In order to enable precise quantification of promoter strengths, we designed a sophisticated measurement system (pCeBiTec18) comprising two independent reporters for accurate normalization. Fluorescence measurements are a common way to determine promoter strengths but are influenced by plasmid copy number and other factors. Having a reference reporter in the plasmid backbone allows correction of many effects. This system was subjected to absolute expression quantification in multiple replicates. Thus, our system equips every iGEM team with a general and easily applicable tool to analyze the expression strength of any promoter-RBS combination. pCeBiTec18 comes along with a detailed protocol for fast and efficient measurement. This system provides an urgently needed universal standard to compare promoter strengths.
Figure 1: Ferritin is suitable for metal recycling, since it can form e.g. iron, silver and gold nanoparticles in its cavity.
Design of the promoter-RBS measurement construct
When working on our project we tested the expression strength of different promoter and RBS combinations for an optimal knock down of the gene expression to a certain level or the optimal expression. To test our various promoter and RBS combinations we constructed a gene construct with which we are able to compare the expression strength of different promoter-RBS combinations. Therefore, we modified the backbone of pSB1C3 by inserting a reporter gene (eCFP BBa_) under the control of a certain Promoter (BBa_), RBS (BBa_) and two terminators (BBa_). This change in the backbone enables the comparison and validation of expression strength of a second reporter gene (mRFP (BBa_)) as insert. The expression of the insert is regulated by a variable promoter-RBS (Library Linkt) combination and terminated by two terminators.
Figure 2: Alignment of the protein sequences of the wild-type and the mutated human ferritin heavy chain. The Alignment was produced with Clustal Omega (Goujon et al., 2010, Sievers et al., 2011).
The expression level of CFP measured through the fluorescence at 435 nm is used as a reference for the measured expression of mRFP at 485 nm.
The differential of the detected signals of both reporter proteins is an easy measurable scaling of the expression strength of different promoter-RBS combinations and enables the comparison even under different cultivation conditions.
Figure 3: Protein structures of the wild-type human ferritin (A, RCSB ID 4oYN) and the mutated human ferritin (B, RCSB ID 3ES3). Despite the mutations of ten amino acids the ferritin retains its shape. The protein structeres were generated with Chimera (Pettersen et al., 2004).
With these changes in the pSB1C3 backbone every iGEM team can fast and easily analyze the expression strength of their used promoter-RBS combination and is also able to compare the results, measured under different cultivation conditions or in different laboratory setups.
Based on our promoter-RBS library we also generated a visualization of expression strengths of different promoter-RBS combinations by which other teams are able to compare their used promoter-RBS complex to the ones tested by us.
Test your own Promoter RBS
A strong promoter leads to an increased transcription level and therefore often to high level of gene expression, but a strong expression is not always the best choice for your construct.
If you cannot decide, whether your choice of promoter and RBS offers the optimal level of gene expression, you can use our new designed vector to test your own promoter-RBS construct.
The only thing you have to do is to exchange the promoter-RBS combination of the vectors insert with your sequence. This can easily be done by restriction digest with XbaI and EcoRI to remove our insert. To insert the construct which should be tested oligonucleotides containing the construct are dimerized. These oligonucleotides contain an overhang to the backbone. Thus, the oligonucleotide dimer could be cloned in the backbone by Gibson assembly.
With the cultures containing this measurement plasmid, you can easily analyze the strength of expression of your promoter-RBS constructs measuring the fluorescence of the mRFP. You can also compare different promoter-RBS combinations by scaling the different mRFP signals through the detected eCFP signal. This even allows you to compare measurements, performed under different cultivation conditions.
Figure 4: Possible applications of nanoparticles produced with ferritin.
Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
