RBS.2 (weak) -- derivative of BBa_0030
>Internal Priming Screening Characterization of BBa_B0031: Has no possible internal priming sites between this BioBrick part and the VF2 or the VR primer.
The 2018 Hawaii iGEM team evaluated the 40 most frequently used BioBricks and ran them through an internal priming screening process that we developed using the BLAST program tool. Out of the 40 BioBricks we evaluated, 10 of them showed possible internal priming of either the VF2 or VR primers and sometime even both. The data set has a range of sequence lengths from as small as 12 bases to as large as 1,210 bases. We experienced the issue of possible internal priming during the sequence verification process of our own BBa_K2574001 BioBrick and in the cloning process to express the part as a fusion protein. BBa_K2574001 is a composite part containing a VLP forming Gag protein sequence attached to a frequently used RFP part (BBa_E1010). We conducted a PCR amplification of the Gag-RFP insert using the VF2 and VR primers on the ligation product (pSB1C3 ligated to the Gag + RFP). This amplicon would serve as template for another PCR where we would add the NcoI and BamHI restriction enzyme sites through new primers for ligation into pET14b and subsequent induced expression. Despite gel confirming a rather large, approximately 2.1 kb insert band, our sequencing results with the VR primer and BamHI RFP reverse primer gave mixed results. Both should have displayed the end of the RFP, but the VR primer revealed the end of the Gag. Analysis of the VR primer on the Gag-RFP sequence revealed several sites where the VR primer could have annealed with ~9 - 12 bp of complementarity. Internal priming of forward and reverse primers can be detrimental to an iGEM project because you can never be sure if the desired construct was correctly inserted into the BioBrick plasmid without a successful sequence verification.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
(Relative to BBa_B0034)
Thessaloniki 2019's Characterization
Aiming at the characterization of the LacZα peptide of β-Galactosidase (Part:BBa_I732006) under the regulation of various RBS parts from the Community RBS Collection of the Registry and two consitutive Anderson Family Promoters (Part:BBa_J23100 & Part:BBa_J23102), Thessaloniki 2019 measured the strength of RBS Part:BBa_B0031, relatively to Part:BBa_B0030, Part:BBa_B0032, Part:BBa_B0033 and Part:BBa_B0034, by conducting a colorimetric β-Galactosidase assay.
β-Galactosidase is an enzyme that is commonly used as a reporter marker to monitor gene expression. It is encoded by the LacZ gene and its function in the cell is to cleave lactose to glucose and galactose. β-Galactosidase assay builds on the α-complementation phenomenon, according to which the LacZ enzyme splits into two peptides, LacZα and LacZω, neither of which is active by itself. Activation of the enzyme occurs when these two peptides reassemble and form a single unit of the Galactosidase enzyme. In E. coli strains such as DH5α and XL1-Blue the mutated LacZω fragment is naturally found in the bacterial genome, so when a vector containing the LacZα fragment is inserted through bacterial transformation, an active form of the β-Galactosidase unit that can cleave its respected substrates can be formed. The strength of a certain RBS, being related to gene expression, can be measured via the expression of this universally used reporter.
LacZ’s activity can be quantified using an artificial substrate such o-nitrophenyl-beta-d-galactopyranoside (ONPG). This synthetic compound is also cleaved to yield galactose and o-nitrophenol which has a yellow color. When ONPG is in excess over the enzyme in a reaction, the production of o-nitrophenol per unit time is proportional to the concentration of beta-Galactosidase. Thus, the production of yellow color can be used to determine enzyme concentration and, therefore, strength of the examined RBS, since its function is immediately related to gene expression.
Miller Units are the units of measurement used β-Galactosidase assays, named after Jeffrey Miller who introduced the protocol concerning the determination of β-Galactosidase activity.
To achieve measurable response of the enzyme’s activity, we inserted the coding sequence for the LacZα fragment (Part:BBa_I732006) into a universal promoter (Part:BBa_J23100) as well as a second promoter (Part:BBa_J23102), followed by a universal RBS (Part:BBa_B0034). Constructs containing the universal promoter were followed by the rest of the RBS parts of the Community RBS Collection available in the iGEM Distribution kit for 2019 (Part:BBa_B0030, (Part:BBa_B0031), Part:BBa_B0032 & Part:BBa_B0033) were also assembled to obtain comparable results. A bi-directional terminator was added (Part:BBa_B0015) and the constructs were inserted into a high copy number pSB1C3 vector. 3A assembly was followed for the creation of all constructs and the produced vector was then transformed and expressed into E. coli DH5α cells.
A detailed version of the protocol we used regarding the β-Glactosidase assay can be found on our wiki here.
Reaction time for sample with vector containing promoter BBa_J23102 was 2 hours, while for the rest of the samples reaction time was 4 hours. Results were obtained using a plate reader measuring in 420nm to detect the yellow colour of o-nitrophenol.
Figure 2. Miller Units of β-Galactosidase assay. Results demonstrate the expression strength of coding sequence LacZα as a result of RBS strength. Highlighted is the output of BBa_B0031. P100 indicates the use of Promoter BBa_J23100 while P102 indicates the use of Promoter BBa_J23102. RBS NN indicates the use of a RBS as in Part:BBa_B00NN.
Figure 3. Miller Units β-Galactosidase assay normalized to BBa_B0034. Results demonstrate the expression strength of coding sequence LacZα as a result of RBS strength in relation to strength of universal RBS BBa_B0034. Highlighted are the outputs of BBa_B0031 and BBa_B0034. P100 indicates the use of Promoter BBa_J23100 while P102 indicates the use of Promoter BBa_J23102. RBS NN indicates the use of a RBS as in Part:BBa_B00NN.