Parts Characterization for Tunable Protein Expression

Abstract

Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression. Constructing targeted genomic variation around genomically integrated fluorescent marker genes enables rapid elucidation of the contribution of specific sequence variants to protein expression. Such an approach can be used to characterize the impact of modifications to the Shine-Dalgarno sequence in Escherichia coli.

Flow-Seq Method: Features and Application in Bacterial Translation Studies

“…A number of studies employing the Flow-seq method have investigated the effect of the sequences of 5’ untranslated regions of different lengths and their individual sites on the efficiency of the reporter fluorescent protein synthesis [ 2 , 39 , 71 , 72 , 73 , 74 ].…”

“…Determining the sensitivity to minor variations in the sequence of the regulatory elements in the 5’ UTR, such as the Shine–Dalgarno sequence, is rather challenging, since minor variations in the 5’ UTR may lead to unpredictable changes in the gene expression level [ 34 , 75 ]. The dependence of the translation efficiency on the 5’ UTR sequence enables efficient and multiplex engineering, provided that the models being built can adequately predict these changes [ 73 ].…”

“…Accordingly, special care should be taken when reapplying the measured expression levels in the bioengineering of metabolic pathways or synthetic biology, since the ribosome binding site depends in large part on the local secondary structure of mRNA. However, whereas the Shine–Dalgarno sequences can be modified by making minimal changes to the secondary structure in a given mRNA region, the relative order of expression level of a particular SD sequence will probably remain intact [ 73 ]. These features are taken into account when using the algorithm in the EMOPEC database, which allows one to test a wide range of gene expression levels, with minimal changes in the SD sequence.…”

“…степени на уровень белка, так как на эффективность трансляции влияет и выбор сайта посадки рибосомы, и, возможно, другие факторы [67]. В ряде работ с применением метода Flow-seq изучено влияние последовательностей 5'-нетранслируемых областей разного размера и их отдельных участков на эффективность синтеза репортерного флуоресцентного белка [2,39,[71][72][73][74].…”

“…Определение чувствительности к незначительным изменениям в последовательности регуляторных элементов в 5'-UTR, таких, как последовательность Шайна-Дальгарно, представляет сложную задачу, поскольку небольшие изменения в составе 5'-UTR могут привести к непредсказуемым изменениям в уровне экспрессии гена [34,75]. Зависимость эффективности трансляции от последовательности 5'-UTR делает возможной эффективную и мультиплексную инженерию при условии, что создаваемые модели способны адекватно предсказывать эти изменения [73].…”