream tasks to show the impact of the nature of biological task on the performance of the model.

PBLAM1-x: standardized transposon tools forhigh-throughput screening.

The engineering of pre-defined functions in living cells requires increasingly accurate tools as synthetic biology efforts become more ambitious. Moreover, the characterization of the phenotypic performance of genetic constructs demands meticulous measurements and extensive data acquisition for the sake of feeding mathematical models and matching predictions along the design-build-test lifecycle. Here, we developed a genetic tool that eases high-throughput transposon insertion sequencing (TnSeq): the pBLAM1-x plasmid vectors carrying the Himar1 Mariner transposase system. These plasmids were derived from the mini-Tn5 transposon vector pBAMD1-2 and built following modular criteria of the Standard European Vector Architecture (SEVA) format. To showcase their function, we analyzed sequencing results of 60 clones of the soil bacterium Pseudomonas putida KT2440. The new pBLAM1-x tool has already been included in the latest SEVA database release, and here we describe its performance using laboratory automation workflows. Graphical Abstract.

A suite of modular, all-synthetic suicide vectors for allelic exchange mutagenesis in multidrug resistant Acinetobacter strains

BackgroundAcinetobacter baumannii is an opportunistic human pathogen that causes a variety of infections in immunosuppressed individuals and patients in intensive care units. The success of this pathogen in nosocomial settings can be directly attributed to its persistent nature and its ability to rapidly acquire multidrug resistance. It is now considered to be one of the top priority pathogens for development of novel therapeutic approaches. Several high-throughput techniques have been utilised to identify the genetic determinants contributing to the success of A. baumannii as a global pathogen. However, targeted gene-function studies remain challenging due to the lack of appropriate genetic tools.ResultsHere, we have constructed a series of all-synthetic allelic exchange vectors – pALFI1, pALFI2 and pALFI3 – with suitable selection markers for targeted genetic studies in highly drug resistant A. baumannii isolates. The vectors follow the Standard European Vector Architecture (SEVA) framework for easy replacement of components. This method allows for rapid plasmid construction with the mutant allele, efficient conjugational transfer using a diaminopimelic acid-dependent Escherichia coli donor strain, efficient positive selection using the suitable selection markers and finally, sucrose-dependent counter-selection to obtain double-crossovers.ConclusionsWe have used this method to create scar-less deletion mutants in three different strains of A. baumannii, which resulted in up to 75% deletion frequency of the targeted gene. We believe this method can be effectively used to perform genetic manipulation studies in multidrug resistant Gram-negative bacterial strains.

Optimized De Novo Eriodictyol Biosynthesis in Streptomyces albidoflavus Using an Expansion of the Golden Standard Toolkit for Its Use in Actinomycetes.

Eriodictyol is a hydroxylated flavonoid displaying multiple pharmaceutical activities, such as antitumoral, antiviral or neuroprotective. However, its industrial production is limited to extraction from plants due to its inherent limitations. Here, we present the generation of a Streptomyces albidoflavus bacterial factory edited at the genome level for an optimized de novo heterologous production of eriodictyol. For this purpose, an expansion of the Golden Standard toolkit (a Type IIS assembly method based on the Standard European Vector Architecture (SEVA)) has been created, encompassing a collection of synthetic biology modular vectors (adapted for their use in actinomycetes). These vectors have been designed for the assembly of transcriptional units and gene circuits in a plug-and-play manner, as well as for genome editing using CRISPR-Cas9-mediated genetic engineering. These vectors have been used for the optimization of the eriodictyol heterologous production levels in S. albidoflavus by enhancing the flavonoid-3'-hydroxylase (F3'H) activity (by means of a chimera design) and by replacing three native biosynthetic gene clusters in the bacterial chromosome with the plant genes matBC (involved in extracellular malonate uptake and its intracellular activation into malonyl-CoA), therefore allowing more malonyl-CoA to be devoted to the heterologous production of plant flavonoids in this bacterial factory. These experiments have allowed an increase in production of 1.8 times in the edited strain (where the three native biosynthetic gene clusters have been deleted) in comparison with the wild-type strain and a 13 times increase in eriodictyol overproduction in comparison with the non-chimaera version of the F3'H enzyme.

Host-Dependent Improvement of GFP Expression in Pseudomonas putida KT2440 Using Terminators of Metagenomic Origin.

Transcriptional terminators are key players in the flow of genetic information, but are often overlooked in circuit design. In this work, we used the Standard European Vector Architecture (SEVA) as a scaffold to investigate the effects of different terminators in the output of a reporter construct expressed in two bacterial species, and found that replacing the conventional T1 and T0 transcriptional terminators of the SEVA vector format with a set of broad-host metagenomic terminators resulted in a significant improvement in the signal of a fluorescent device in Pseudomonas putida KT2440 but not in Escherichia coli DH10B. Our results suggest that replacing the default set of terminators present in the SEVA specification may be an useful strategy for fine-tuning circuit expression in P. putida, which can be leveraged for the development of new devices with improved performance in this microbial host.

Vectorized PoCA algorithm for faster image reconstruction for Muon Tomography

Muon Tomography is a promising technique for detecting the presence of high atomic number (Z) material, using naturally available cosmic muons. The reconstruction algorithms used in Muon Tomography need to process a considerably high number of muon tracks to reconstruct the image of the object under test. This makes the image reconstruction process computation-intensive and time-consuming. The algorithms to process the muon tracks are event-based algorithms, where all the events are independent and can be processed in any order. The traditional form of parallelism tries to distribute a small segment of work to multiple computing cores, but does not utilize the data level parallelism. The event-based algorithm can be processed on vector architecture and can provide a path to better utilize the data level parallelism. This approach provides another level of parallelism on standard computers without the need for specialized hardware. In this paper, we have tried to exploit the vector architecture provided by the current generation of CPUs to implement the vectorized version of Point of Closest Approach (PoCA) reconstruction algorithm for Muon Tomography. Results are presented using different vector instruction sets, for single-precision and double-precision calculations.

HPCG on long-vector architectures: Evaluation and optimization on NEC SX-Aurora and RISC-V

Accelerators are becoming a key component to improve efficiency in High-Performance Computing systems (HPC). While GPU based systems are widely used to accelerate HPC workloads, new systems based on long-vector architectures are rapidly gaining popularity. The development of optimized math libraries becomes fundamental to achieve high performance in those emerging vector architectures. This paper focuses on the optimization of the HPCG benchmark, which comprises four fundamental kernels found in many numerical applications. We target two relevant long-vector architectures like the NEC Vector Engine and the RISC-V ’V’ vector extension. Compared to the well-tuned proprietary solution, our open HPCG implementation achieves a 1.6% improvement in performance on the NEC Vector Engine and achieves near maximum memory bandwidth utilization in the two evaluated RISC-V vector accelerator designs.

SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes.

The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard-but not its entirety-has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms.

Acceleration with long vector architectures: Implementation and evaluation of the FFT kernel on NEC SX‐Aurora and RISC‐V vector extension

SummaryNovel architectures leveraging long and variable vector lengths like the NEC SX‐Aurora or the vector extension of RISCV are appearing as promising solutions on the supercomputing market. These architectures often require re‐coding of scientific kernels. For example, traditional implementations of algorithms for computing the fast Fourier transform (FFT) cannot take full advantage of vector architectures. In this article, we present the implementation of FFT algorithms able to leverage these novel architectures. We evaluate these codes on NEC SX‐Aurora , comparing them with the optimized NEC libraries; and in a prototype of a RISC‐V core with a vector processing unit. We present the benefits and limitations of two approaches of RADIX‐2 FFT vector implementations. We show that our approach makes better use of the vector unit of the NEC SX‐Aurora , reaching higher or equal performance than the optimized NEC library. More generally, we prove the importance of maximizing the vector length usage of the algorithm, taking advantage of the FFT properties to reduce long‐latency vector operations, and reordering the instructions according to the specific hardware features to boost the performance of FFT‐like computational kernels.

Basicsynbio and the BASIC SEVA collection: software and vectors for an established DNA assembly method.

Standardized deoxyribonucleic acid (DNA) assembly methods utilizing modular components provide a powerful framework to explore designs and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use and enables hierarchical assemblies through an idempotent format. These features enable applications including pathway engineering, ribosome binding site (RBS) tuning, fusion protein engineering and multiplexed guide ribonucleic acid (RNA) expression. In this work, we present basicsynbio, open-source software encompassing a Web App (https://basicsynbio.web.app/) and Python Package (https://github.com/LondonBiofoundry/basicsynbio), enabling BASIC construct design via simple drag-and-drop operations or programmatically. With basicsynbio, users can access commonly used BASIC parts and linkers while designing new parts and assemblies with exception handling for common errors. Users can export sequence data and create instructions for manual or acoustic liquid-handling platforms. Instruction generation relies on the BasicBuild Open Standard, which is parsed for bespoke workflows and is serializable in JavaScript Object Notation for transfer and storage. We demonstrate basicsynbio, assembling 30 vectors using sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA vector collection is compatible with BASIC and Golden Gate using BsaI. Vectors contain one of six antibiotic resistance markers and five origins of replication from different compatibility groups. The collection is available via Addgene under an OpenMTA agreement. Furthermore, vector sequences are available from within the basicsynbio application programming interface with other collections of parts and linkers, providing a powerful environment for designing assemblies for bioengineering applications. Graphical Abstract.

SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria

BackgroundCyanobacteria are photosynthetic autotrophs that have tremendous potential for fundamental research and industrial applications due to their high metabolic plasticity and ability to grow using CO2 and sunlight. CRISPR technology using Cas9 and Cpf1 has been applied to different cyanobacteria for genome manipulations and metabolic engineering. Despite significant advances with genome editing in several cyanobacteria strains, the lack of proper genetic toolboxes is still a limiting factor compared to other model laboratory species. Among the limitations, it is essential to have versatile plasmids that could ease the benchwork when using CRISPR technology.ResultsIn the present study, several CRISPR-Cpf1 vectors were developed for genetic manipulations in cyanobacteria using SEVA plasmids. SEVA collection is based on modular vectors that enable the exchangeability of diverse elements (e.g. origins of replication and antibiotic selection markers) and the combination with many cargo sequences for varied end-applications. Firstly, using SEVA vectors containing the broad host range RSF1010 origin we demonstrated that these vectors are replicative not only in model cyanobacteria but also in a new cyanobacterium specie, Chroococcidiopsis sp., which is different from those previously published. Then, we constructed SEVA vectors by harbouring CRISPR elements and showed that they can be easily assimilated not only by conjugation, but also by natural transformation. Finally, we used our SEVA-Cpf1 tools to delete the nblA gene in Synechocystis sp. PCC 6803, demonstrating that our plasmids can be applied for CRISPR-based genome editing technology.ConclusionsThe results of this study provide new CRISPR-based vectors based on the SEVA (Standard European Vector Architecture) collection that can improve editing processes using the Cpf1 nuclease in cyanobacteria.

Beyond fluorescein: Use of fluorescent protein calibrants for direct and absolute quantification of protein production in synthetic biology

While inter-lab calibration standards are approaching mainstream usage in synthetic biology, such calibrations are not in fact sufficient for absolute protein quantification required for modelling synthetic circuits. Fluorescein-based calibration of plate reader and flow cytometry instruments allows the measurement of green fluorescent protein (GFP) in synthetic cells to graduate from arbitrary units to calibrated units, but retains important caveats. Fluorescein is only a good calibrant for green FPs, leaving other FPs uncalibrated, and only provides conversions to units of brightness, not to molecule numbers. Ideal assay calibrants in molecular biology consist of the same molecule as the one to be measured – in this case, a purified preparation of the appropriate fluorescent protein. Here we show that by using purified FP calibrants, all protein species in a synthetic circuit can be quantified in absolute terms using no advanced instrumentation. We develop a SEVA (Standardised European Vector Architecture)-based expression vector that allows the high-level production of soluble protein and describe a straightforward 2-day protocol for the purification of micrograms of FP, followed by a calibration that relates fluorescence activity to protein mass. We validate this protocol by calculating conversion factors for a panel of commonly-used FPs including superfolder GFP, mCherry, mScarlet-I and mTagBFP2 on multiple laboratory instruments, and use these calibrations to debug synthetic circuits. We also demonstrate that the suspected bias of the presence of mCherry on OD600 measurements is real, but in practice requires extreme overexpression (~100,000 proteins per cell) to have a meaningful impact on cell density estimates.