Molecular Legos: Enter the BioBrick!

The world of a genetic engineer can be pretty stressful at times.

Photo by Aarón Blanco Tejedor on Unsplash

You are dealing with a blueprint in which each letter is barely half a nano-metre large, and depending on the organism, there can be millions to billions of these in just one cell. It is the raw un-simplified code, similar to binary on computers, so you can trust that it will be a headache introducing, removing, or substituting certain genes into an organisms genome — never mind building new genetic circuits. Unfortunately, unlike computer code, if there is any mistake, it is often irreversible, and likely expensive too.

This leads to the question, why would a field with such a tedious and error-prone research process flourish so well? Well, there have been ways to simplify and even hasten the research process. This has been done by introducing a new way to code in certain DNA sequences in larger reliable blocks. You can trust that this block of DNA code will work because it has worked for others, and you can quicken and even automate the process in which these sequences are introduced. This amazing new advancement is known as a BioBrick.

First introduced by researchers at MIT in 2003, Biobricks are essentially a standard for DNA parts, associating certain functions to these stands. These allow us to categorize these parts into specific groups based on what they do. We also refer to the BioBrick standard as RFC [10].

Hopefully as we progress through the article, you’ll start to associate these molecular bricks with a common childhood toy that I think we all know: Legos.

Photo by Ryan Quintal on Unsplash

They are a child favorite, an enemy to bare feet, and the perfect analogy to our dive into BioBricks. There are Legos of different sizes and shapes, but they are all capable of connecting to each other, and even more powerfully, there are some that fit perfectly into specific structures. Like these small plastic bricks, some DNA sequences are of different sizes, but they are able to fit together, and function optimally in certain positions within a sequence as they have definitive purpose.

How are BioBricks obtained?

Across all species that walk, swim, fly, or stand in one place in our planet, there is often a set way each may choose to express a trait. This could be growth rate, metabolic reaction intensity and frequency, or structural development. Since these processes could be similar across these organisms, the genetic sequences involved in expressing these traits could be too. When sequencing an organisms genome or trying to identify specific genes through reverse translation (using the amino acids in a protein to determine what DNA bases were involved) or using BLAST algorithms (using a suspected similar known gene in a known organism to figure out the unknown gene) new biobricks can be obtained.

New biobricks can also be constructed by combining smaller biobricks into a large structure that works to fulfill a specific function. Looking back at Legos, maybe you brought some together to build a Lego house. Multiple of these Lego houses could go together to make a Lego town. (PICTURE)

These new bricks can then be registered on repositories like the iGEM Registry of Standard Biological Parts or the BioBricks Foundation. So far, on the iGEM Registry of Standard Biological Parts alone, over 20,000 BioBricks have been identified.

→ Interesting Note: Every person who participates in the international Genetically Engineered Machines (iGEM) competition must submit a new BioBrick, a pretty clever way to increase the size of the database.

I think you may be starting to get why BioBricks are instrumental to the efficiency and development of fields such as genetic engineering. However, besides increasing efficiency and speed with work, there are other reasons they were created:

• Teaching genetic circuit design: It simplifies the process by putting it in larger chunks, guaranteeing any newbie quicker understanding
• Practicing the art of building genetic circuits: BioBricks make it easier and quicker to build genetic circuits, so it is a faster way to get accustomed to what is otherwise a pretty difficult skill to pick up.
• Progressing this field of science: It makes it easier to store data about various important parts of our genetic code, allowing for easier sharing of this data, and quicker communication between researchers.

How do different parts go together?

There are 5 general DNA parts intrinsic to the structure of a gene that should allow for constitutive expression (constant production of the product), you will never fail to see them:

• A Promotor: It is the site for RNA transcriptase binding, initiating transcription.
• An RBS/Kozak Sequence: The site at which a ribosome will bind to on RNA, beginning translation.
• The Coding Sequence: The part of the gene that will code for the protein in translation. It usually also includes the start codon.
• Stop codon: This part puts an end to translation
• The Terminator: This part puts an end to transcription by the RNA transcriptase

RFC 10: iGEM Handbook

Additionally, these parts are often sandwiched under six cut sites (three on each side of the whole DNA sequence, with one repeated on both ends). The sites are:

• EcoRI
• NotI
• XbaI
• SpeI
• PstI

These allow for the isolation of the sequence by restriction enzymes, an important process needed to transfer these parts from one organism to another in molecular cloning.

The cut sites that precede the sequence are known as the “prefix”, and the cut sites that follow it are known as the “suffix”.

An example sequence of the RFC10 standard

Prefix                                     Suffix
5' - GAATTC GCGGCCGC T TCTAGA G ...part... T ACTAGT A GCGGCCG CTGCAG - 3'
     EcoRI    NotI      XbaI                  SpeI     NotI   PstI

Considering the DNA parts above, as well as the cut sites, we have a complete BioBrick

Usually, you’ll see these standard parts go in the order listed to enable the proper transcription and translation of your desired protein. However, there are many more ways you can structure biobricks to come up with novel metabolic functions.

Limitations:

BioBricks as a model does have a few setbacks however, these usually arise as we try to get to more complex structures. These mainly appear in Constructing protein fusions, and the Prefixes and Suffixes.

When considering the construction of protein fusions (two or more proteins essentially glued together to perform a more specific and niche function based on the function of its individual parts), the BioBrick method falls short and may need to be changed to form the proteins. The scar sequence (the cleavage section) doesn’t allow for the binding of another protein sequence post translation by containing a stop codon (a sequence that signals the stop of translation in a mRNA sequence). To allow for the coding of another protein, one would have to attach it to the main protein being coded for, adding on complexity that may result in error in translation.

Fusion Protein: Wikipedia

Moreover, the cut sites at the prefix and suffix should be taken into consideration, as they are not exactly the most rare in sequences. This could be particularly dangerous in longer gene sequences that you may want to use. Restriction enzymes will always respond to cut sites that they are complementary to, meaning that if it happens to fall in a gene sequence, it will be cleaved. While this can be avoided by using mutations that would not interfere with the resultant amino acid coded, this is often a time consuming process.

Is the BioBrick standard the only one being used?

To address some of the limitations that the BioBrick standard holds, other standards have been pioneered. These are often not too different, featuring only slight changes to the prefix and/or suffix of the BioBrick. These standards include:

• RFC [12]: This is also referred to as the BB-2 Standard. It was made to allow for protein assembly by moving the stop codon in the scar sequence further down. A downside of the method is that there are not a lot of parts within the standard registry that are compatible with the standard
• RFC [21]: This is also known as the Berkeley standard, and is also used for protein assembly. However, unlike the BB-2 method, different cut sites are used (namely BamHI and BglII)
• RFC [23]: This method, also known as the Silver Standard, was pioneered to allow for fusion proteins. This is achieved by shortening the scar sites to six base pairs. This allows for two proteins to be joined together while the frame of the proteins remains constant
• RFC [25]: This is also known as the Freiburg standard, it is also used for the construction of fusion proteins by modifying the prefix and suffix sequences.

A wrap up, and what’s next?

From this short article, we learned about the basics of genetic engineering, and what allows for the complex feats that people are able to achieve in synthetic biology. The relatively simple framework are the brick and mortar (or Legos if you prefer) of genetic circuit design.

Next, I’ll be covering the high level language we use to understand genetic circuits: Synthetic Biology Open Language (SBOL)

Sources:

Because a lot of great people are the true synthesizers of the knowledge that’s helped me understand this :)

Help:Standards — parts.igem.org

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