Difference between revisions of "Part:BBa I732006"

Revision as of 00:58, 21 October 2019

lacZ alpha fragment

In strains with lacZ-omega (lacZ N-terminal deletion mutant) like DH5alpha, DH10B and Top10, lacZ-alpha restores the beta-galactosidase activity.

Sequence and Features

Characterization of lacZalpha in a cell-free chassis

In order to improve the characterization of the lacZalpha fragment, we characterized it in a T7-M15 cell lysate to see whether we obtain high levels of absorbance to use beta-galactosidase and alpha complemetation as our downstream reporter scheme in further experiments. M15 cells have a lacZ delta mutation which makes them encode a form of beta-galactosidase lacking residues 11-41. Beta-galactosidase produced without those residues is missing a small part and is thus not functional.

But if this mutated form of beta-galactosidase is brought together with the missing lacZ alpha part (which we express in the lysate in this case), the two will connect and form a functional beta-galactosidase part. In fact beta-galactosidase is a tetramer, it needs for units of LacZalpha and the rest to assemble in order to function. The figure below shows the expression of a functional beta-galactosidase in T7-M15 cells upon the assembly of the differents alpha and omega parts.

Improvements

This part was improved with a T7 promoter and terminator : BBa_K2203002 . This part is also used as a reporter in the following composite parts which are toehold switches : BBa_K2203003 , BBa_K2203004 , BBa_K2203005 , BBa_K2203006 .

Thessaloniki 2019's 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 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.

Results

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.