This year we made the following contributions to the iGEM community:
1) Evaluating an enhanced growth medium to increase the plasmid DNA yields of low copy number vectors.
As part of our project, we needed to subclone fragments into low copy number vectors with pSC101 ori. Obtaining sufficient amounts of plasmid DNA using standard miniprep kits can be challenging in such cases. To address this, we consulted the literature in search of an easy, cost-effective, and efficient method to enhance yields. We came across a study (Wood WN, Smith KD, Ream JA, Lewis LK. Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells. J Microbiol Methods. 2017 Feb;133:46-51. doi: 10.1016/j.mimet.2016.12.016. Epub 2016 Dec 23. PMID: 28024984; PMCID: PMC5286560) which suggested that cultivating cells in LB medium with elevated levels of yeast extract can significantly improve plasmid DNA recovery.
To test this hypothesis, we prepared two types of media: regular LB medium (10 g Tryptone, 10 g NaCl, and 5 g Yeast Extract per liter) and LB medium with increased yeast extract levels (10 g Tryptone, 10 g NaCl, and 24 g Yeast Extract per liter). Subsequently, we initiated overnight cultures of E. coli DH10B cells carrying the low copy number SC101 ori-based vector pSB4K5 (kanamycin resistance, using half the regular amount of the antibiotic). On the next morning, we isolated pDNA from both cultures using the Wizard Plus SV Minipreps DNA Purification system (Promega) following the provided protocol with no modifications. While pelleting the cells in the initial step, we observed that the LB medium with increased levels of yeast extract had higher cell densities and more intensive red colour.
Impacts of the elevated yeast extract levels on the cultivation of cells carrying pSB4K5
Encouraged by this initial observation, we proceeded with the procedure, ensuring that cell pellets of approximately equal sizes were used for both conditions. However, to our surprise, upon completing the procedure and measuring the yields with the Quantus fluorimeter (Promega), no difference was evident; both growth mediums yielded results in the range of 15-20 ng/μl. We repeated the experiment two more times with new cultures and obtained identical results. Our conclusion is that the suggested medium is not effective for purifying low copy number plasmid backbones from the Registry, such as pSB4K5-SC101. Nevertheless, it is apparent that increased levels of yeast extract can be advantageous when producing chromoproteins from low copy number vectors, as illustrated in the figure above for mRFP1.
2) Building an expression construct for a dual gene-specific mutator system capable of introducing transition mutations at consistent frequencies within a target sequence in vivo
To achieve the most important of our goals for this year, we had to improve the efficiency of the NDM-2&5 MBL enzyme via directed evolution. To introduce the required random mutations in vivo, we decided to utilize a dual gene-specific mutator system that was available for ordering from Addgene in the form of 3 separate plasmids (Seo D, Koh B, Eom GE, Kim HW, Kim S. A dual gene-specific mutator system installs all transition mutations at similar frequencies in vivo. Nucleic Acids Res. 2023 Jun 9;51(10):e59. doi: 10.1093/nar/gkad266. PMID: 37070179; PMCID: PMC10250238). To examine its efficiency, we built a test construct using the following parts of the Registry: T7 promoter and medium RBS (BBa_K525998) and amilGFP (BBa_K592010).
Test construct for T7 driven in vivo mutagenesis
In our experiments, this combination has demonstrated its effectiveness, reducing the required compounds when compared to the original paper, which utilized the promoter region from a pET vector, necessitating also the addition of IPTG for efficient mutagenesis.
3) Measuring the efficiency of the blaVIM-2 part from the registry for ampicillin degradation
The iGEM Bulgaria 2022 team constructed an expression vector featuring a strong constitutive promoter, a medium RBS, and the blaVIM-2 coding sequence (BBa_K4364001). They subsequently demonstrated that cell-free extracts from E. coli strains harboring this construct could effectively degrade carbapenems in water samples. In our work this year, we characterized the same construct by employing the cell-free extract to address ampicillin contamination. When applying 500 μl of the extract to a sample containing 1 mg of ampicillin and incubating it at 37°C for 45 minutes, a noticeable reduction in antibiotic levels could be observed using the agar well dilution method.
Diminishing ampicillin levels following VIM-2 treatment – the agar wells were loaded with serial 2-fold dilutions
4) Technical Report: Microfluidic Chip Incubator for Synthetic Biology Students
Introduction
The Microfluidic Chip Incubator is designed to maintain an optimal temperature of 37°C for cell cultures. This is crucial for various cellular processes and reactions that are most efficient at this temperature. The device aims to create a stable environment that mimics the human body's temperature, facilitating scientific research, medical diagnostics, and pharmaceutical studies that require specific temperature conditions for cell cultivation.
Objective
The primary goal is to provide a stable and controlled environment for cell growth, thereby accelerating the data collection process for cell growth studies. This is achieved by creating "ideal conditions" for the cells to thrive.
Components and Functionality
Schematic of the elements of the purposed Incubator principle is shown in Figure 1.
Figure 1 CAD Schematic of the disassembled parts of the geometry. 1- Incubator Lid; 2 - Aluminum Plate for direct heating; 3 - Compartment for the heating element and sensor; 4 - Space for the temperature controller.
A suitable heating element was selected to generate the required heat. The latter is placed together with a thermal sensor, beneath the metal plate (2), which is used to transfer the heat to the microfluidic chip. Aluminum was chosen for its even heat distribution and good conductivity. A relay is used to turn the heating element on and off based on the temperature readings (4). It acts as an electromechanical switch controlled by an electrical signal. The temperature is measured via a thermistor (Negative Temperature Coefficient type) is used to measure the temperature. To simplify the setup, a pre-assembled temperature controller was chosen. This controller integrates all the necessary electrical components, including the relay and operational amplifiers.
When the temperature in (3) rises above the set point, the thermistor's resistance decreases, causing the relay to turn off the heating element. Conversely, when the temperature falls below the set point, the relay turns on the heating element. The precise dimensions of the parts are given in the appendix below.
Device Fabrication
Some of the components mentioned previously were manufactured utilizing a fused deposition modeling (FDM) 3D printer, whereas for specific elements like the compartment housing the heating element and sensor (referred to as component 3), we opted for a resin-based 3D printer. This deliberate choice of 3D printing technologies provided us with the versatility to customize the fabrication process to the unique demands and attributes of each component. Moreover, for component 3, we selected a specialized type of resin. This decision was motivated by our objective to minimize heat loss through the walls of the compartment, thus ensuring the highest level of measurement precision.
Figure 2 During the fabrication process
Microfluidic chip
The microfluidic chip comprises a single, sizable chamber with dimensions of 40mm x 40mm x 0.8mm. This chamber serves as the platform for collecting and monitoring bacterial growth. The chip operates on the principle of capillary loading, where a sample is introduced through a 3mm x 5mm inlet and is drawn into the chamber by capillary pressure. Two outlets are integrated into the chip to provide a controlled exit for captured air in the chamber.
Conclusion
The Microfluidic Chip Incubator is an efficient and reliable device for maintaining cells at an optimal temperature of 37°C. Its design ensures quick and even heating, making it an invaluable tool for students in synthetic biology for their research and experiments.
Figure 3 An image showcasing the operational engineered device designed for cell incubation, featuring a microfluidic chip on top of it.