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1 Introduction to Part Assembly: Building with BioBrick TM Objective: To simulate a standard method for assembling biological parts. Intended Learning Outcomes: 1. Students will be able to distinguish and properly use synthetic biology terms: BioBrick TM part, assembly, digestion, ligation, plasmid backbone; 2. Students will become familiar with a standard strategy for assembling a new composite part from two part samples, into a single plasmid backbone; 3. Students will visualize the process of 3A Assembly by simulating the process using common materials; and 4. Students will be able to articulate the mechanics involved in digestion and ligation of assembly parts. Introduction Restriction enzymes and their function as cleaving molecules produced by bacteria to break down, or restrict, foreign DNA were discovered in the 1960s. Since then, the field of genetic engineering has relied on the ability of these enzymes to recognize and cut at specific locations along the DNA molecule called restriction sites. Different restriction enzymes have their own recognition site, but a restriction site in general, is a short, palindromic base-pair sequence. A palindrome is a word, phrase, number, or other sequence of symbols that reads the same forward or reversed. A DNA palindrome is a sequence of nucleotides in which the top strand read from 5' to 3' is the same as the bottom strand read from 5' to 3'. For example, the following restriction enzymes are considered palindromes. Can you explain why? Design Test Build

2 EcoRI SpeI 5... G ^ A A T T C A ^ C T A G T C T T A A ^ G T G A T C ^ A... 5 PstI Xbal 5... C T G C A ^ G T ^ C T A G A G ^ A C G T C A G A T C ^ T... 5 Figure 1. Common restriction enzymes and their associated cut sites indicated by ^. When EcoRI cuts at its restriction sites, the resulting strands will leave single stranded tails called sticky ends that can easily rejoin to complementary single strands with the help of DNA ligase. Not all restriction enzymes cleave in a way that forms sticky ends. Some cut the DNA strand directly across from each other and therefore produce what are known as blunt ends. In synthetic biology, the process of restriction enzyme digests and ligations are employed for the specific purpose of building novel devices from standard parts. The abstraction hierarchy is a language that synthetic biologists use to refer to specific DNA sequences that code for specific functions. This language successfully distinguishes between DNA, parts, devices, and systems, so that just like the letters of the alphabet, DNA sequences can be stitched together to make parts which can further be connected into more complex composites in the form of devices and systems that code for specific, reliable outputs. The ability to build complex systems requires a method for reliably, assembling the desired constituent parts as well as a warehouse for managing and storing available parts.

3 Figure 2. (above) The actual BioBrick part, device, or system is located between the prefix and suffix while the plasmid backbone is the sequence that starts with the suffix, ends with the prefix, and includes the origin of replication and antibiotic resistance marker. A high copy plasmid DNA backbone produces a high yield in culture to make assembly easy ( parts.igem.org/plasmid_backbones/assembly). Luckily, the Registry of Standard Biological Parts ( serves as a repository for standardized genetic parts that are used in the assembly of devices and systems. There seem to be an infinite number of ways these parts can be designed, but in order to ensure the compatibility of the parts available through the Registry, the BioBrick TM standard has been adopted and is widely used. BioBricks TM are standard biological parts of DNA sequences that have a prefix and suffix so that all BioBrick TM parts are compatible, making assembly of these parts easy. Although assembly cannot be guaranteed under this protocol, the standardization of parts and protocol increases the likelihood that components will be reliably synthesized into a composite part. BioBrick parts introduce the engineering principles of abstraction and standardization into synthetic biology. 3A assembly stands for three antibiotic assembly, and relies on three-way ligation between the two parts of interest and the backbone vector - all of which have a different antibiotic resistance to help in colony selection. This positive and negative selection reduces the number of incorrect plasmid assemblies that give rise to colonies after transformation. Overview Why would you want to combine parts? Well, imagine that you have designed a system that is predicted to operate as described in the Introduction to Genetic Circuits module. The components in this Figure 3. Schematic of how component parts ( A and B here) will be delivered on 2 different plasmid backbones conferring different antibiotic resistances. cassette include a Tet promoter, a ribosome binding site, and the coding sequence

4 Part A. Part B. Figure 4. Notice that the assembly product (lower plasmid) is a composite of Parts A and B and also confers a different antibiotic resistance; this is the foundation for 3A Assembly - 3 (different) A(antibiotics). for green fluorescent protein. After searching through the Registry of Parts, you discover that the Tet promoter and the RBS you want to use in your device is available as a single part, as well as the green fluorescent protein coding region, and may arrive in 2 separate agar stabs of live bacteria (E. coli); that is, each component has been transformed into live bacteria, and you will have to first grow more bacteria under the proper antibiotic resistance conditions, and then miniprep your cells to extract the desired plasmids for assembly. Your plasmids with the desired BioBrick components may also arrive as purified plasmid DNA samples which will eliminate the growth and mini-prep steps in your assembly. By employing the steps outlined in 3A Assembly, the parts you want to combine into the same plasmid (Part A, Part B, and the backbone) will first undergo a restriction enzyme digest and then undergo a ligation to chemically combine the parts of interest. Once the plasmid has been built, it will then be transformed into the desired chassis, usually E. coli. In this activity, you will first model the assembly method described using paper plasmids. Your end result should be a plasmid that contains the parts necessary for expressing green fluorescent protein once transformed into a bacterium. In the second part of this activity, you will be provided with a different modeling kit that you will use to solve a persistent microbiology lab problem. As you work with your partner(s), discuss the reasons for your part selections, and be prepared to use your whiteboard to address the following questions: 1. Describe each part of your genetic construct. 2. Did other teams have the same design?

5 BioBrick identification code 3. Why did your team choose these parts over other parts? 4. What alternative sequence of parts might give us similar results? 5. Is there a way to actually test our design? Procedure Exercise 1. Modeling 3A Assembly of BioBricks 1. You will be provided with 3 sheets with 6 strips of double stranded DNA. The sheets have been designed to correspond to specific BioBrick parts which are labeled on the templates as BBa_R0040 (Part A), BBa_E0240 (Part B), and the destination plasmid. Each sheet is similarly formatted to include information about the BioBrick construct. Shaded regions denote sequences of significance including tetracycline repressible promoter (in blue), origin of replication (in purple), and kanamycin resistance (in red). Order the strips will be taped together vertically Part identification information. This sequence is the code for a kanamycin resistance plasmid backbone that is carrying the sequence for the tetracycline repressor promoter which is flanked by a prefix and suffix consistent with the BioBrick standard. See the example to the left. 2.Cut out each BioBrick along the dotted lines, keeping sure that the order stays the same. It might help to label the back of each strip you cut with its corresponding order number. 3.Tape the strips of paper DNA together so you have a vertical strip of paper made from 6 smaller strips. Tape the ends of this vertical strip of linear, paper DNA into a circle so that the top of strip 1 and the bottom of strip 6 are touching. You have now built the first BioBrick part. 4.Repeat steps 2-3 for the other BioBrick and the plasmid backbone. Make sure not to mix up the papers. 5.Once you have assembled 3 circular, paper plasmids, scan along the DNA sequence of BBa_R0040 (coding sequence for a constitutive promoter; one that is on all the time) until you find

6 the EcoRI site (refer to the list from the introduction above for the sequence). 6. Once you have identified the EcoRI cut site, use scissors to simulate what this restriction enzyme will do to the phosphodiester backbone by cutting just between the G and the first A of the restriction site on both strands. Do not cut all the way through the strip. Remember that EcoRI cuts the backbone of each DNA strand separately. 7. Now separate the hydrogen bonds between the cut sites by cutting between the nucleotides in this EcoRI site. Separate the two pieces of DNA. Look at the new DNA ends produced by EcoRI. Are they sticky or blunt? Write EcoRI on the cut ends. 8. Next, find the SpeI site on this strip of linearized DNA and repeat the procedure described above. Are the new ends sticky or blunt? Label the new ends SpeI, and discard the kanamycin resistant (kanr) backbone. Check for Understanding: Describe what you think was accomplished by cutting at these 2 restriction sites on this plasmid. 9. Repeat this exercise with your next BioBrick component by scanning along the DNA sequence of BBa_E0240 (coding sequence for a composite part that includes an RBS, green fluorescent protein open reading frame, and a double terminator) until you find the XbaI site (refer to the list from the introduction above for the sequence). In this case, simulate the activity of XbaI in the same way you did with EcoRI. Are these ends sticky or blunt? Label the new ends XbaI. 10.Next, find the PstI site on this strip of linearized DNA and repeat the procedure described above. Are the new ends sticky or blunt? Label the new ends PstI, and discard the ampicillin resistant (amr) backbone. Check your Understanding: Describe what you think was accomplished by cutting at these 2 restriction sites on this plasmid. 11.Repeat the restriction digest by cutting the plasmid backbone, psb1c3, only once at EcoRI and PstI sites. Label the new ends EcoRI and PstI. Check your Understanding: Describe what you think was accomplished by cutting at these 2 restriction sites on this plasmid backbone. 12.By examining your products, you should now have successfully cut out a promoter (BBa_R0040) and a composite part containing an RBS, the open reading frame for

7 GFP, and a double terminator (BBa_E0240). In addition you should have a linear strip of DNA that confers chloramphenicol antibiotic resistance. Check your Understanding: Examine these components. Is there anything interesting about these parts? 13.An enzyme called DNA ligase can rejoin single stranded DNA by reforming phosphodiester bonds between nucleotides. For DNA ligase to work, two nucleotides must come close together in the proper orientation for a bond (the 5' side of one must be next to the 3' side of the other). Check your Understanding: Do you think your products can be reconnected using DNA ligase? What makes you think so? 14. Using tape, secure the BioBrick parts together and then attach them to the chloramphenicol backbone. Check your knowledge: Describe what you think was accomplished in this step. 15. If you reasoned that the newly constructed plasmid now has the same prefix and suffix as the original parts along with a mixed site, and that this new arrangement is now referred to as a composite part, you would be correct 16.In the space provided, sketch the process of 3A Assembly that you modeled. Feel free to simplify your model in a way that makes sense to you. On the circle that follows, draw the components of your final assembly.

8 My Interpretation of 3A Assembly My Final 3A Assembly Plasmid Exercise 2. Designing a Plasmid 1. Now that you have simulated what happens think about this... during 3A Assembly,

9 Is it possible to design a system that can program E.coli to produce different compounds that smell fragrant? Since scents can both act as natural reporters a n d have a diverse array of applications, is it possible to design a system with known parts that could improve the workplace environment for microbiologists working with Escherichia coli since E. coli produce a natural foul scent? 2. You have been provided with a set of simple BioBrick models to work with in this exercise. Each model is a linearized version of a designated BioBrick construct. See the example below for details: 3. When these linear DNA plasmids are cut out and taped into a circle, they will look like RBS Double terminator sequence GFP coding sequence ampicillin resistance this: GFP ampr 4. With your partner(s), determine which parts you might use to reasonably answer the question Linear from BioBrick above. plasmid Remember, the goal is to use the parts provided to design and construct (using 3A Assembly) a model plasmid that could be transformed into E. coli to solve the problem of a stinky lab 5. Keep the following in mind as you work: a. You are limited by the parts provided in this kit; b. Feel free to consult The Registry of Biological Parts ( Catalog) for more information on the parts in your kit; c. Make sure you think about when you want your bacteria to produce this smell during your brainstorming session; Cut Sites Linear BioBrick plasmid

10 d. Include a list of your parts and how they function as well as a description of the predicted behavior of the bacteria once your newly designed construct has been ported into the cell culture. 6. This should help you organize your work: Preliminary Research. Do a little research and investigate what the parts can do. Then, decide as a group if the part is useful in helping you solve this problem. (Note: Leave off the extra a and b when searching in the Registry.) Part ID Number BBa_J45992 BBa_R0040a BBa_R0040b BBa_E0240 BBa_J45199 BBa_J45099 BBa_Q04401a BBa_Q04401b psb1c3 What part does it code for? What does it do? Can we use it? (Y/N) As a team, design at least 3 different genetic constructs using the parts from above in different combinations. Then, record a prediction about how you think this circuit would work in a cell. Then, rate your design on a scale of 1-5, 5 being the most reasonable design for this problem, with justification. Possible Combination 1. Part 1 Part 2 Part 3 Final Construct

11 Part 1 Part 2 Part 3 Final Construct Predicted Cell Behavior: Design Rating (circle): Justification for Rating: Possible Combination 2. Part 1 Part 2 Part 3 Final Construct Predicted Cell Behavior: Design Rating (circle): Justification for Rating: Possible Combination 3.

12 Part 1 Part 2 Part 3 Final Construct Predicted Cell Behavior: Design Rating (circle): Justification for Rating: 7. Now, build your final plasmid using 3A Assembly. Illustrate how your team employed 3A Assembly to construct your novel design in the space provided. How we Assembled these Parts 8. Then, draw your final construct on the circle below. Be prepared to share your unique work with your classmates.

13 What do you think? Now that you have simulated what happens during 3A Assembly, and have designed your own system to solve a pervasive microbiology lab problem, do you think your system has real potential in the lab? In other words, does it work? How can we answer this question? Extension 1. Additional paper BioBrick DNA sequences have been included in this activity if you would like to make additional plasmids using 3A Assembly. 2. Alternatively, you might consider having students demonstrate how this assembly method works by have them wear the name tags for each component involved in 3A Assembly. As they are demonstrating the process, they will be evaluated on their ability to articulate the mechanics of the 3A assembly process. 3. Students could produce a Voki for 3A Assembly ( 4. Use the 3A Assembly Kit distributed by igem to prepare a composite part. 5. The method you modeled in this activity is only one method of DNA assembly. Gibson, Golden Gate, and MoClo are 3 other leading assembly methods available

14 for piecing together genetic parts. In your group, pick one of these methods. Do some research, and compare it to 3A Assembly that you modeled. Consider the advantages and disadvantages of each method, and report back to the class what your group discovered. Going Further. Restriction Maps This is a restriction map of the circular plasmid psb1c3. This plasmid contains 3,139 base pairs. There is an EcoRI site at base pair The locations of other restriction sites are shown on the map. The numbers after the enzyme names tell at which base pair that enzyme cleaves the DNA. If you digest psb1c3 with EcoRI, you will get a linear piece of DNA that is 3,139 base pairs long. 1. What would be the products of a digestion with the two enzymes EcoRI and PstI? 2.What would be the products of a digestion with the two enzymes XbaI and SpeI? 3.What would be the products of a digestion with the four enzymes EcoRI, XbaI, SpeI, and PstI? 4. If you took the digestion products from question 1 and digested them with SpeI, what would the products be? Acknowledgements: This activity is intended to be used in conjunction with the 3A Assembly kit provided by igem Headquarters to all high school teams participating in the igem HS Jamboree (MIT, 2006). The technical information for BioBrick parts and constructions used in this activity were obtained from the Registry of Biological Parts as submissions by former collegiate igem Teams. Assembly of parts using the paper plasmid constructions was inspired by an introductory plasmid lab entitled The E. coli Insulin Factory (author, unknown).