BioBuilding: Synthetic Biology for Students: Lab 4

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PDF of this page Lab 4: What a Colorful World Simplifying assumptions about "the cell" are brought into question when different strains are transformed with DNA that makes them grow in colorful ways. Source Acknowledgements: Objectives By the conclusion of this laboratory investigation, the student will be able to: Define and properly use synthetic biology terms: chassis, system, device, minimal cell, sensor, color generator. Define and properly use molecular genetics terms: operon, gene expression, bacterial transformation. Explain the role of chassis in synthetic biology and engineering. Conduct and interpret the results of a bacterial transformation. Introduction One potential use of engineered bacteria is as indicator of toxic substances. Bacterial sensing systems have been designed for arsenic and lead. Bacteria are cheap and easy to produce and store. This reduces the need for expensive and technologically complex chemical tests. The bacteria are also much more sensitive to the toxin levels. However, there is one potential drawback. The bacteria respond to the toxin metabolically. This means we may be able to detect a change in pH or other indicator of metabolism. This requires further equipment such a pH indicator. Sensors have been linked by synthetic biologists to other forms of output such as the green fluorescent protein. However, this also requires further equipment such as a fluorescent light. This reduces the practicality in impoverished areas of the world, the very areas most at risk for arsenic or lead contamination. The 2009 Cambridge iGem team took up the challenge to design an indicator that could be used without additional technology. They designed color generator devices that could be linked to sensors. E Coli don’t naturally produce color but other bacteria do, so they designed “e chromi” to have a gene that produces pigment if the gene is activated. One pigment they used is Violacein, a pigment produced by genes originally found in Chromobacterium violacein. These genes can be engineered to produce purple and green. The violacein operon consists of five genes which metabolize L-tyrosine. Expression of all five genes will produce a purple pigment. However, removal of the third gene in the sequence will cause the cell to metabolize the L-tyrosine into a green pigment. These pigments are easily visible to the naked eye. This device could be linked to a biosensor for a toxin and the bacteria will turn color in response to the toxin concentration. Why not just use the Chromobacterium? Synthetic biologists like to use E coli because it is well understood and easy and safe (if proper strains are used) to work with. Synthetic biologists refer to the host cell as the chassis. For an engineered genetic system to function in a chassis, the chassis must supply the cell with energy, materials for protein synthesis and materials those proteins will use when they function. The chassis will take care of all the material needs the system needs to function to the engineer’s specifications. The better the chassis is understood, and the better it can provide materials for the engineered system, the better the results. By primarily using one chassis, synthetic biologists are managing complexity. Using a standard chassis, allows engineers from many labs across the world to compare results. We do this in everyday life when we buy bananas or bell peppers. We simply call them bananas or bell peppers. But actually many varieties mixed together in the store. But is it really important that we are aware of this when we shop? As long as the taste is similar, does it matter what variety of peppers you use? You can think of your computer and its operating system as a chassis. We can think of Macs and PCs as different chassis, though in computer lingo they are known as platforms. However, to manage complexity, they are engineered so that most word processor files written on one platform can be viewed and edited on the other. There was a time in the past when this was not the case, but the computer companies have agreed on certain standards so that users wouldn’t be lost in the complexity. Cars, however, are a different story. A car is a highly engineered system of interconnected parts. While many of these parts are similar but they must be tailored to the size and function of the car. So, while the chassis of a truck, a GTO muscle car and a Toyota hybrid are different, so are many of the internal parts that make up the engine and the drive train. We might be able to move a radio from a truck chassis to a sports car chassis, but not much else. The car manufacturers are comfortable with this complexity and it has little effect on the user of the car. Synthetic biologists, Anthony Forster and George Church, are working to further remove the complexity from their engineered systems by creating what are known as minimal cells. The idea is to design a cell that contains just the minimum genome to maintain its existence. These cells will only be able to survive on special media and all of their metabolic functions will be well characterized. Another example of research into this idea was published by Craig Venter in May of 2010. His lab replaced the genome of a bacterial cell with a fully synthesized genome and were able to produce bacteria that expressed the synthetic genome. As appeal as these chassis are for synthetic biology, the work has a way to go before they can be in general use. So, until minimal cells or synthetic cells are a viable option, biologists have long used E coli as a chassis for experiments as a way to manage complexity. It turns out that two different strains are primarily used. One strain is known as K-12 and the other B. Both strains are known to be safe and have been effectively used for genetic experiments for almost 100 years. The differences between these strains seem to minor. Most are related to metabolism and none would seem likely to affect the color generator system. You can read about the interesting history of these strains here. So, one would imagine that both strains of E coli containing the violacein device would behave similarly. But is this true? Are we sure that these two strains will produce the same color intensity? Imagine that a group of engineers are manufacturing an arsenic sensor in E coli whose purple color changes intensity as a function of arsenic level? Now imagine that a second group of engineers are also doing this? What if they were using different strains of E coli? Are we sure that the pigment will be expressed the same in a different chassis? Would you put a V-8 engine from a Lexus into a Mercedes chassis? Would the engine behave the same? Would the car? In this lab you will transform bacteria from two different strains of E coli, in other words, two different chassis. Our strain 1 is a B strain, while our strain 2 is a K12 strain. You will insert plasmids containing the violacein device. One set of plasmids has the purple version of this device while the other has the dark green version. Otherwise, the plasmids are the same. Will we see the same intensity of colors? Or will the chassis have an effect on the color? Data Table In your lab notebook, you will need to construct a data table as shown below. (These may be provided) Procedure For AmpR version For each pair of transformations, patch 1 cm x 1 cm of 4-1A (= NB437, = BL21(DE3)) and 4-2A ( = NB 438, = ER2738) onto LB, 37° overnight. Best done night before next steps. Resuspend each patch in 200 ul TB+10%DMSO DNA was miniprep'd from NB446 or NB451. Pellet resuspended in 40ul H2O Tx'd 5 ul DNA into 75 ul cells, ice 5' then 42° 90 seconds. Add 0.5 ml of LB and plate 250 ul on LB+Amp. For KanR version For each pair of transformations, patch 1 cm x 1 cm of 4-1 (= NB452, = NEBexpress, BL21 derivative) and 4-2 ( = NB 438, = ER2738) onto LB, 37° overnight. Best done night before next steps. Resuspend each patch in 200 ul TB+10%DMSO DNA was prep'd from NB442 (pPRL) or NB443(pGRN). Stocks are btw 0.4 and 0.5 ug/ul Tx'd 5 ul each DNA into 75 ul cells, ice 5' then 42° 90 seconds. Add 0.5 ml of LB. Outgrow 30' and plate 250 ul on LB+Kan. Calculations Lab Report I. Introduction Provide a brief introduction describing the field of synthetic biology. Briefly describe the purpose of the lab. What are we trying to do here? Presume that a reader of your lab report has not read the assignment. What is the role of the chassis? How does chassis effect the expression of a genetic system? How might synthetic engineers modify the relation between a chassis and an engineered genetic system to reduce the chassis effect on the system? Why important to engineer a minimal cell? What are advantages/concerns of engineering a minimal cell How might we test for the differences in the chassis that may be affecting a genetic system? You may find helpful information here and here. II. Methods You do not have to rewrite the procedure. Explain why you did each step of the protocol. III. Results Present the data tables in clear format. Present drawings of each slide. Describe the results: Describe the appearance of each plate. Are the colors different? Are the colonies different in number, size and/or shape? What was the transformation efficiency for each plate? Does it differ between the strains? IV. Discussion Draw a conclusion: Do the color generators produce the same results in different chassis? Justify your answer. Analyze the data: Be sure to discuss how each part of the experiment and results adds to your conclusion. Are we sure that the transformation worked? What do the controls that lacked plasmid and the controls that had ampicillin negative plates tell us? Discuss errors and other reasons for data variability. Use your results to explain why it is important for synthetic biologists to fully characterize the chassis used in an engineered system. Navigation BioBuilding: Synthetic Biology for Students BioBuilding: Synthetic Biology for Students: Lab 1 BioBuilding: Synthetic Biology for Students: Lab 2 BioBuilding: Synthetic Biology for Students: Lab 3 BioBuilding: Synthetic Biology for Students: Lab 4 BioBuilding: Synthetic Biology for Students: Essay BioBuilding: Synthetic Biology for Students: Design Assignment BioBuilding: Synthetic Biology for Students: Glossary [to biobuilder]