CHE.496/2008/Responses/a4

Genetic Programming

• Discussion leader: Dan Tarjan (Discussion guide)

Patrick Gildea's Response

• Idempotent Vector Design for Standard Assembly of Biobricks
  • The purpose of this article was to make a case for the standardization of assembly techniques for biobricks to ensue that structural elements of the biobricks remain the same as all the biobricks transform into the module as a whole. In addition, the article goes through the standard sequence they chose as a basis for biobrick components - on the upstream end by EcoRI and XbaI restriction sites, and on the downstream end by SpeI and PstI restriction sites. For a module to be inserted into an assembly vector – specific restriction enzymes are used to cut the DNA (in the regulatory sequence) and insert the biobrick components at the restriction sites that have been cut. In order for this technique to be useful, the assembly vector cannot contain any other sites that the restriction enzymes could cut into. I am not 100% sure, but the restriction enzyme discussed in the article is applicable to a broad spectrum of organisms that can be used to insert biobrick modules. This is important because as a standard, this technique has to apply to many different types of assembly vectors. This article is relevant for the 2008 project because we need to understand how to fit in our biobrick module into the assembly vector without changing the structure or function of our biobrick components. For example, accidental creations of ATG start codons that could potentially start mutations, as well as other scenarios. Furthermore, this area of standardization needs to be explored – the article mentions different assembly vectors that add different functionalities to the assembly process (such as the NOMAD system). Research into different assembly vectors and investigations into what functionalities, benefits/disadvantages could provide a better basis of standardization.

• Genetic parts to program bacteria
  • The purpose of this article is to discuss how biobricks are being used to program organisms via cell-based sensors and circuits. In other words, frameworks for synthetic biology (akin to VLSI for electronics in the 1980’s) are being developed in order to design biological systems with process and data abstraction in mind. A list of genetic parts and their description is given, which is very valuable for looking at parts that program bacteria (Table 1). The common types of tools are discussed: sensors and how they work with different types of inputs (i.e. u.v. light, cytoplasmic regulatory proteins); circuits such as switches, inverters, logic gates, etc. The descriptions and their use of different tools used to govern the behavior of a bacterial organism is the most valuable part of the paper because it gives us all a run-through on the different tools that can be used and ideas for more complex systems can arise from thinking about the different ways these tools can be used in different combinations. These concepts of the tools can be applied toward design ideas of programming bacteria for a specific purpose. In other words, we can use the knowledge in this paper as a source of general knowledge to be applied to whatever project design we come up with. Another part of the paper that is especially beneficial is the debugging and tuning of complex systems with different techniques described. For the project in 2008, we may have to use one of these techniques depending on what we design. Knowing what tools to apply to a problem is half the solution of solving a problem.
• Patrick Gildea 16:08, 27 January 2008 (CST):

Kevin Hershey's Response

• Idempotent Vector Design for Standard Assembly of Biobricks
  • This articles primary purpose is to illustrate the design components of the biobricks. It begins by discussing the aspect of standardization using the analogy of a screw, where standardizing the thread of a screw helped the industry tremendously. The article then gets to the meat of the topic, discussing the design of biobricks. The most important design is the location of the restriction enzymes. The standard format is as follows: Eco RI, Xba 1, insert, Spe 1, Pst 1. This allows users to 'plug and play' devices into the biobrick as long as they keep the sequences standard. The advantage to this format is that if an insert is added to an existing biobrick, it cannot be broken again. In other words, if the Eco RI and Xba 1 are chosen to be ligated, the sequence is lost, and the biobrick does not have a risk of being fragmented after it has been constructed.
• Genetic parts to program bacteria
  • In this article, Christopher Voigt discusses the engineering currently being done in synthetic biology. That is to say, some examples of how synthetic biology is currently being used and created. His figure showing the different switches and sensors is very useful, especially as a precursor to using the Standardized Registry of Biological Parts. Voigt then goes into detail of different two component systems, sensors and inputs, genetic circuits, and actuators. Voigt then goes into detail on debugging and tuning. The main point from this article is the ability of synthetic biologists to build complex systems from their standardized parts.
• User:KPHershey 12:01, 27 January 2008 (EST)

George McArthur's Response

• Idempotent vector design for standard assembly of biobricks
  • This article, by Tom Knight (sort of the father of synthetic biology), is primarily about BioBrick theory and construction (with some information regarding the motivation behind this design). In a nutshell, synthetic biology requires the use of standard parts. In our case (i.e., synthetic biology practiced by iGEM and the Registry), BioBricks provide the standard. BioBricks utilize standard antibiotic resistances and restriction enzyme sites. The main idea is that the insert (which usually contains a promoter, ribosomal binding site, coding region, and at least one termination) is flanked by a prefix and suffix that contain the standard restriction enzyme sites. The prefix includes EcoRI and XbaI while the suffix includes SpeI and PstI (in that order). This serves as the basis for the genetic engineering that is done in the lab.
• Genetic parts to program bacteria
  • Chris Voigt lists various parts that have been developed to perform in bacteria in this nice introduction to synthetic biology as a programming science. Individual genetic parts can be combined to create more complex systems. That is, cells can be programmed to perform tasks. Using sensors (input), internal circuitry (biochemical logic gates), and actuators (outputs such as the production of a certain chemical), a complex system can be designed and constructed in bacteria like E. coli.
• GMcArthurIV 17:19, 27 January 2008 (CST)

Eyad Lababidi's Response

• Genetic Parts
  • This article really opened up my eyes to the possibilities of parts that could be used in cells. I never realized the capabilities were already so broad, but what i do not understand is if certain chemicals are the inputs and outputs for each part that is acting as an actuator or a circuit or an input device then isnt there a limit on the number of different chemicals you can modify and have to carefully pay attention to the effects of the changing environment on capability of the parts, which explains why characterization is so important but to characterize the parts in endless amounts of different situations due to different chemical levels seems quite impossible. This also explains why the article explains that simple biosynthesized cells have been realized while complex cells will have a much harder time to be completed successfulle.

• Idempotent Vector Design
  • I definitely agree that the standardization of biosynth will create the next industrial revolution because it will open up doors of understanding in a field that has had a lot of smoke and mirrors for the inner workings of cells. With standardization of not only cut sites but also process it makes the products interchangeable with other users and cheap to produce. Having the process standardized also makes it easier for new users to know what not to do in cases liek repeating code patterns that should not be repeat in order to complete the biosynthetic process. I did not understand the explanation on how the cuts actually happen and where the new genes are inserted and i hope that we can go over that during discussion.

• Eyad Lababidi 16:29, 27 January 2008 (CST)

George Washington's Response

• Idempotent Vector Design for Standard Assembly of Biobricks
  • This article described, in short, the importance of standardizing DNA assembly, followed by detailed description of the processes involved. I couldn't completely follow the fine details of their experimental design, but it gave a decent idea of what was involved in developing the standard. For instance, I liked the example of EYFP, and the need for point mutations to eliminate its PstI site. The standard presented in the article seems an effective construct, as its unique front and back restriction sites and formation of mixed sites upon ligation allow relatively simple concatenation of any number of component genes, so long as they do not contain copies of these restriction sites within them. However, as aforementioned, point mutations should be able to eliminate any issues with these. In any case, techniques such as these should be useful in the construction of our own vectors for the competition. Standard assembly techniques will save untold weeks of experimentation and failure in the seemingly simple transformation of a few genes. By mastering these techniques (or at least achieving some modicum of proficiency), we should be able to expedite our research and focus on our experiments, not our experimental methods.

• Genetic parts to program bacteria
  • This article described several systems that have been developed for use in cell systems. It enumerated members of the classes of component: sensors, genetic circuits, and actuators. Knowing well many of the devices described will be incredibly useful in developing our own systems, as will many not described. Being familiar with the state-of-the-art in component design is going to be essential if we are to succeed. The article emphasized standard organisms, promoters, etc. in the creation of reliable, designable, predictable systems. One of the consequences of this will be much more usable transfer diagrams, which describe response curves for output given input into the components. If these can be created for standard organisms with standard conditions, they can be predicted in synchrony with other parts, which can be combined, in silico, to design a complete system without the endless experiments that would otherwise be needed to analyze synergy. The most important thing to know is that familiarity with these types of systems must be acquired such that when our team realizes a need for some function, we can immediately reference the literature and obtain the part we need quickly. We cannot afford to have to scour the literature every time we need something new.
• George Washington 21:56, 27 January 2008 (CST)

Brandon Freshcorn's Response

• Genetic parts to program bacteria
  • Voigt introduces the reader to the “most robust genetic parts that have been reused in different designs” which have been broken down into three categories. These categories include sensors (inputs), genetic circuits (processing), and actuators (specific outputs- such as apoptosis). This article made me realize how closely synthetic biology relates to my previous linear systems and signals class where we modeled various chemical, biological, and electrical phenomena. The author even specifically writes about the availability of transfer functions for certain genetic parts. With this new insight, we could easily program and model our own genetic circuit using the methods found in a linear systems and signals course.

• Idempotent Vector Design for Standard Assembly of Biobricks
  • Knight starts with discussing the motivation for the standardization of assembly techniques of DNA sequences- “the widespread ability to interchange parts, to assemble sub-components, to outsource assembly to others, and to rely extensively on previously manufactured components”. This standardization is achieved through the use of only certain types of restriction sites (EcoRI and XbaI on the 5’ side and SpeI and PstI on the end) and further achieved by removing other restriction sites of this type by point mutation. This standardization allows for the ligation of components during which a mixed SpeI/XbaI site is formed, but since this new sequence is no longer a restriction site, the sequence cannot be further cleaved.

--Brandon S. Freshcorn 07:55, 29 January 2008 (CST)