Difference between revisions of "Team:UI Indonesia/InterLab"

Revision as of 09:54, 26 July 2018

InterLab Studies

Introduction

In the field of engineering, repeatable and reproducible measurements are important to obtain valid and reliable results. However, these have been proven difficult to achieve as there are differences in environmental condition of laboratories, individuals conducting the measurements, instruments being used, and other sources of variability. Eventually, this could lead to hindrance of advancements in engineering, including synthetic biology.

For past several years, iGEM Measurement Committee has been working on this issue by encouraging registered iGEM 2018 teams to participate in annual InterLab study. Since its introduction in 2014, the study has been conducted four times, making this year’s study to be the fifth one. The goal of this study is to minimize possible sources of variability in laboratory measurements and thus allowing synthetic biology to attain its full potential as a tool for improving quality of life.

InterLab study is mainly focused on fluorescence measurements as one of widely utilized protocols in synthetic biology studies. Data obtained from such measurements are often reported in different units, or processed in different methods, thereby hampering fluorescence data comparison. Hence, iGEM Measurement Committee introduces a standardized protocol for green fluorescence protein (GFP) expression level measurement. Previous studies showed that variability of the measurements can be significantly reduced by calibrating measured absolute fluorescence units of expressed GFP against known concentration of florescent molecule. However, when the procedure is carried out against a population of cells, the cell number in given sample makes great variability among measurements. This is due to the values of total cell number used to calculate mean expressed GFP per cell are obtained from optical density (OD), which is a subject to large variability.

Therefore, in this year’s InterLab study, participating 2018 iGEM teams are encouraged to help iGEM Measurement Committee in investigating whether more direct method in expressing total cell number for fluorescence calculation, such as absolute cell count or colony-forming units (CFUs), are better than OD to reduce variability in bulk measurement. We proudly announce our participation in 5th InterLab study for the first time ever, in the hope that our results may contribute to the improvements in synthetic biology. Our members contributing in this InterLab study are shown in Figure 1.

Materials and Equipments

Some of the materials for this study are obtained from 2018 DNA Distribution Kit. Other materials and equipment are provided by Institute of Human Virology and Cancer Biology (IHVCB), UI.

Materials from 2018 DNA Distribution Kit
• LUDOX CL-X (45% colloidal silica suspension) stock
• Microspheres (silica beads suspension) stock ~ 4.7 x 108 microspheres
• Sodium fluorescein stock
• Devices (parts in pSB1C3 plasmid backbone, all dried in Distribution Kit Plate): negative control, positive control, test device 1, test device 2, test device 3, test device 4, test device 5, and test device 6.

Materials and equipment provided by IHVCB, UI:
• ddH2O
• 1x phosphate buffered saline (PBS), pH 7.4-7.6
• 96-well plate (clear plate with flat bottom)
• 96-well plate reader (GloMax®– Multi Detection System, Figure 2). Specifications: can measure both absorbance and fluorescence, no settings for pathlength correction and temperature adjustment, has four installed filters and two customizable filter holders in six-position filter wheel, reads the samples from top of the plate
• Competent cells of Escherichia coli strain DH5α
• Luria-Bertani (LB) media, liquid and agar
• Chloramphenicol (dissolved in absolute ethanol at concentration of 25 mg/mL, when added into LB media it should be at ratio 1:1000)
• 50 ml Falcon tubes
• Incubator (set at 37oC)
• 1.5 ml microtubes
• Bucket with ice
• Micropipettes and tips

Methods

Safety cautions

During laboratory works, we took extra precautions to minimize potential harm. We wore personal protective equipment (PPE) such as laboratory safety gowns with head cover, masks, and gloves (Figure 3, left). For procedures that require aseptic technique such as bacterial inoculation, we conducted the experiments in biosafety cabinet (BSC) to prevent contamination (Figure 3, right). We also managed every infectious waste according to our laboratory’s standard operating procedures, such as decontaminating all media used for bacterial growth with 10% sodium hypochlorite overnight before being thrown into appropriate container and autoclaving them (121^oC, 20 minutes) along with other infectious wastes. In addition, to ensure validity of our results, we used same instrument settings, 96-well plates, and volumes of sample added to the plates in entire protocol whenever possible.

Part 1: Calibration

Before we proceeded to Part II and III of the protocol, we had made three calibrations as follows:

• Calibration #1: this calibration is aimed to obtain a conversion factor to transform absorbance at 600 nm wavelength (Abs₆₀₀₆₀₀ as would be measured with spectrophotometer due to Abs₆₀₀ data are volume dependent. First, we made four replicates of LUDOX CL-X and ddH₂O in 96-well plate (each well contains 100 uL volume). We then measured Abs₆₀₀ of the samples and recorded them. The Abs₆₀₀ data were calculated for their respective mean and then subtracted each other to obtain corrected Abs₆₀₀. OD₆₀₀ measured with spectrophotometer (from reference) was divided with corrected Abs₆₀₀ to get the conversion factor.

OUR PROJECT

To be added

RESULTS AND DISCUSSIONS

To be added

Team UI Indonesia
igemui2018@gmail.com

@@ Line 160: / Line 160: @@
    <h3><b>Materials and Equipments<b></h3>
    <h5>Some of the materials for this study are obtained from 2018 DNA Distribution Kit. Other materials and equipment are provided by Institute of Human Virology and Cancer Biology (IHVCB), UI.
+<br>
 <br>
 <i>Materials from 2018 DNA Distribution Kit</i>
@@ Line 182: / Line 183: @@
 <br>•	Micropipettes and tips
 </h5><br>
-  <h5>Tar chemoreceptor mediates E. coli movement away from nickel and cobalt (repellent molecules), and towards aspartate and maltose (attractant molecules). Its cytoplasmic domain is associated with two proteins, CheW and CheA. CheW mediates signal transduction from Tar chemoreceptor to CheA, while CheA has a kinase domain which autophosphorylates its own histidyl residue. Tar chemoreceptor undergoes conformational change upon repellent molecule binding, leading to CheA activation and thus transfers its phosphoryl group to CheY, a regulatory protein that phosphorylates FliM in basal body of bacterial flagellum. These processes eventually lead the bacterium to swim smoothly away from repellent substance. On the other hand, attractant molecule binding into Tar chemoreceptor inhibits CheA and thus phosphorylation of CheY and FliM will not happen. This causes the bacterial flagellum to rotate in opposite direction and facilitates the bacterium to swim towards attractant substance.</h5><br>
-   <h3><b>LuxAB-eYFP Fluorescence Resonance Energy Transfer (FRET) System<b></h3>
+   <h3> Methods </h3>
-   <h5>Basically, a molecule is excited to higher energy state when it absorbs a photon energy. This molecule relaxes back to ground state when the energy is emitted back to the environment or transferred into another molecule. FRET is a phenomenon in which non-radioactive energy is transferred from excited donor molecule to acceptor molecule via dipole-dipole interactions. Molecules involved in this phenomenon are called fluorophores as they emit fluorescence according to their respective emission spectrum after absorbing higher photon energy. The fluorescence emission spectrum of donor fluorophore must overlap with the absorption and emission spectrum of acceptor fluorophore for FRET to occur. Furthermore, the efficiency of energy transfer is highly influenced by the physical proximity of interacting fluorophores, being the most efficient at several nanometers. Hence, FRET can be applicated to study the distance of macromolecules such as proteins at molecular level.</h5><br>
+  <h5><i>Safety cautions</i></h5>
-   <h5>LuxAB and eYFP are one of the most widely studied paired fluorophores. In this case, LuxAB is the donor fluorophore as it emits cyan colored light with relatively high energy (peak emission at 490 nm). eYFP serves as the acceptor fluorophore when in close contact with LuxAB, as it absorbs high energy from LuxAB that is overlapped with its own absorption spectrum and emits yellow colored light with lower energy (peak emission at 530 nm). To be utilized in macromolecules interaction studies, LuxAB and eYFP should be incorporated with the molecules of interest. When the molecules of interest are in contact, energy transfer between LuxAB and eYFP will happen and its efficiency can be measured with fluorescence-lifetime imaging microscopy method.</h5><br>
+   <h5>During laboratory works, we took extra precautions to minimize potential harm. We wore personal protective equipment (PPE) such as laboratory safety gowns with head cover, masks, and gloves (Figure 3, left). For procedures that require aseptic technique such as bacterial inoculation, we conducted the experiments in biosafety cabinet (BSC) to prevent contamination (Figure 3, right). We also managed every infectious waste according to our laboratory’s standard operating procedures, such as decontaminating all media used for bacterial growth with 10% sodium hypochlorite overnight before being thrown into appropriate container and autoclaving them (121<sup>o</sup>C, 20 minutes) along with other infectious wastes. In addition, to ensure validity of our results, we used same instrument settings, 96-well plates, and volumes of sample added to the plates in entire protocol whenever possible. </h5><br>
+   <h5><i>Part 1: Calibration</i></h5>
+  <h5>   Before we proceeded to Part II and III of the protocol, we had made three calibrations as follows:</h5><br>
+  <h5>•	Calibration #1: this calibration is aimed to obtain a conversion factor to transform absorbance at 600 nm wavelength (Abs<sub>600</sub) data from plate reader into comparable OD<sub>600</sub> as would be measured with spectrophotometer due to Abs<sub>600</sub> data are volume dependent. First, we made four replicates of LUDOX CL-X and ddH<sub>2</sub>O in 96-well plate (each well contains 100 uL volume). We then measured Abs<sub>600</sub> of the samples and recorded them. The Abs<sub>600</sub> data were calculated for their respective mean and then subtracted each other to obtain corrected Abs<sub>600</sub>. OD<sub>600</sub> measured with spectrophotometer (from reference) was divided with corrected Abs<sub>600</sub> to get the conversion factor.   </h5><br>
 </div>