Team:KCL UK/Design
Project Design
Overview:
Our team aimed to create and test sRNA based translational regulation system in order to fine tune gene expression for therapeutic and synthetic biology application. We started with the sRNA molecule construct which inhibits GFP reporter gene translation by binding to its mRNA molecule in the region spanning the Ribosomal Binding Site (RBS) and the first two codons of the GFP gene. To test our system in E.coli, we designed two plasmids systems. First, we created a fluorescent reporting system using pSB4K5 plasmid backbone, which contains the pSC101 origin of replication. Then we created the pSB1C3 plasmid backbone containing pUC plasmid derived origin of replication and expressing our sRNA molecules. Both plasmids have compatible origin of replication.
During this work we utilised pSB4K5 plasmid backbone for two GFP reporter systems BBa_K608010 and BBa_K608011, both having BBA_J23110 promoter, either BBa_0032 or BBa_0034 ribosomal binding sites and the BBa_E0040 GFP parts. Our sRNA constructs had pSB1C3 plasmid backbone, BBa_J23100 promoter, mRNA target binding region, against either BBa_B0032 or BBa_B0034 RBSs and the sRNA scaffold derived from the E.coli sRNAs GcvB, Spot42 and RprA. In addition to these sRNA constructs we also created three negative control constructs where target binding regions were replaced with the transcription terminator T1 from the E.coli rrnB gene. We first tested the GFP expression in E.coli cells with both plasmids expressing GFP reporter gene and corresponding sRNAs. Positive bacterial cells were selected with two antibiotics (Chloramphenicol and kanamycin) and inhibition of the GFP expression was monitored during bacterial growth for five hours using 96 well plate reader.
Our work has shown that sRNAs complementary to the start codon of the GFP mRNA and having the target binding region spanning at least 12 nucleotides towards the RBS are sufficient to completely inhibit protein translation. This work has demonstrated great capacity to create a very effective system for fine tuning gene expression in bacteria.
Our Design:
1. Creating a fluorescent reporting system:
Firstly, we needed to create a reporting system to allow us to visualise changes in gene expression. To do so, we used the plasmid BioBrick pSB4K5 part to create two plasmid fluorescent reporter system, one plasmid containing part pSB1C3 and another plasmid containing pSB4K5. This part contains part BBa_I50042 which is a pSC101 replication origin and is compatible with the minimal pUC-derived high copy replication origin found in part BBa_I50022 composite part pSB1C3 and thus suitable for E.coli transformation simultaneously with two plasmids. This resulted in our BioBrick pSB4K5_BBa_K2968010 and BioBrick pSB4K5_BBa_K2968011. Both of these parts contain the GFP gene as well as the ribosomal binding sites. What distinguishes the two parts is the strength of the ribosomal binding sites; pSB4K5_BBa_K2968010 contains contains medium promoter from the constitutive promoter family combined with strong ribosomal binding sites (PR4) and GFP. Hence, they target against either BBa_B0032 or BBa_B0034 RBSs respectively. pSB4K5_BBa_K2968011 contains medium promoter and a strong ribosomal binding site (RP5). These parts were created using BBa_K2968013 and BBa_K2968014 respectively.
2. Synthesising the sRNA BioBricks:
We have created nine total sRNA BioBricks. Three of these BioBricks are negative controls and do not contain any sRNA. The remaining six are plasmids that contain the sRNA and underwent double transformation with the plasmid containing the fluorescent reporter system to demonstrate the effect of the sRNA on gene expression. To do so, we used the plasmid backbone pSB1C3, which is a high copy number plasmid backbone. Our sRNAs GcvB, Spot 42, and RprA were synthesised into the pSB1C3. Each sRNA was used to produce three BioBricks; one as the negative control, one with complementarity to BBa_K2968013 (hence the medium strength ribosome binding site), and one with complementarity BBa_K2968014 (hence the strong ribosomal binding site). For the GcvB sRNAs BioBricks, this was done using GcvB_BBa_E0040_BBa_B0032 and GcvB_BBa_E0040_BBa_B0034 which showed complementarity to BBa_K2968013 and BBa_K2968014 respectively. For the Spot42 sRNA BioBricks, this was done with Spot42_BBa_E0040_BBa_B0032 and Spot42_BBa_E0040_BBa_B0034 respectively. For the RprA sRNA BioBricks, this was done with RprA_BBa_E0040_BBa_B0032 and RprA_BBa_E0040_BBa_B0034 respectively. This allowed us to demonstrate the effect of different sRNAs on gene expression with genes of varying degrees of ribosomal binding site.
3. Double transformation and carrying out our measurements:
Once we had synthesised our plasmids, they underwent double transformation. This was carried out for all of the sRNA plasmids. Hence, we sRNA BioBrick underwent double transformation with both BioBrick pSB4K5_BBa_K2968010 and BioBrick pSB4K5_BBa_K2968011. We transformed XL1-Blue E.coli cells with both the reporter plasmids and the sRNA targeting the first two codons of the GFP gene simultaneously. We repeated this transformation with the same sRNA parts but with 3 different promoter strengths. In order to measure the level of the GFP protein fluorescence in E.coli with each sRNA BioBrick, we measured the optical density and GFP Fluorescence of XL1 Blue E.coli cell cultures harboring both the fluorescent reporter plasmid and the sRNA BioBrick. Each experiment was performed in duplicate with three different sRNA BioBricks; GcvB, Spot42 and RprA.
4. Creation of our software tools:
Our software tools use the data produced by the wet lab constructs to determine their usability in the construction of viral capsids. This is done via our model, which was incorporated into the second software tool, CapsidBuilder. The model determines the translation efficiencies of our constructs, or models the translation initiation process. In doing so, one can determine which of our constructs (hence which promoter and ribosomal binding site) can be used to construct novel viral capsids.
