Overview
To make contribution for future iGEM teams, we did some useful work. First, we added some new documentation learned from literatures to an existing part K4167660 (Part: BBa K4167660 ), as well as new data of this part collected from our experiments. We have documented them on the Part’s Main Page on the Registry. In addition, we created a 3D printed hardware for our detector which would be used in refrigerator for detecting bacteria to remind people cleaning or disinfecting refrigerator. All of which are shown as follows:
1. Improvement of the existing part BBa_K4167660
1.1 New documentation of BBa_K4167660 collected from literatures
LacZ protein, also known as β-galactosidase, is an enzyme encoded by the lacZ gene of Escherichia coli. β-Galactosidase (β-gal) has been widely used as a transgene reporter enzyme. This enzyme can hydrolyze lactose and its derivatives (such as X-gal) to produce galactose and glucose. Due to its catalytic activity and easy detection characteristics, lacZ protein is widely used in molecular biology and genetic engineering, especially as a reporter gene.
1.1.1 The main structural characteristics of LacZ protein
(1) Tetrameric structure: LacZ protein is a homologous tetramer with a molecular weight of approximately 116 kDa per subunit. The four subunits interact to form a stable tetramer structure, which is essential for their catalytic activity.
(2) Active site: Each subunit contains an active site that can bind to substrates and catalyze reactions.
(3) Substrate binding pocket: A substrate binding pocket is formed around the active site, which specifically recognizes and binds lactose or its analogues.
Fig.1 Structural simulation diagram of LacZ protein.
1.1.2 The main functions of LacZ protein
LacZ protein has multiple important functions in lactose metabolism and molecular biology experiments in E. coli:
(1) Lactose metabolism: In E. coli, lacZ protein hydrolyzes lactose to produce galactose and glucose, providing the carbon source and energy required for cellular metabolism.
(2) Reporter gene: Due to its catalytic activity, lacZ is often used as a reporter gene. For example, in the blue white screening, as a substrate, X-gal is hydrolyzed by lacZ to produce a blue product, which is used to distinguish between recombinant and non-recombinant clones.
(3) Gene expression analysis: By measuring lacZ activity, the regulatory mechanism and level of gene expression can be studied.
1.1.3 LacZ protein activity assay method
The following methods are commonly used to study the activity and function of lacZ protein:
(1) ONPG decomposition experiment:
Principle: Using ONPG (ortho nitrophenyl-β-D-galactosidase) as a substrate, lacZ catalyzes its decomposition to produce the yellow product ortho nitrophenol.
Step: React the cell lysate with ONPG and measure the absorbance changes using a spectrophotometer.
Result: The change in absorbance is directly proportional to the activity of lacZ.
(2) X-gal color rendering experiment:
Principle: X-gal generates blue insoluble products under lacZ catalysis, which are easy to observe with the naked eye.
Step: Cultivate bacteria containing the lacZ gene on a medium containing X-gal, and determine lacZ activity by observing blue colonies.
Result: Blue colonies indicate active expression of the lacZ gene.
(3) Fluorescent substrate experiment:
Principle: Using fluorescently labeled substrates (such as MUG), lacZ catalyzes the generation of fluorescent products.
Step: Mix the substrate with the sample and measure the fluorescence intensity using a fluorescence spectrophotometer.
Result: Fluorescence intensity is directly proportional to lacZ activity.
Fig. 2. Being a reporter gene, β-Gal staining in the whole brain and in the kidney of LacZ transgenic mice.
(A) The whole brain showed very strong lacZ expression. In the cere bellum, strong lacZ signals are present in granular layer and white matter. In the brainstem, strong lacZ expression is also detected (Left panel). Enlargement of the area within the rectangle (Right panel). (B) In the kidney, strong expression of lacZ was observed in the tubules, all the cells of the medulla, and the papillae of the kidney (kidney cross-section (left panel), enlarged view of medulla(right panel)). Weaker expression was detectable in the pelvis, glomeruli, and blood vessels.
References
[1] https://www.bilibili.com/read/cv36137131/?jump_opus=1
[2] Zhang GJ, Chen TB, Connolly B, Lin SA, Hargreaves R, Vanko A, Bednar B, Macneil DJ, Sur C, Williams DL. In vivo optical imaging of LacZ expression using lacZ transgenic mice. Assay Drug Dev Technol. 2009 Aug;7(4):391-9. doi: 10.1089/adt.2009.0195.
1.2 New data of BBa_ K4167660 collected from our lab
In our experiment, to identify whether the LacZ gene of recombinant plasmid express or not，both the complementary fragment of RNA aptamer and the plasmid pET-28a-Esch-lacZ were transformed into BL21DLacZ strain (with LacZ deletion). After culture 12 h with fresh LB medium containing Kanamycin and Ampicillin, the LacZ protein (β-galactosidase) was purified using 6x His tag column for identification. The SDS-PAGE electrophoresis result was shown in Fig. 3.
Fig.3 The SDS-PAGE result shows expression and purification of LacZ protein. M: Marker, 1: All supernatant proteins of BL21DLacZ strain, 2: All supernatant proteins of BL21DLacZ strain transformed with pET-28a-Esch-LacZ and the complementary fragment of E. coli aptamer, 3: Purified LacZ protein from the supernatant proteins of sample 2.
Return to index
2. Creation a 3D printed hardware for our detector
We created a 3D printed hardware for our detector which would be used in refrigerator for detecting bacteria to remind people cleaning or disinfecting refrigerator. The printed hardware is a box which accommodates main board, breadboard, Dupont wire, RGB color sensor and cell free system hydrogel constructed in our project.
Firstly, we drafted a hardware sketch that outlines its external dimensions, wall thickness, internal partitions, and lid requirements, etc., which is shown in the following Fig.4.
Fig.4 A draft of a hardware sketch.
Then we used the software Blender 4.3.0 to build a 3D model (Fig.5).
Fig.5 The front cover of Blender software we used.
Using this software, we construct a basic cube based on external dimensions, adjust wall thickness by inserting surfaces, construct a basic box by excavating and inserting surfaces, create internal structure by subdividing the bottom surface, construct partitions by pulling up subdivided rectangles, project a circle on the side and excavate a circular hole, and finally automatic vertex adsorption and triangulation. The 3D model under construction is shown bellows (Fig.6 and Fig.7) .
Fig.6 The 3D model under construction.
Fig.7 The 3D model displaying its appearance size.
After completion building of 3D model, we printed it. In order to understand the functions of each part, we use Fig.8 and Fig.9 for illustration.
Fig.8 The functions of each part of printed 3D model.
Fig.9 The printed 3D model with iGEM sticker and our team’s name.
For the application scenarios of this 3D model, please refer to our hardware page.
Return to index