CHE.496/2009/Responses/a4

Engineering Principles

• Discussion leader: Maria

(Thaddeus)'s Response

• Idempotent Vector Design for Standard Assembly of Biobricks
  • Describes the usefulness and construction of Biobricks as well as the formatting of the Standard Assembly of Biobricks.
  • Consists of a double stranded DNA strand with four unique restriction sites flanking the gene of interest.
  • Each construct can be cut to either form a front or rear insert or vector.
  • After each round of cutting and inserting new inserts into the vector the construct will be identical to the construct before the new piece was inserted, allowing the process to be repeated in the exact same way.
  • Bases in between restriction sites were chosen carefully to prevent unwanted methylation and cutting of consruct.
  • Contain standard primers for the verification of sequence.
  • This article will be useful for the VGEM team because we will be using biobricks to create our machine and will be creating new biobricks as part of the project.

• Genetic parts to program bacteria
  • The article describes how cells can be programed by using simple devices that form circuits and complex behaviors when combined properly. It also describes the most successful parts.
  • Each circuit or device can only be used once in a biological machine because the biochemical reactions take place in the same space. This will require redundancy of parts to create complex machines.
  • Response of circuit is dependent on what state in the cell cycle the cell is. Devices must resist this dependence.
  • Sensors allow cells to gain information about the external environment.
  • Genetic circuits interpret and process the information from the sensors.
  • Actuators control cell behavior.
  • Debugging can be achieved either by replacing a non-compatible part of a protein with a known part or by applying selective pressure to a sensitive part of the machine under mutagenisis until a functioning machine evolves.
  • This article will be useful to the VGEM team because it breaks down the standard parts of a machine and identifies their function. We will be using these parts to construct our machine.

Thaddeus Webb 19:58, 18 February 2009 (EST)

Rohini's Response

Tom Knight’s article, “Idempotent Vector Design for Standard Assembly of Biobricks” discussed the method of standard vector assembly using the biobrick composition technique. Knight along with authors of various articles previously assigned for this class stress the necessity of having a standard set of reliable engineering mechanisms. By utilizing a standard and universal assembly technique, research in building biological systems from different genetic components can be greatly benefited. With the construction of standards, researchers can use previously manufactured components from other systems, outsource parts to various assemblies and be able to interchange parts between systems. Knight details the two step process of making a biobrick which is: a) prefixing a recipient with a donor component and b) post-fixing an insertion of a donor fragment into a recipient vector. The example used to illustrate this procedure is the transformation of the LacZα gene into a standard component that can be then used to test the effectiveness of the cloning technique. Knight also mentioned various challenges he faced dealing with his research. I did not realize that one has to be very selective in picking the restriction enzymes to cut the DNA sequence. The enzymes have to be able to withstand the environment of the experiment and other specificities. Overall, Knight’s research goal was to transform component parts during the assembly reaction while ensuring that distinct structural elements of the component remain unaltered. As a result, the component can be placed within a library of previously assembled components and made accessible for future complex assemblies.

I think researchers have found some interesting and revolutionary scientific applications that have the potential to advance the current treatments used within the field of medicine. I was amazed after reading about how scientists are working on discovering ways to create more operational commands in bacteria such as: synthesizing anti-malarial and cancer fighting drugs. They were able to begin working on their research only after realizing microorganisms can be programmed using synthetic constructs of DNA set with operational tasks. Voigt’s article, “Genetic Parts to Program Bacteria” discussed present challenges dealing with genetically engineering microorganisms. The two most prevalent problems are: cells can evolve and could potentially mutate and genetic parts interfere with one another within a system which means a genetic circuit can only be used once in design.

The information present in the two articles can be applied to the VGEM project. If we can work with standard components already available in the biobrick library then we can deal with fewer complexities.

Rohini Manaktala 8:10 p.m., 18 February 2009

Patrick's Response

• Idempotent Vector Design for Standard Assembly of Biobricks
  • Tom Knight’s article illustrates the concept of standards for biobricks in the form of restriction sites in order to make different biobricks compatible with each other. Each biobrick consists of a plasmid vector of double stranded DNA containing the desired gene of the biobrick that is flanked on the upstream end by EcoRI and XbaI restriction sites, and on the downstream end by SpeI and PstI restriction sites. This standard is applied for every biobrick in the registry because it allows for uniformity of biobricks so they can be simply pieced together like legos. The paper goes through biobrick assembly procedure, the composition technique where you simply add another biobrick on the upstream or downstream end depending on the restriction enzyme that is used. However, this paper does not discuss the Triple assembly procedure that was used this past summer, which allows for the simultaneous insertion of three biobricks in a plasmid vector through the use of 3 different antibiotics and their corresponding resistances on each biobrick component. New members on the team may find the example 0.5 useful as a thought exercise on constructing a biobrick. This article is relevant mainly for the new members on the team in order to introduce them to the biobrick standards – the restriction enzymes used. As well as to introduce to them the idea of designing and constructing plasmids based on these enzymes.
• Genetic parts to program bacteria
  • This article discusses various biological “devices” that have been designed as logic devices such as the repressilator. The goal is to develop a framework where the idea of scalability can be applied to biological devices. Among the devices discussed in the article are: sensors and their inputs, promoters, aptamers, switches, logic gates, etc. I found Table 1 to be very useful to study as many sensors and their functionality is described. This is extremely helpful since it is possible in the future we may build a network of devices that may incorporate a sensor in this table – I found the graphs particularly interesting as the slopes for the input versus output for each device (for example: Tet, Lac, and Ara) where their reaction rates vary despite being somewhat similar in design. This behavior is described as the transfer function and is elaborated in box 1, where the behavior of how the output changes as a function of input . The variation for devices that are similar in construction is due to cell-to-cell variation, fluctuations owing to small numbers of molecules, and noise in transcription and translation. The paper discusses in length how inducers affect the behavior of the cell, for example, cytoplasmic regulatory proteins. Also mentioned in the paper is RNA aptamers, which are small RNA molecules that changes conformation when bound to a protein, thereby changing the function of the protein. This is similar to the idea of the genetic attenuator which knocks off the transcript in a gene sequence at a rate based on their termination efficiency. If I were to optimize a gene sequence, what would serve best? Either the genetic attenuators or some other method that directly interact with the enzyme? Offhand, the genetic attenuator would be easier to implement and easier to control, yet it is food for thought.
• Patrick Gildea 17:59, 19 February 2009 (EST):

Joe's Response

• Idempotent Vector Design for Standard Assembly of Biobricks
  • BioBricks:
  • Are DNA sequences of defined structure and function
  • Designed to be used as vector parts to construct biological systems in living cells
  • Consist of six restriction sites for restriction enzymes
  • The restriction sites between two parts are cut by the restriction enzymes for the chaining process
  • Have upstream and downstream sequences that are not considered true parts of the brick
  • Registry of thousands of parts is maintained
  • Demonstrates standardization in synthetic biology
  • Three levels: parts (like protein or promoter), devices(collection of parts), and systems(performing some defined function)
  • Are the basis for the iGEM competitions
  • We will be using them over the summer, so we need to be familiar with them

• Genetic parts to program bacteria
  • Gives examples of the way microorganisms can be programmed using DNA constructs
  • Sensors, genetic circuits, and communication components have been designed
  • Problems: molecular interactions promote interference and make isolation difficult, and cells are alive, so they can mutate or react to a change in environment
  • One interesting concept was the genetic switch to control gene expression. The design is simple yet the it can be quantified and optimized through various methods such as RT (real time) PCR
  • The logic gates and dynamic circuits also looked interesting, but I tend to like electrical engineering anyway
  • One key point was on debugging and tuning complex systems. It is clear that standardization and computer design will help accelerate system construction but there will still be problems connecting the parts to get a functioning system
  • Lots of data exists on metabolic pathways, so one possibility for the summer might be programming the metabolic pathways of cells in order to obtain a product from proteins.

Joe Bozzay 17:08, 19 February 2009 (EST)

Maria

Bio bricks

• descriptive article: meant to familiarize us with the methods
• discusses the difference between inserts and vectors
• describes regulatory sequenes and how they are cut

Spel/Xbal cannot be cut by enzymes

• NOMAD – only one type of insert
• minimum genes needed for proper cutting (~8)
• verification primers are used for PCR
• RNA binding site and terminator much be included (can be added by primer)

Genetic parts to program bacteria

• sensoprs and inputs
  • communication
  • identify microenvironment
• spatial patterns : quorum sensing: negative feedback
• record images
• form biofilm in response to uv light

We should build a list of techniques (ie repressors which are uv dependent) that could be applied to other expression pathways.

• Maria Fini 18:35, 19 February 2009 (EST):