Protein engineering
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Automated Design of Synthetic Ribosome Binding Sites To control Protein Expression.
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Protein engineering
- 1. Automated Design of Synthetic Ribosome Binding Sites To Control Protein Expression - Howard M Salis, Ethan A Mirsky & Christopher A Voigt Presented By: Anshu Raj Dahal Mahamudul Hasan Bhuyan
- 2. Aim of the study Problems Microbial engineering for fine control over protein expression. • Mutations (codes will optimize the translation process and will aid in the efficiency to industries) Solution Algorithm to predict the synthetic RBS sequence that provides a target translation initiation rate on a proportional scale instead of depending upon old libraries. Algorithm will measure free energy and sequenced dependent energy that occur during RNA folding and hybridizaton- Forward engineering.
- 3. Thermodynamic Models • r ∝ exp(−βΔGtot ) • They postulate that initiation of translation involves interaction between an activated 30S ribosomal subunit and the 5'-terminal region of a messenger RNA already folded in a specific secondary structure.
- 4. Thermodynamics of translation initiation (a) The ribosome translates an mRNA transcript and produces a protein in a multistep process: the assembly of the 30S complex (box), initiation, elongation, termination, and the turnover of ribosomal subunits and other factors. (b) The thermodynamic free energy change during 30S complex assembly is determined by five molecular interactions that participate in the initial and final states of the system. The Watson-Crick base pairs and G:U wobbles (red lines) are shown.
- 5. Plasmid & Vector • The Luria-Bertani (LB) media was used. • The expression system is a ColE1 plasmid with chloramphenicol resistance (derived from pProTet, Clontech). • E. coli DH10B strain. • For RFP expression
- 6. Forward and Reverse Engineering
- 7. Transcription factors and RBS
- 8. AND Gate Genetic Circuit • The AND gate genetic circuit is composed of three plasmids: pBACr-AraT7940, pBR939b and pAC-SalSer914 with kanamycin, ampicillin and chloramphenicol resistance markers, respectively.
- 9. AND Gate Genetic Circuit
- 10. Summary • The protein expression level can be optimized through: - changing nucleotide sequence (playing with the sequence) in the mRNA , - transcription factors - promoters - Shine Dalgarno sequence -spacers and the information of the start codon.
- 11. Thanks for being with us !
Editor's Notes
- Summary: Same linear relationship is obtained for the 29 synthetic sequences as compared with 28 natural RBS , though the target ΔGtot value was -7.1 – 16 kcal/mol and RBS sequences varied from 16 to 35 nucleotides, and also highly dissimilar. The more negative ΔGtot the expression value is higher.
- Translation Initiation Rate Testing: Two chimeric proteins were constructed that fused the first 27 nucleotides from commonly used transcription factors to an RFP (TetR27-RFP and AraC27-RFP). We then designed 23 synthetic RBSs with ΔGtot targets ranging from −8.5 to 10.5 kcal/mol . That means altered expression level is achieved with the combination of targeted ΔGtot value and a transcription factor. The linearity is achieved once we have desired ΔGtot and protein coding sequence (upper-left, R2 = 0.62; lower-right, R2 = 0.51) , (lower-left, R2 = 0.04; upper-right, R2 = 0.02). Accurate protein coding sequence is required to predict ΔGtot . TF-RFP represents the three chimeric proteins altogether. -8.5 to 10.5 is the range of ΔGtot for 23 synthetic RBS. Fig. a. for two chimeric proteins.
- Fig. a . And gate circuit ( represent the activation of gfp reporter gene ) , it activates gfp reporter when both Ara and Sal promoter are sufficiently induced. Fig. b. Fitness curve ( a curve relating 9 experimentatl values, 9 are synthetic RBS and the predicted ΔGtot with the Pbad promoter ). Optimal region of ΔGtot for Pbad promoter is -1.17 (+/- ) 2 kcal/mol ( that means for optimal function of the Pbad promoter ΔGtot of -1.17 is needed ). The accuracy of the circuit’s logic (referred to as its fitness) is highest when the maximum expression level from the PBAD promoter is optimized to a value between underexpression and overexpression. Otherwise, when the promoter is underexpressed, gfp expression is never turned on; and when the promoter is overexpressed, transcriptional leakiness causes gfp expression even when an input is absent. Our Fig. c Arabinose and salicylate present in the medium induce the promoters Ara and Sal respectively to regulate gfp gene.