Team:KU ISTANBUL/Engineering
Engineering
A. Introduction to Experiments
Reflectin Proteins
Cephalopods (octopuses, squids, cuttlefish, etc.) often have an adaptation of demonstrating camouflage as a result of adaptive transparency. Some of the cephalopod species can vanish from the environment with their optical adaptation capabilities. Cephalopods owe these camouflage skills to their ability to change optical properties by how light is transmitted, absorbed, and reflected by their skin [1]. Specifically, reflectivity on their tissues are achieved by stacking flat, insoluble, structural platelets by alternating layers of high and low refractive index in iridocytes [2]. These alternate arrangements, called Bragg reflectors, create a thin-film interference pattern which is the reason for reflection of incident light from the tissue [1]. In aquatic animals, reflector platelets generally consist of purine crystals, particularly guanine and hypoxanthine; however, cephalopod reflector platelets contain reflectin proteins instead of these purine crystals. Reflectin proteins found in cephalopods are responsible for transparency abilities by employing structural coloration and iridescence [3, 4].
Optical characterization of these proteins is an important effort for studies involving reflectin proteins. However, it is hard to characterize the refractive index of these proteins since there are multiple Bragg stacks with unknown variation in spacings, refractive indices, and orientations in a typical tissue of a chalepod. Hence,the results of different studies can vary greatly although the refractive index of these proteins are generally found higher than ~1.40, up to ~1.62 [1, 2, 4]. High refractive index of reflectin proteins allows possible use in biotechnology as a novel biomaterial which can be used to design a resonator from proteins to create fully biological lasers.
We will be using following reflectin proteins:
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Reflectin A1 obtained from Loligo pealeii
Figure 1: Upper: The oval squid , WaterFrame / Getty Images.[5] Bottom Left: Hawaiian bobtail squid (Euprymna scolopes) lit up by luminescence, DOUG PERRINE/MINDEN PICTURES Bottom Right: Hawaiian bobtail squid (Euprymna scolopes), DOUG PERRINE / SEAPICS.COM
Silicatein Proteins
Silicateins are a group of enzymes that catalyze the formation of a biosilica layer with a high refractive index rate, and therefore crucial for our experiments [6]. Silicateins are mostly found in the axial filaments of the marine sponges. These proteins have a similar sequence to the mammalian cathepsin L, which is a protease and catalytic triad hydrolase. Due to the structural characteristics of silicateins, they can be considered as analogous to the regular catalytic triad hydrolases.[7]
Silicate molecules have a high refractive index that would make them a good material for turning cells into a functional resonator. In order to build a resonator with silicate molecules, Silicatein must be secreted out of the cell by the help of Silicic acid and the Sodium Silicate must be given to the bacterial medium. Silicatein polymerases Sodium Silicate onto the surface of the bacteria and covers the all bacterial cell wall with a bioslicate layer[8].
Figure 2: NOAA Office of Ocean Exploration / Wikimedia Commons [9]
Fluorescent Proteins
Fluorescent Proteins (FPs) are capable of emitting light when excited by laser beams and they could act as reporter proteins that could be attached to other proteins. When FPs absorb a photon, electrons jump from their ground state to the excited state and emit a photon while returning back to their ground state. The emission of the photons enables scientists to observe the attached cells and proteins. Researchers can check the presence of target structures by using these proteins, and can use them as a gain medium which is a main component of a laser. Main properties of FP’s are their excitation and emission spectra, brightness and maturation time. Depending on their emission spectra, fluorescent proteins are classified as green, blue, red, yellow or orange. [10]
Fluorescence proteins can target specific locations inside the cell by help of intermolecular tags. This allows tracking expression and localization of various recombinant proteins and therefore can be a useful tool for controlling or simply measuring the expression rates of proteins. If the production of fluorescent proteins is visible in the cell, scientists assume the target gene is also expressed by the cell.
Structure of Green Fluorescent Protein(PDB ID:1GFL)
Plasmids
We used 1 constitutive promoter and 1 inducible promoter for our e.coli plasmid designs. As we are going to coexpress 2 plasmids in e.coli we picked different ori’s for e.coli plasmids. We also picked 1 constitutive promoter and 1 inducible promoter for e.coli to not force e.coli cells. For yeast and e.coli plasmids we picked different selection markers for the ones we want to coexpress to make selection effective.
LipA signal sequence+Reflectin A1
TorA+Reflectin A1
ompA+Silicatein
Pir3+Reflectin A1(Anchor domain protein(PDB ID:2jwh)+Reflectin A1 is going to be inserted in the same way to the backbone)
Pir3+Silicatein
sfGFP(Yeast Design)(designs are made in the same way for other FPs)
sfGFP(E.Coli)(designs are made in the same way for other FPs)
B. Experiments with E. coli
Experiment #1
Aim
We want to express silicatein-a in E.Coli BL21 Star strain to observe if there are any inclusion bodies formed.
Introduction
In this experiment, ‘Silicatein a’ will be expressed in BL21 Star strain of E. coli and checked for expression rates and formation of any inclusion bodies with His Tag purification and later running an SDS-Page gel.
Materials & Methods
Competent E. coli BL21 Star cells are transformed with pET 28 a(+) (Kanamycin resistance with IPTG inducible T7 promoter) plasmid containing the silicatein-alpha insert. Transformed bacterial cells are incubated in SOC medium for one hour at 37 ºC and centrifuged to increase the bacterial cell concentration. Concentrated and transformed cells are planted into erlenmeyer flasks containing liquid LB and Kanamycin. Bacterial cultures are induced with 1mM IPTG and flasks are incubated in a shaking incubator overnight at room temperature (18 ºC).
Bacterial cells are lysed by help of digestive enzymes and sonication according to the cell lysis protocol. Lysed bacterial cells are centrifuged and proteins in lysate purified by using standard chromatography methods. Nickel column is used to purify proteins with 6x His tag by immobilized metal affinity chromatography (IMAC). After protein purification, SDS-PAGE is applied to check the expression levels and formation of possible inclusion bodies.
Expected Outcome
There is a low possibility that there will be inclusion bodies formed according to similar experiments made before [8,11].
Discussion
We are not expecting any issues with the expression of ‘silicatein a’ protein with the current plasmid vector and the E. coli strain we are planning to use. However, if there are any issues with the expression, we are planning to switch the plasmid vector to pQE30 and our E. coli strain to M15. There is a study where the ‘silicatein alpha’ is expressed successfully with the vector above [8]. In the same study, it is shown that there is no inclusion body occurrence, this indicates that we can switch to their expression system if we face problems as such.
Experiment #2
Aim
We want to express silicatein-a with membrane fusion protein (OmpA) [8] and silicatein-a in E.Coli Bl21 Star strain to observe proteins expressed in the construct stick to the membrane and work properly [8].
Introduction
In order to check if there is silicate on the surface of the bacteria, Rhodamine dye will be used because it binds to silicate. When Rhodamine is given to the medium, the dye will bind the surface of the bacteria if there is silicate on the surface, which will be visible under a fluorescence microscope [11].
Materials & Methods
Competent E. coli BL21 Star cells are transformed with plasmid containing silicatein-a with membrane fusion protein (OmpA). Transformed bacterial cells are incubated in SOC medium for one hour at 37ºC and centrifuged to increase the bacterial cell concentration. Concentrated and transformed cells are planted into tubes with liquid LB containing Ampicillin. Bacterial culture in tubes is induced with 1mM IPTG and the tubes with bacteria are incubated in a shaking incubator overnight at 37ºC
The overnight cell culture is processed according to Polysilicate layer in silicatein expressing protocol.
Rhodamine will be added into the bacterial culture following rhodamine123 staining protocol and observed under fluorescence microscope at 488nm [12]. Additionally, samples will be prepared for SEM imaging with ZEISS EVO LS15 SEM according to the protocol.
Expected Outcome
After treating bacteria with silicic acid the bacteria will have a silicate covering on its outer membrane without problems such as no expression, folding or inclusion bodies etc. We expect to see rhodamine dye binding the surface of the bacterial cell wall.
Discussion
If rhodamine is not observed on the surface of the bacteria, this would indicate that bacteria did not express the protein or silicic acid did not work as expected. In this case, the expression system could be changed in order to check if the expression of the silicatein is working fine with other vectors. Additionally, SEM images will be taken with ZEISS EVO LS15 SEM (available at KUYTAM, Koc University) to check incorporation of the silicate molecules to the surface. However, in Experiment #1 we already checked the working expression system for silicatein-a if experiment #1 worked this experiment will likely to work as well.
Experiment #3
Aim
In this experiment, we plan to express Silicatein-a+Membrane Fusion Protein (OmpA) and various FPs simultaneously in E.Coli BL21 strain to generate a biolaser by using FPs as the gain medium and Silicatein for constructing the resonator around the cell membrane.
Introduction
Fluorescent proteins are crucial in this experiment because they have identical properties of commercial laser gain mediums. For that reason, they will be expressed in the Bacteria to construct a complete laser mechanism along with a Poly-silica resonator.
Materials & Methods
Competent E. coli BL21 Star cells are transformed with plasmid containing silicatein-a with membrane fusion protein (OmpA) and a plasmid with selected FP insert. Transformed bacterial cells are incubated in SOC medium for one hour at 37 ºC and centrifuged to increase the bacterial cell concentration. Concentrated and transformed cells are planted into tubes with liquid LB containing Ampicillin and Kanamycin. Bacterial cultures in tubes are induced with 1 mM IPTG and tubes with bacteria are incubated in a shaking incubator overnight overnight at 37ºC
The overnight cell culture is processed according to the polysilicate layer in silicatein expressing protocol After the Polysilicate layering is finished cells are taken to measure lasing qualities.
Expected Outcome
With the silicatein on the surface of the bacteria and FPs in the cytoplasm, we are expecting to see a functioning biolaser when the gain medium is stimulated with proper wavelength light.
Discussion
The best results for this experiment could be achieved only if there is not any problem with expression and translocation of Silicatein in the bacterial cell. In other words, this experiment is dependent on the positive outcome of ‘Experiment #2’ otherwise it would not work since the major element of the experiment is the generation of a resonator via silicatein protein. It is not expected to see a problem in the expression of FPs because FP expression in E. coli is a well researched and optimized field [10] hence the outcome of this experiment is less likely to be affected by a problem during FP production. The result would determine if E. coli biolasers with Silicatein resonator is possible and therefore this experiment is one of the major experiments of this project.
Experiment #4
Aim
We want to express reflectin A1 protein in E.Coli Bl21 Star strain to observe if we see any inclusion bodies of the protein. Also, we want to observe if the proteins are unfolded when they form inclusion bodies.
Introduction
In this experiment we will be covering the cell with reflectin A1 protein.
Materials & Methods
Competent E. coli BL21 Star cells are transformed with plasmid containing the reflectin A1 insert. Transformed bacterial cells are incubated in SOC medium for one hour at 37 ºC and centrifuged to increase the bacterial cell concentration. Concentrated and transformed cells are planted into erlenmeyer flasks containing liquid LB and Kanamycin. The bacterial cultures are induced with 1mM IPTG and incubated in a shaking incubator overnight at room temperature (18 ºC).
Bacterial cells are lysed by help of digestive enzymes and sonication according to the cell lysis protocol. Lysed bacterial cells are centrifuged and proteins in lysate purified by using standard chromatography methods. Nickel column is used to purify proteins with 6x His tag by immobilized metal affinity chromatography (IMAC). After protein purification, SDS-PAGE is applied to check the expression levels and formation of possible inclusion bodies.
Expected Outcome
We expect to see reflectin proteins to form inclusion bodies inside the cell [13]. 2011 Cambridge team directly tried to express reflectin A1 in E. coli but it formed inclusion bodies. Instead of locating reflectin proteins into the cytoplasm they tried to export reflectin to the periplasm of bacteria.
Discussion
We expect to see reflectin A1 forming inclusion bodies inside the cell with the current vector backbone and host cell we use [13]. We plan to try low-medium expression vectors to see how it affects the formation of inclusion bodies in the future. We also plan to observe the proteins that form the inclusion bodies are folded or unfolded. Another possible outcome is not seeing any inclusion bodies which is great for our overall experiment.
Experiment #5
Aim
In this experiment, we are going to express reflectin A1 protein with the TorA and LipA [13] tag in E.Coli BL21. Our goal is to transport reflectin A1 to periplasm without inclusion bodies. Also, we want to fill the periplasm with reflectin A1 to use it as an organic resonator.
Introduction
In this experiment we will try to translocate reflectin A1 with a torR or LipA tags onto the periplasm of E.Coli.
Materials & Methods
Competent E. coli BL21 Star cells are transformed individually with plasmid containing reflectin A1 protein with membrane fusion protein torR and in another plasmid with LipA. Transformed bacterial cells are incubated in SOC medium for one hour at 37ºC and centrifuged to increase the bacterial cell concentration. Concentrated and transformed cells are planted into tubes with liquid LB containing Ampicillin. The bacterial cultures in tubes are induced with 1mM IPTG. The tubes with bacteria are incubated in a shaking incubator overnight at 37ºC. Incubated cells will be compared with their condition in the beginning according to their optical qualities.
Expected Outcome
We expect to see reflectin A1 proteins getting transported to the periplasm and filling it without inclusion bodies.
Discussion
The best outcome is to see the cell periplasm filled with reflectin A1 which can work as a resonator. However, if we can’t see full coverage of folded reflectins inside the periplasm, we are planning to use different secretion tags. If we can’t have success with other tags, we are only going to use reflectin A1 proteins as a resonator in yeast.
Experiment #6
Aim
In this experiment, we will try to express reflectin A1 protein tagged with TorA and LipA[13] additionally we will overexpress FPs inside the E.Coli BL21 strain. We want to see FPs acting like a gain-medium whereas reflectin acting like a resonator.
Introduction
In this experiment, we will try to translocate reflectin A1 onto the periplasm of E.Coli and over-express FP inside the cell to create a biolaser.
Materials & Methods
Competent E. coli BL21 Star cells are transformed with plasmid containing silicatein-a with membrane fusion proteins torR or LipA and a plasmid with selected FP insert. Transformed bacterial cells are incubated in SOC medium for one hour at 37 ºC and centrifuged to increase the bacterial cell concentration. Concentrated and transformed cells are planted into tubes with liquid LB containing Ampicillin and Kanamycin. The bacterial cultures in tubes are induced with 1 mM IPTG. The tubes with bacteria are incubated in a shaking incubator overnight overnight at 37ºC
In the end, bacterial cells will be measured for their lasing qualities.
Expected Outcome
We expect to see FPs getting properly expressed without inclusion bodies. We also expect to see the periplasm filled with reflectin proteins.[13,14]
Discussion
This experiment solely depends on the success of Experiment #5. If Experiment #5 gives the expected results and we fail to get the expected outcome out of Experiment #6, that means expressing FP is the problem. As a plan B, we are planning to use different types of FPs to overcome the problem. However, if we can’t get any success, we are planning to insert organic dyes which can act as a gain medium. If none of our solutions works, we are only going to use reflectin proteins as a resonator in yeast.
Experiment #7
Aim
We want to insert fluorescent synthetic dyes into E. coli and after that we want to express reflectin A1+torA, reflectin A1+LipA and silicatein-alpha+ompA sequentially in different experiments.
Introduction
We have to cover the e.coli with reflectin A1 proteins in order to create a functional resonator. We will overexpress reflectin fused with the given proteins in order to cover the cell with reflectin proteins.
We will synthesize silicatein with given proteins to export them out of the cell. E. coli culture will be in silicic acid and silicatein-a will polymerase the silicate and cover the E. coli with a poly-silicate layer.
We will first insert fluorescent synthetic dyes to use them as gain mediums. After that we will induce the given protein synthesis with galactose.
Materials & Methods
Trypan blue dye is inserted into E. coli BL21 Star cells according to dye insertion protocol. E. coli BL21 Star cells made components are transformed individually with plasmid containing reflectin A1 protein with membrane fusion protein torA,in another plasmid with LipA and silicatein-alpha+ompA inserts. Transformed bacterial cells are incubated in SOC medium for one hour at 37ºC and centrifuged to increase the bacterial cell concentration. Concentrated and transformed cells are planted into tubes with liquid LB containing Ampicillin. The bacterial cultures in tubes are induced with 1mM IPTG. The tubes with bacteria are incubated in a shaking incubator overnight at 37ºC.
Expected Outcome
We expect to see reflectin A1 proteins and silicatein with given proteins to get expressed properly when a synthetic fluorescent dye is inserted before.
Discussion
There are no studies that tried this experiment so we are not sure if we will have the expected outcome. However, if we have problems in expression or exportation of reflectin A1 and silicatein, we will try a second experiment. In the second experiment we will express silicatein and reflectin proteins.
C. Experiments with S. cerevisiae
Experiment #8
Aim
In this experiment, we aim to express reflectin A1 protein in S. cerevisiae (Baker’s yeast) to determine if reflectin proteins are expressible in yeast cells.
Introduction
In experiments done by Cambridge IGEM team in 2011 [13] bacterial expression is observed; however, due to the inclusion of body formation, the proper folding of the Reflectin A1 protein is not proven. Expression of Reflectin proteins in yeast could solve the problems during expression.
Materials & Methods
Yeast cells are transformed with PMG1(Gal1-GST) plasmid containing reflectin A1 insert according to the Yeast Transformation protocol (S. Elledge). Transformed yeast cells are transferred into liquid SC minus His and Leu + Raffinose/Galactose medium and incubated at 30 ˚C for 2 days. Incubated yeast cells are lysed and prepared for Nickel column chromatography.
Expressed proteins are purified by using standard chromatography methods. Nickel column is used to purify proteins with 6x His tag by immobilized metal affinity chromatography (IMAC). After protein purification, SDS-PAGE is applied to check the expression levels.
Expected Outcome
We expect to see a proper expression of reflectin A1 proteins with the yeast cells, our current expression vector and protocols.
Discussion
If protein expression is not observed after His Tag purification and SDS Page, the most likely problem would be caused by a problem in the plasmid itself. In order to solve a problem of this scale the best solution would be to repeat the transformation of the plasmid or switch expression vector if there is a complication between plasmid vector and the target gene. If the problem is not solved after changing expression vector, the problem could be caused by a reflectin itself and in this situation it would be problematic to go on with the same yeast strain or yeast as organism to express reflectin A1.
Experiment #9
Aim
We aim to express and translocate reflectin A1 proteins onto the cell wall of yeast. We are planning to achieve that by synthesizing reflectin together with selected cell wall and membrane proteins.
Introduction
We will overexpress reflectin A1 tagged with a variety of proteins[15] to translocate the reflectin proteins to the cell wall or membrane of yeast.
Materials & Methods
Yeast cells are transformed with plasmid containing reflectin A1 with selected protein inserts according to the Yeast Transformation protocol (S. Elledge). Transformed yeast cells are transferred to liquid SC minus His and Leu + Raffinose/Galactose medium and incubated at 30 ˚C for 2 days. Yeast cells will be checked for their optical qualities if any changes occur in the diffraction rates of the cells.
Expected Outcome
We expect reflectin A1 to be translocated onto the membrane or the cell wall successfully.
Discussion
If one of the tags we fused to reflectin A1 does not work, we will switch to another protein until we achieve our goal or find the best experimental result. The first tag we will try is the pir3 synthesized with reflectin A1. If that tag doesn’t work, We will try the second option, pir 4.[16] If it doesn’t work, we will try the third option: Glycosylphosphatidylinositol-anchored Domain from a Trypanosome Variant Surface Glycoprotein tag.[17]
The anchor protein attaches itself to the cell membrane, and the other proteins attach to the cell wall which increases the chances of reaching the aim of this experiment. Suitable fusion of both anchor domain and reflectin, along with the other two tag-reflectin A1 structures; we will try to cover up the entire surface of the cell membrane or the cell wall.
Experiment #10
Aim
We aim to express and translocate reflectin A1 onto the cell wall of yeast, also express FPs to generate a fully functioning biolaser.
Introduction
We will overexpress reflectin A1 tagged with a variety of membrane or cell membrane proteins of yeast. As these proteins are exported to the cell wall and attach there, hence it will carry and locate reflectin proteins onto the cell wall. In this experiment, reflectin A1 proteins will act as a resonator as it will cover the cell wall and FPs will act as a resonator.
Materials & Methods
Yeast cells are transformed with both plasmid containing reflectin A1 with selected protein inserts and plasmid containing selected FPs according to the Yeast Transformation protocol (S. Elledge). Transformed yeast cells are transferred to liquid SC minus His and Leu + Raffinose/Galactose medium and incubated at 30 ˚C for 2 days. Yeast cells will be checked for their lasing properties if there are any.
Expected Outcome
We expect to see reflectin A1 proteins covering the cell wall of yeast. We also expect to see overexpressed FP inside the cell.
Discussion
The result of this experiment is dependent on the success of Experiment #9. If we can’t see the expected results in this experiment even if Experiment #9 is successful, that means the expression of the FPs we use creates a dysfunctionality. For Plan B, we are going to try different types of FPs and expression plasmids.
Experiment #11
Aim
In this experiment we aim to express silicatein-a successfully in yeast.
Introduction
In this experiment ‘Silicatein a’ will be expressed in yeast and checked for expression rates via SDS-PAGE.
Materials & Methods
Yeast cells are transformed with PMG1(Gal1-GST) plasmid containing silicatein-a insert according to the Yeast Transformation protocol (S. Elledge). Transformed yeast cells are transferred into liquid SC minus His and Leu + Raffinose/Galactose medium and incubated at 30 ˚C for 2 days. Incubated yeast cells are lysed and prepared for Nickel column chromatography. Expressed proteins are purified by using standard chromatography methods. Nickel column is used to purify proteins with 6x His tag by immobilized metal affinity chromatography (IMAC). After protein purification, SDS-PAGE is applied to check the expression levels.
Expected Outcome
We expect to successfully express silicatein-a in yeast with our current expression vector.
Discussion
We are not expecting any issues with the expression of ‘silicatein a’ protein with the current plasmid vector and the yeast we are planning to use. However, if there are any issues with the expression, we are planning to switch the plasmid vector we currently use.
Experiment #12
Aim
In this experiment, we aim to express silicatein-a with a variety of cell wall or membrane proteins of yeast. The main goal of this experiment is to see the polymerisation of silicic acid components. (mainly silicate) We want the poly-silicates to cover the cell wall or membrane and act as a resonator for our biolaser.
Introduction
Silicateins are enzymes that catalyze the formation of biosilica which has a high diffraction rate[6].This is important because we can use biosilica as a resonator if we can cover the cells with it. We will overexpress silicatein-a tagged with a variety of proteins to translocate the silicatein-a onto the cell wall or membrane of yeast. Yeast culture will be in silicic acid and silicatein-a will polymerase the silicate and cover the yeast with a poly-silicate layer.
Materials & Methods
Yeast cells are transformed with plasmid containing silicatein-a with selected membrane and cell wall protein inserts according to the Yeast Transformation protocol (S. Elledge). Transformed yeast cells are transferred to liquid SC minus His and Leu + Raffinose/Galactose medium and incubated at 30 ˚C for 2 days. Same protocol for silicate application will be used for yeast. Additionally rhodamine123 stating protocol will be used in case silicate works with yeast cells.
Expected Outcome
We expect to see the silicatein-a’s attach to the cell wall and catalyze the polymerisation of silicates. We also expect to see these poly-silicates covering the cell wall and acting as a resonator for our biolaser.
Discussion
There are no studies that tried what we are trying to do on yeast. However, there are some successful and similar approaches to what we are trying to achieve. However, none of these experiments is done by using yeast. If the pir3 tagged silicatein-a doesn’t stick to the cell wall, we are planning to use two different tags in two different experiments.The first tag we will try is the pir4 synthesized with reflectin A1[16]. If that tag doesn’t work, We will try the second option: Glycosylphosphatidylinositol-anchored Domain from a Trypanosome Variant Surface Glycoprotein tag[17].
These three tags are well secreted endogenous proteins, which increases the chances of reaching the aim of this experiment. Suitable fusion of both 2JWH anchor domain and reflectin A1, along with the other two tag-silicatein structures; we will try to stick the silicatein-a to the cell wall.
Experiment #13
Aim
In this experiment, we aim to express silicatein-a, translocate it onto the cell wall of yeast, and express FPs. The main goal of this experiment is to see the polymerisation of silicic acid components (mainly silicate) and have FPs in the cytoplasm to generate a fully functioning biolaser.
Introduction
In this experiment, we aim to express silicatein-a with a variety of cell wall or membrane proteins of yeast. We are going to check if the silicatein-a attaches to the cell wall of the yeast. Yeast culture will be in silicic acid and silicatein-a will polymerase the silicate and cover the yeast with a poly-silicate layer as a resonator. Additionally, FPs will be expressed and used as a gain medium in the cytosol to generate a biolaser.
Materials & Methods
Yeast cells are transformed with both P425-Gal1a (Leu2) plasmid containing silicatein-a with selected protein inserts and P423-Gal1a (HIS3) plasmid containing selected FPs according to the Yeast Transformation protocol ( S. Elledge). Transformed yeast cells are transferred to liquid SC minus His and Leu + Raffinose/Galactose medium and incubated at 30 ˚C for 2 days.
Expected Outcome
If Experiment #12 works properly, we expect to obtain lasing from our setup, and have a functional biolaser.
Discussion
This experiment’s success solely depends on Experiment #11. If we conduct Experiment #11 successfully and fail to do Experiment #12, that means FPs cause a problem in the cell. We will use different types of expression plasmids and different types of FPs according to results of the experiments to overcome any problems.
Experiment #14
Aim
We want to insert fluorescent synthetic dyes into yeast and after that we want to express reflectin A1+pir3, reflectin A1+pir4, reflectin A1+Glycosylphosphatidylinositol-anchored Domain from a Trypanosome Variant Surface Glycoprotein, silicatein-alpha+pir3, silicatein-alpha+pir4, silicatein-alpha+Glycosylphosphatidylinositol-anchored Domain from a Trypanosome Variant Surface Glycoprotein sequentially in different experiments[16,17]
Introduction
We will overexpress reflectin A1 fused with the given proteins in order to cover the cell with reflectin.
We will synthesize silicatein with given proteins to export them out of the cell. Yeast culture will be in silicic acid and silicatein-a will polymerase the silicate and cover the yeast with a poly-silicate layer.
We will first insert fluorescent synthetic dyes to use them as gain mediums. After that we will induce the given protein synthesis with galactose.
Materials & Methods
Yeast cells are stained with DAPI prior to transformation of any plasmids. Competent yeast cells are transformed with inserts containing reflectin A1+pir3, reflectin A1+pir4, reflectin A1+Glycosylphosphatidylinositol-anchored Domain from a Trypanosome Variant Surface Glycoprotein, silicatein-alpha+pir3, silicatein-alpha+pir4, silicatein-alpha+Glycosylphosphatidylinositol-anchored Domain from a Trypanosome Variant Surface Glycoprotein according to Yeast Transformation protocol ( S. Elledge). Transformed yeast cells are transferred to liquid SC minus His and Leu + Raffinose/Galactose medium and incubated at 30 °C for 2 days.
Expected Outcome
We expect to see reflectin A1 and silicatein-a with given proteins to get expressed properly when a synthetic fluorescent dye is inserted before.
Discussion
There are no studies that tried this experiment so we are not sure if we will have the expected outcome. However, if we have problems in expression or exportation of reflectin A1 and silicatein, we will try a second experiment. In the second experiment we will express silicatein and reflectin with given proteins and then we will insert the synthetic dyes.
D. Lasing Experiments
Experiment #15 - Lasing from Stationary Phase E.coli Cells
We are going to use samples obtained from Experiment #6 for this experiment. We will initially continue to grow E. coli cells, obtained from Experiment #6, in LB, spin down and resuspended in PBS. PBS will be stopping the growth of these cells. The cells will be fixed on a slide by pipetting them under 3% agarose-TAE pads. These cells will be taken on the sample place in a petri dish on our optical setup. Fluorescent proteins in these cells will be excited by a laser light at their excitation peak with a pulse width of 5-8 ns, generated by a Nd:YAG laser and tuned to the pump wavelength by an optical parametric oscillator (Spectra Physics). The emission from the sample will be collected by a microscope objective and coupled with Andor Newton Cooled Spectroscopy Camera. Details of the optical setup can be found in the hardware page.
Experiment #16 - Lasing from Stationary Phase Yeast Cells
We are going to use samples obtained from Experiment #13 for this experiment. We will initially stop the growth of yeast cells obtained from Experiment #13 by resuspending them with PBS. We will fix the cells on agarose pads. These cells will be taken on the sample place in a petri dish on our optical setup. Fluorescent proteins in these cells will be excited by a laser light at their excitation peak with a pulse width of 5-8 ns, generated by a Nd:YAG laser and tuned to the pump wavelength by an optical parametric oscillator (Spectra Physics). The emission from the sample will be collected by a microscope objective and coupled with Andor Newton Cooled Spectroscopy Camera. Details of the optical setup can be found in the hardware page.
Experiment #17 - Lasing from Growing E.coli cells
We are going to use samples obtained from Experiment #6 for this experiment. We will keep these cells in an incubator and let them grow and divide during our experiment. We will first express silicatein or reflectin A1 and then we will start expressing FPs. We will be using an incubator integrated to our optical setup. Details of this configuration of our optical setup can be found in the hardware page.
Cells obtained from Experiment #6 will be taken into the incubator on a petri dish. Fluorescent proteins in these cells will be excited by a laser light at their excitation peak with a pulse width of 5-8 ns, generated by a Nd:YAG laser and tuned to the pump wavelength by an optical parametric oscillator (Spectra Physics). We will be pumping the fluorescent proteins in a cyclic manner. Thus we will be pumping the sample for a short period of time in a pumping cycle. We have determined the exact time to pump the sample in terms of growth time of cells and maturation time of fluorescent proteins as can be seen in our model. The emission from the sample will be collected by a microscope objective and coupled with Andor Newton Cooled Spectroscopy Camera. Details of the optical setup can be found in the hardware page.
Experiment #18 - Lasing from Growing Yeast Cells
We are going to use samples obtained from Experiment #13 for this experiment. We will keep these cells in an incubator and let them grow and divide during our experiment. We will first express silicatein or reflectin A1 and then we will start expressing FPs. We will be using an incubator integrated to our optical setup. Details of this configuration of our optical setup can be found in the hardware page.
Cells obtained from Experiment #13” will be taken into the incubator on a petri dish. Fluorescent proteins in these cells will be excited by a laser light at their excitation peak with a pulse width of 5-8 ns, generated by a Nd:YAG laser and tuned to the pump wavelength by an optical parametric oscillator (Spectra Physics). We will be pumping the fluorescent proteins in a cyclic manner. Thus we will be pumping the sample for a short period of time in a pumping cycle. We have determined the exact time to pump the sample in terms of growth time of cells and maturation time of fluorescent proteins as can be seen in our model. The emission from the sample will be collected by a microscope objective and coupled with Andor Newton Cooled Spectroscopy Camera. Details of the optical setup can be found in the hardware page.
E. Experiment Plans for Future Applications
Experiment #19 - Lasing from Red Blood Cells
As red blood cells have high refractive index and an oval shape, they are great candidates for being a resonator for a biolaser [18]. We will use Acridine Orange (AO), a known fluorescent dye which is now an FDA investigational drug as a gain medium [19]. As acridine orange binds to the Hemoglobin (the protein which is abundant in red blood cells) we will insert acridine orange inside the red blood cells and they will bind to the hemoglobin [20]. Then we will excite these dyes with a pumping source which can give out 502 nm laser (acridine orange’s max. excitation). After these experiments we hope to see lasing coming out of red blood cells.
Experiment #20 - Obtaining Lasing from Diatoms
In terms of shape, diatoms can be classified as centric and pennate diatoms. Centric diatoms have a radially symmetrical shape whereas pennate diatoms have a bilaterally symmetrical shape. Even if the most of the diatoms are pennate, we will use a centric diatom due to its distinct radial symmetry.
Since the refractive index of diatom silica frustule is n=1.46 [21] it is possible to get lasing out of it with just staining it with a synthetic dye. Thalassiosira pseudonana will be used in our experiments since it is a centric diatom, which will increase our chance of getting lasing due to its shape, and it is widely used and studied[22]. It is proven that using 1 wt % of sulforhodamine (SRh) is effective as a gain medium.[23] In our experiments, we will use the same dye.
Experiment #21 - Observing Whispering Gallery Modes of Oocytes
The structure of the oocytes are divided into; the nucleus, the cytoplasm, the mitochondria, and the zona pellucida. The physical properties of the oocytes gives advantage to sustain the electromagnetic resonances. Optical features of oocytes like refractive index ,which is not comprehensively studied in the literature, makes a possible model cell line for the biolasers.
Zona Pellucida is a semi-transparent coat around the mammalian oocytes and the embryos. The thickness varies around 13µm and the diameter varies around 150µm between the species. This layer is composed of the interconnected glycoproteins by non-covalent interactions[24].
Especially after fertilization, one of the phenomena states that oocytes gain elastic property and changes surface proteins. This phenomenon not only increases the refractive index [25] of the zona region but also reduces surface roughness. As mentioned before, the properties of the oocytes differ between the species. It is important to choose feasible oocytes to obtain better lasing.
Experiment #22 - Obtaining Lasing from Oocytes
The absorptive properties of the oocytes is one of the important points. The absorbance spectra should be known for selected oocytes. In the literature, it is stated that FTIR spectroscopy results of the different oocytes, some regions present high absorbance like amino acids that involve DNA [26]. When we consider the visible and near infrared spectra, which is between 532 to 769 nm, the Rhodamine B dye absorbs lower wavelength effectively [27].
Rhodamine B dye has a characteristic to diffuse quickly which can be distributed in cytoplasm in a short time. The limiting factor is also it’s easy-diffusing property. There is a potential to diffuse surrounding the medium. Even the fertilization is stimulated after the uptake of the synthetic dye, zona pellucida is not able to preserve it. Instead of synthetic dyes, chemical dyes can be implemented such as quantum dot coating. This method is likely suitable to generate WGM [28, 29, 30, 31].
Quantum Dot/ Polyelectrolyte Coating Procedure
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Suspend embryo with the cationic poly allylamine hydrochloride
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Rinse the embryos and extract the supernatant.
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Repeat the first two steps then add anionic poly acrylic acid. (In this step we are expecting that zona pellucida layer is coated by polyelectrolyte coat regardless zona regions’ charge)
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Amino group specific quantum dots are added to the mixture. Then EDC and NHS should be added to the mixture to facilitate carboxyl-to-amine crosslinking.
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Rinse the mixture. Unbound quantum dots should be eluted.
Bovine oocytes are coated with quantum dots. In A, cytoplasm and zona region excited with visible wavelength with the emission from the cytoplasm excited using 532 nm CW laser. In B when the white light source is omitted, quantum dots can be observed. This figure is taken from the PhD Thesis named “Biological Cell Resonators”.[32]
In the thesis, the group concluded that WGM can be observed with quantum dot coating.
Experiment #23 - Random Laser Experiments
We are going to express and purify fluorescent proteins from E.coli BL21. After we purify the FPs, we will create an aqueous %5 FP solution. After we create the solution, we will drip it on a glass slide and heat it inside a 50 °C oven. After the water is evaporated, cracked FP stain will be obtained. As FPs are luminescent proteins, we can use them as a gain medium & resonator at the same time to obtain random lasing.[33]
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