BME494s2013 Project Team3

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Overview & Purpose E-coli may be used as a factory to produce materials like plastic By modifying the input and output for the Lac switch, it may be possible to produce materials such as plastics. The switch could be triggered by another environmental factor other than [IPTG], and instead of producing GFP, more useful materials like plastic could be an output. Currently, production of plastics is a very energy intensive process, and by using bacteria for the production, we can save energy and limit waste into the environment. The IPTG-input/fluorescent protein-output is proof of concept for the production of synthetic materials such as plastic being created by bacteria. In the original project, a synthetic compound was utilized to trigger the metabolic pathway for the degradation of lactose. Using synthetic biology, we can splice together different genetic features to create an entirely new metabolic response. We can find a promoter that responds to a different input. In the natural Lac-operon, the cell produces proteins for the breakdown of lactose. We modified the natural process by initiating the production of GFP instead of the functional proteins found in the original process. It seems that if we are able to control our protein output, we can produce synthetic products as well. Background Diagram of Lac operon and how it functions without lactose present (top) and with lactose present (bottom) The natural Lac-operon has 2 controls that tightly regulate the production of the proteins necessary for the breakdown of lactose. In the presence of glucose, the Lac Operon inhibits the production of those proteins. When glucose is present, the lac repressor is bound to the operator, prohibiting the transcription of the proteins. In the presence of lactose, lactose is able to bind to the lac repressor initiating a conformation change in the repressor protein, causing it to release and allow for transcription. This, however, is not the only control in place. It is incredibly energy intensive to produce the proteins necessary for the breakdown of lactose, so when there is glucose present, the glucose will be metabolized first, even with some lactose present. This is accomplished by the presence of a second regulatory device, cyclic AMP (cAMP). cAMP is present only when there are very low levels of glucose found in the environment. cAMP serves as an activator and binds to RNA polymerase allowing for transcription of the output proteins. In the synthetic [IPTG] induced Lac-operon, IPTG serves the same function as lactose, it binds to the repressor causing a conformational change, allowing for transcription of proteins. In the natural Lac-Operon, the proteins were functional, and necessary for the breakdown of lactose. In our synthetic system, green fluorescent protein is produced instead. This protein serves as an indicator, allowing for visual verification that the lac switch is operational. Design: Our genetic circuit OUR GENE SWITCH: AND gate Gene Switch: Both [IPTG] AND [Low Glucose Levels] conditions must be met for GFP production Truth table describing inputs required to produce an output The functionality of our genetic switch resembles that of an "AND" logic gate: the device requires two conditions to be true in order for an output to be produced. One conditional requirement is that IPTG must be present in the device's environment. When IPTG is present, it binds to the LacI repressor, thus allowing for transcription to continue. The other conditional requirement is that glucose levels in the device's environment must be low. Glucose levels inversely affect production of cyclic AMP (cAMP): when glucose levels are low, cAMP production increases and when glucose levels are high, cAMP production decreases. cAMP binds to catabolite activator protein (CAP) to form the CAP-cAMP complex. In order for this complex to form, cAMP must be present and, thus, glucose levels must be low. The CAP-cAMP complex is an input that our device requires in order to produce an output. In the natural lac operon, the CAP-cAMP complex leads to enhanced activation of gene expression from the lac operon. However, if glucose is present, cAMP levels will in turn be low and the host will preferentially metabolize glucose even if lactose is present. Building: Assembly Scheme GENERAL ASSEMBLY: To form the build the lac switch, the group used Type IIs Assembly, which allows the the parts to be assembled in one step. For this type of assembly, forward and reverse primers needed to be created and placed in the system so as to create sticky ends that can bind various parts together in a specified order. Additionally, site-directed mutagenesis (1) need be performed so as to remove the BsmBI cut-site in the promoter region. To put the pieces together, PCR is implemented, which allows all the parts to be replicated thousands of times to yield a pure product. Subsequently, digestion and ligation is implemented, during which BsmBI cuts the DNA fragments and creates complementary overhangs that anneal via base pairing. (1) SITE-DIRECTED MUTAGENESIS: Using these particular parts, site-directed mutagenesis needed to be performed. Because the K418003 promoter region's amino acid code contained a BsmBI cut site, the system would naturally cut off at that site, thus thwarting the system. Using site-directed mutagenesis, the team cut located the CGTCTC BsmBI cut-site sequence and sought to replace the first C with a G as this strategy allowed for the sequence to maintain its same functionality without breaking off. Since C, and the two CC base pairs that preceded it, coded for proline, it was admissible to change the C into a G because CCG still coded for proline. Once this promoter was altered, primers were created for the mutation as well as the promoter itself, and they were both added to the PCR assembly. LIST OF PARTS AND THIER RESPECTIVE PRIMERS: Diagram of assembly of parts (left) and diagram of what those parts signify in system(right) pSB1A3 – BioBrick assembly plasmid: -Forward Primer : 5'-cacaccaCGTCTCaactagtagcggccgct -Reverse Primer: 5'-cacaccaCGTCTCatctagatgcggccgcg K418003 - composite Lac promoter inducible by IPTG: -Forward Primer: 5'-cacaccaCGTCTCatagattgacagctagctca -Reverse Primer:5'-cacaccaCGTCTCatgtgtgtgctcagtatctt R0010 – Operator: -Forward Primer: 5'-cacaccaCGTCTCacaatacgcaaaccgc -Reverse Primer: 5'-cacaccaCGTCTCatttctgtgtgaaattgtta B0030 – Strong RBS: -Forward Primer: 5'-cacaccaCGTCTCattaaagaggagaaa -Reverse Primer: 5'-cacaccaCGTCTCaaattttctcctctttaat K259006 – Composite part made from green fluorescent protein (GFP) and double terminator: -Forward Primer: 5'-cacaccaCGTCTCaatgcgtaaaggagaa -Reverse Primer: 5'-cacaccaCGTCTCatagtaaataataaaaaagc Mutagenesis Primers: -Forward Primer: 5’-agctgttgccGgtctcactgg -Reverse Primer: 5’-ccagtgagacCggcaacagct The Polymerase Chain Reaction table of substituent amounts and cycle order Reagent Lac-promotor Composite Operator RBS GFP/double terminator Composite Vector Template DNA 0.2 μL 0.2 μL 0.2 μL 0.2 μL 0.2 μL 10 μM Forward Primer 1.0 μL 1.0 μL 1.0 μL 1.0 μL 1.0 μL 10 μM Reverse Primer 1.0 μL 1.0 μL 1.0 μL 1.0 μL 1.0 μL 2x GoTaq Green Mix 25 μL 25 μL 25 μL 25 μL 25 μL H2O 22.8 μL 22.8 μL 22.8 μL 22.8 μL 22.8 μL Total Volume 50 μL 50 μL 50 μL 50 μL 50 μL Image of hypothetical PCR reaction showing the smaller parts (operator/promoter) moving faster through the gel and the larger parts (vector) moving slower through the gel The above table used the following thermal cycling properties: 95°C, 3 min. [95°C, 15 sec; 55°C, 15 sec; 72°C, 30 sec] x 30 72°C/ 3 min. 4°C/ infinity Table of volume of parts used and cycle order during digestion process Reagent Assembly Negative Control PCR fragment 1 (vector) 1.0 μL 1.0 μL PCR fragment 2 1.0 μL None PCR fragment 3 1.0 μL None PCR fragment 4 1.0 μL None PCR fragment 5 1.0 μL None 10x T4 Ligase 0.25 μL 0.25 μL BsmB1 0.5 μL 0.5 μL H2O 4.25 μL 8.25 μL Total Volume 10 μL 10 μL The above table used the following thermal cycling properties: [45°C, 2 min.; 16°C, 5 min.] x 25 60°C, 10 min. 80°C, 20 min. 4°C, infinity Testing: Modeling and GFP Imaging A LAC SWITCH MODEL Network diagram of our device showing variables and parameters used by Ceroni, et al. IPTG and LacI bind together to form a dimer (D). In the presence of the dimer and the activated promoter (AP), green fluorescent protein is produced (GFP). We used a previously published synthetic switch, developed by Ceroni et al., to understand how our system could potentially be modeled and simulated. Mathematical modeling is a method of representing via the language of math how a system is expected to behave. The various components of a system, referred to as system parameters, are assigned variable names. These variables hold values that can be adjusted during testing without incurring what would otherwise be additional production costs. Modeling is very beneficial in that multiple tests and simulations can be performed on the system so that a predictable pattern of behavior can be developed, thus reducing production costs. We modeled our device after the Ceroni switch, using similar variables and parameters to represent the various components and processes of our device. The graphic to the right depicts these components and the variables used to represent them in a mathematical model. The following table contains the variables and parameters used both in our model and the Ceroni et al., model. Variables and parameters used by both our device's model and the Ceroni model. Variable Description Units I IPTG Concentration mM G Concentration of GFP protein molecules/cell LF Free LacI molecules molecules/cell LI LacI molecules bound to IPTG molecules/cell MG mRNA molecules of GFP molecules/cell ML mRNA molecules of LacI molecules/cell λG/L Protein degradation rate minutes-1 λMG/L mRNA degradation rate minutes-1 αG GFP rate of synthesis minutes-1 AL LacI rate of synthesis minutes-1 αMG GFP transcription rate minutes-1 αML LacI transcription rate minutes-1 AN INTERACTIVE MODEL We used a model of the natural Lac operon to understand how changing the parameter values changes the behavior of the system. The parameters that we used are shown in the following table: Variables and parameters used in MATLAB to simulate system behavior Variable Description mu dilution of input (IPTG) - [IPTG]/cell volume, with IPTG diffusion gamma_M degradation of M - [mRNA] for Bgal gamma_B degradation of B - [Bgal] output protein K half maximum of transfer function alpha_M production rate of M alpha_B production rate of Bgal (output) cell bacterial cell volume (cell = 1) Image 1: Concentration of Bgal over time; input IPTG at 0.32 mM Image 2: Sustained output of Bgal over time; input IPTG at 0.0639 mM Image 3: Lowest observable output of Bgal over time; input IPTG at 0.021 mM When interacting with the MATLAB model, we were able to analyze three outputs: concentration of IPTG over time, concentration of β-galactosidase (Bgal) over time, and mRNA concentration over time. Our focus was on how the concentration of Bgal changed over time as we adjusted the input concentration of IPTG. The default concentration of IPTG was set to 0.32 mM. The Bgal concentration over time when the input IPTG was set to this value can be seen in Image 1 to the right. Next, we adjusted the concentration of IPTG to attempt to find a value that would allow for sustained output of Bgal over time. We determined the best estimate of IPTG concentration to sustain a relatively constant output of Bgal over time was 0.0639 mM of IPTG. At this concentration of IPTG, the sustained output value of Bgal was approximately 2.54 x 10-4 mM. The resulting data can be seen in Image 2 on the right. Finally, we adjusted the concentration of IPTG to attempt to find a value that would result in no Bgal being produced. We were able to determine that the lowest value of IPTG concentration that did produce a Bgal output was 0.021 mM of IPTG. Any concentration of IPTG below this value resulted in the lowest observable output of Bgal: approximately 0.4 x 10-5. However, even at this concentration of IPTG, trace amounts of mRNA were still present in the system. These amounts decreased immediately after our model started to run but were still high enough to continue to initially drive transcription. Thus, an initial spike of Bgal output was observed at 0.021 mM of IPTG. Shortly after almost all mRNA had degraded, the total amount of Bgal had degraded to the minimum threshold value of 0.4 x 10-5. Because the mRNA in the system never completely degraded, we never observed a complete lack of Bgal output. The resulting data can be seen in Image 3 to the right. COLLECTING EMPIRICAL VALUES TO IMPROVE THE MODEL Image 4: Average GFP production over time Image 5: Max GFP production rate We explored how one technique, imaging via microscopy could be used to determine the production rate of an output protein, in this case GFP in yeast, could be used to determine a "real" value for maximum GFP production rate under our own laboratory conditions. To accomplish this, we used ImageJ to identify specific cells in a series of images that ranged from maximum GFP output to virtually no GFP output. We tracked these cells throughout the image series and used ImageJ selection tools to give us an average brightness, or simulation of GFP output, per cell per image. We used these brightness values to generate a plot in MATLAB that showed a rough value of how the GFP output in our cells increased over time. Applying a best fit curve to our data, we found that a cubic equation generated the closest fit to our data. These data can be seen in Image 4 to the left. The equation that best represented our data was: y = -0.011x3 + 0.4x2 - 2.1x + 1.5 To find the maximum rate of GFP production, we took the derivative of our best fit equation. This gave us the equation: y' = -0.033x2 + 0.8x - 2.1 Plotting this equation in MATLAB gave us Image 5 on the left. By taking the derivative of our quadratic, we were able to find the maximum GFP production rate of 2.75 mM/hour. Ideally, the GFP production rate measured by this method could be entered as a value for GFP rate of synthesis in the Ceroni et al. model. Human Practices Details some of the stakeholders of synthetic biology within society PLASTIC PRODUCTION: By switching the promoters at the beginning of the system to respond to some other input, and changing the output protein into one that could initiate cell monomors to combine and replicate, the system could potentially produce valuable bioplastics. Because today the the production of plastics is very energy intensive, using engineered biological cells to produce them may prove to be environmentally, if not commercially, viable. ADDITIONAL APPLICATIONS: Cancer Research: If we were to make the switch dependent on a tumor antigen input, the system could potentially detect a cancer cell and switch on, at which point it could produce synthetic antibodies as outputs, which could potentially speed up the host's immunologic response. Additionally, researches could experiment using different output proteins to see which ones would be most effective in neutralizing cancer cells while reducing damage to neighboring cells. Information Technology: Additionally, because DNA can hold thousands of combinations of amino acids, the system may provide a quick, safe, and cheap way to store data. By manipulating logic gates to respond to user-initiated inputs, the cells could act like biological computers that could record their activities for a very long time. Even when the cell dies, its DNA could be retrieved and its data analyzed. Our Team Your Name My name is Jennifer Sherwood, and I am a senior majoring in chemical engineering. I am taking BME 494 because I am interested in learning new ways to engineer biology. An interesting fact about me is that I enjoy playing golf and creating stained glass lamps in my spare time. Randle Kuehner My name is Randle Kuehner, and I am a part-time senior majoring in biomedical engineering. I am taking BME 494 because synthetic biology sounded incredibly interesting and I didn't really know anything about it prior to this semester. An interesting fact about me is that, from Phoenix, I drove to the Arctic Ocean at Deadhorse, AK and back in July 2010. Cristian Cirjan My name is Cristian Cirjan , and I am a Junior majoring in Biomedical Engineering. I am taking BME 494 because I see synthetic biology as a new and exciting area of study that could significantly affect the way we live in the future. An interesting fact about me is that I was born in Romania and I got to America off a travelling visa my family won in an annual visa lottery. Works Cited 1. Brown, W., Ralston, A. & Shaw, K. (2008) Positive transcription control: The glucose effect. Nature Education 1(1). Web. 2. Ceroni, Francesca, et al. "Rational design of modular circuits for gene transcription: A test of the bottom-up approach." Journal of Biological Engineering (2010). 3. Church,George M. "Repelling Viruses, Reviving Mammoths." The Wall Street Journal. The Wall Street Journal, 19 Oct. 2012. Web. 4. OpenWetWare contributors. Haynes:TypeIIS Assembly [Internet]. OpenWetWare, ; 2013 Feb 25, 18:20 UTC [cited 2013 Mar 18]. Available from: http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&oldid=679110. 5. Parts Registry: http://partsregistry.org/Main_Page 6. Lewis, M. The lac repressor, C. R. Biologies 328 (2005) 521–548.