Team:Thessaloniki/Parts

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Parts

Parts

Overview

Part Name Type Description Length
BBa_B0031 Basic Characterization of the LacZα peptide of β-Galactosidase under the regulation of various RBS parts from the Community RBS Collection of the Registry and two consitutive Anderson Family Promoters. 14bp
BBa_I732006 Basic Characterization of the LacZα peptide of β-Galactosidase under the regulation of various RBS parts from the Community RBS Collection of the Registry and two consitutive Anderson Family Promoters. 14bp
BBa_K1477014 Composite Characterization of the LacZα peptide of β-Galactosidase under the regulation of various RBS parts from the Community RBS Collection of the Registry and two consitutive Anderson Family Promoters. 432bp
BBa_K3304000 Basic - Device Input Gate with p65 consensus sequence 97bp
BBa_K3304001 Basic - Device Join Gate for the p65 specific CRN 93 bp
BBa_K3304003 Basic - DNA Reporter complex for output TCC 27bp
BBa_K3304006 Basic - DNA Fork Gate of the formal CRN 114 bp
BBa_K3304005 Basic - DNA DTG auxiliary strand 37 bp
BBa_K3304006 Basic - DNA BTD auxiliary strand 27 bp
BBa_K3304007 Basic - DNA NTB auxiliary strand 27 bp
BBa_K3304008 Basic - DNA Input TBB for our Join Gate 27 bp
BBa_K3304009 Basic - DNA Input TAA for our Join Gate 27 bp
BBa_K3304010 Basic - DNA Input strand TRR for our Join Gate 27 bp
BBa_K3304011 Basic - DNA Strand Displaced from input TBB - BTR 27 bp
BBa_K3304012 Basic - DNA Strand RTQ that links Join and Fork Gates 27 bp
BBa_K3304013 Basic - DNA CTB auxiliary strand 27 bp
BBa_K3304014 Basic - DNA ITC auxiliary strand 27 bp
BBa_K3304015 Basic - DNA Output TCC 27 bp
BBa_K3304105 Basic - Device Input Gate with ELK1 sequence 91 bp
BBa_K3304106 Basic - Device Join Gate for the ELK1 specific CRN 87 bp
BBa_K3304107 Basic - DNA Fork Gate of the ELK1 CRN 108 bp
BBa_K3304108 Basic - DNA BTD auxiliary strand for our ELK1 Input Gate 27 bp
BBa_K3304109 Basic - DNA NTB auxiliary strand for our ELK1 Input Gate 27 bp
BBa_K3304110 Basic - DNA Input TBB for our ELK1 Join Gate 27 bp
BBa_K3304111 Basic - DNA Input TAA for our ELK1 Join Gate 27 bp
BBa_K3304112 Basic - DNA Input strand TRR for our ELK1 Join Gate 27 bp
BBa_K3304113 Basic - DNA RTQ auxiliary strand- ELK1 that links Join and Fork Gates 27 bp
BBa_K3304114 Basic - DNA CTB auxiliary strand for ELK1 27 bp
BBa_K3304115 Basic - DNA ITC auxiliary strand for ELK1 27 bp
BBa_K3304116 Basic - DNA Output TCC for ELK1 27 bp
BBa_K3304117 Basic - DNA Strand Displaced from input ELK1 TBB - BTR 27 bp
BBa_K3304118 Basic - DNA Single stranded auxiliary strand for our ELK1 Input Gate - DTG 37 bp
BBa_K3304100 Composite Expression strength measurement vector EYFP/ECFP paired 1895bp

Characterization

Goal

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 expression of the coding sequence of the LacZα fragment under the regulation of different RBS parts Part:BBa_B0031, Part:BBa_B0030, Part:BBa_B0032, Part:BBa_B0033 and Part:BBa_B0034, by conducting a colorimetric β-Galactosidase assay.

Methods

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 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 1: β-Galactosidase assay. "B00NN" indicates the RBS part (as in Part:BBa_B00NN) used with promoter BBa_J23100, while "J23102" indicates the use of a vector containing promoter BBa_J23102 with universal RBS BBa_B0034. Increased strength of expression is indicated by yellow colour.
Figure 2 Miller Units of β-Galactosidase assay. Results demonstrate the expression strength of coding sequence LacZα as a result of RBS strength. 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 is the output of 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.

Improvement

Goal

Designing and experimentally validating a foundational advance project that aims at a better understanding of the transcription factors’ binding to absolute DNA sequences, we accept the significance of deeply embedding the regulation of gene expression.

Regarding the construction of regulatory devices, identification of the appropriate Promoter - RBS combination is of pivotal importance. Inspired by iGEM Bielefeld_CeBiTec 2018 team, who through testing the strength of a single promoter, a single RBS and their combination, designed a promoter-RBS library and a suitable measurement system to analyze the expression strength of the chosen promoter-RBS combination, we concluded in improving their part by choosing an advanced fluorescent protein match for the measurement.

It has already been stated that the measurement of a vector carrying two reporter genes is being conducted via normalization of the measured promoter - RBS’ strength expression to a constant expression level of the reference reporter gene. The fluorescent proteins that are going to be encoded by the aforementioned reporter genes ought to fulfill four criteria. Those criteria regard its bright fluorescence signal, its spectrally distinguishable excitation and emission, its similar maturation rates with the fluorescent protein tested and its similar DNA sequence close to the upstream promoter.

Evaluating the alternative fluorescent proteins by these criteria, we concluded inserting in our construct the EYFP fluorescent protein whose emission and absorption spectra do not interact with their ECFP’s equivalents. Additionally, the EYFP and ECFP fluorescent proteins have very similar sequences as well as maturation half-lives.

After selecting the fluorescent protein that aimed to replace the mRFP in the BBa_K2638560 part, we performed the following experiments to quantitatively describe the expression strength of the different promoter-RBS combinations.

Methods

Aiming at improving the BBa_K2638560 part by replacing the mRFP with an EYFP fluorescent protein, we conducted experiments including the cloning and creation of the new composed part in a PSB1C3 vector, the transformation and overnight incubation in DH5aplha competent cells and the measurement of the chosen colonies’ fluorescence using a Real-Time PCR.

Best Part Collection

Part Name Type Description
BBa_K3304000 Basic - Device Input Gate with p65 consensus sequence
BBa_K3304001 Basic - Device Join Gate for the p65 specific CRN
BBa_K3304003 Basic - DNA Reporter complex for output TCC
BBa_K3304004 Basic - DNA Fork Gate of the formal CRN
BBa_K3304005 Basic - DNA DTG auxiliary strand
BBa_K3304006 Basic - DNA BTD auxiliary strand
BBa_K3304007 Basic - DNA NTB auxiliary strand
BBa_K3304008 Basic - DNA Input TBB for our Join Gate
BBa_K3304009 Basic - DNA Input TAA for our Join Gate
BBa_K3304010 Basic - DNA Input strand TRR for our Join Gate
BBa_K3304011 Basic - DNA Strand Displaced from input TBB - BTR
BBa_K3304012 Basic - DNA Strand RTQ that links Join and Fork Gates
BBa_K3304013 Basic - DNA CTB auxiliary strand
BBa_K3304014 Basic - DNA ITC auxiliary strand
BBa_K3304015 Basic - DNA Output TCC
BBa_K3304016 Composite Amil Cp with BsaI recognition sites that insert PvuII
BBa_K3304101 Basic Primer T(7)>A
BBa_K3304102 Basic Primer C(10)>T
BBa_K3304103 Basic Primer T(6)>A
BBa_K3304104 Basic Primer SNPForward