Artificial gene synthesis

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https://en.wikipedia.org/wiki/Artificial_gene_synthesis 

 

DNA Double Helix

Artificial gene synthesis, or gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing. This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.

Synthesis of the first complete gene, a yeast tRNA, was demonstrated by Har Gobind Khorana and coworkers in 1972. Synthesis of the first peptide- and protein-coding genes was performed in the laboratories of Herbert Boyer and Alexander Markham, respectively. More recently, artificial gene synthesis methods have been developed that will allow the assembly of entire chromosomes and genomes. The first synthetic yeast chromosome was synthesised in 2014, and entire functional bacterial chromosomes have also been synthesised. In addition, artificial gene synthesis could in the future make use of novel nucleobase pairs (unnatural base pairs).

Standard methods for DNA synthesis

Oligonucleotide synthesis

Main article: Oligonucleotide synthesis

Oligonucleotides are chemically synthesized using building blocks called nucleoside phosphoramidites. These can be normal or modified nucleosides which have protecting groups to prevent their amines, hydroxyl groups and phosphate groups from interacting incorrectly. One phosphoramidite is added at a time, the 5' hydroxyl group is deprotected and a new base is added and so on. The chain grows in the 3' to 5' direction, which is backwards relative to biosynthesis. At the end, all the protecting groups are removed. Nevertheless, being a chemical process, several incorrect interactions occur leading to some defective products. The longer the oligonucleotide sequence that is being synthesized, the more defects there are, thus this process is only practical for producing short sequences of nucleotides. The current practical limit is about 200 bp (base pairs) for an oligonucleotide with sufficient quality to be used directly for a biological application. HPLC can be used to isolate products with the proper sequence. Meanwhile, a large number of oligos can be synthesized in parallel on gene chips. For optimal performance in subsequent gene synthesis procedures they should be prepared individually and in larger scales.

Annealing based connection of oligonucleotides

Usually, a set of individually designed oligonucleotides is made on automated solid-phase synthesizers, purified and then connected by specific annealing and standard ligation or polymerase reactions. To improve specificity of oligonucleotide annealing, the synthesis step relies on a set of thermostable DNA ligase and polymerase enzymes. To date, several methods for gene synthesis have been described, such as the ligation of phosphorylated overlapping oligonucleotides, the Fok I method and a modified form of ligase chain reaction for gene synthesis. Additionally, several PCR assembly approaches have been described. They usually employ oligonucleotides of 40-50 nucleotides long that overlap each other. These oligonucleotides are designed to cover most of the sequence of both strands, and the full-length molecule is generated progressively by overlap extension (OE) PCR, thermodynamically balanced inside-out (TBIO) PCR or combined approaches. The most commonly synthesized genes range in size from 600 to 1,200 bp although much longer genes have been made by connecting previously assembled fragments of under 1,000 bp. In this size range it is necessary to test several candidate clones confirming the sequence of the cloned synthetic gene by automated sequencing methods.

Limitations

Moreover, because the assembly of the full-length gene product relies on the efficient and specific alignment of long single stranded oligonucleotides, critical parameters for synthesis success include extended sequence regions comprising secondary structures caused by inverted repeats, extraordinary high or low GC-content, or repetitive structures. Usually these segments of a particular gene can only be synthesized by splitting the procedure into several consecutive steps and a final assembly of shorter sub-sequences, which in turn leads to a significant increase in time and labor needed for its production. The result of a gene synthesis experiment depends strongly on the quality of the oligonucleotides used. For these annealing based gene synthesis protocols, the quality of the product is directly and exponentially dependent on the correctness of the employed oligonucleotides. Alternatively, after performing gene synthesis with oligos of lower quality, more effort must be made in downstream quality assurance during clone analysis, which is usually done by time-consuming standard cloning and sequencing procedures. Another problem associated with all current gene synthesis methods is the high frequency of sequence errors because of the usage of chemically synthesized oligonucleotides. The error frequency increases with longer oligonucleotides, and as a consequence the percentage of correct product decreases dramatically as more oligonucleotides are used. The mutation problem could be solved by shorter oligonucleotides used to assemble the gene. However, all annealing based assembly methods require the primers to be mixed together in one tube. In this case, shorter overlaps do not always allow precise and specific annealing of complementary primers, resulting in the inhibition of full length product formation. Manual design of oligonucleotides is a laborious procedure and does not guarantee the successful synthesis of the desired gene. For optimal performance of almost all annealing based methods, the melting temperatures of the overlapping regions are supposed to be similar for all oligonucleotides. The necessary primer optimisation should be performed using specialized oligonucleotide design programs. Several solutions for automated primer design for gene synthesis have been presented so far.

Error correction procedures

To overcome problems associated with oligonucleotide quality several elaborate strategies have been developed, employing either separately prepared fishing oligonucleotides, mismatch binding enzymes of the mutS family or specific endonucleases from bacteria or phages. Nevertheless, all these strategies increase time and costs for gene synthesis based on the annealing of chemically synthesized oligonucleotides.

Massively parallel sequencing has also been used as a tool to screen complex oligonucleotide libraries and enable the retrieval of accurate molecules. In one approach, oligonucleotides are sequenced on the 454 pyrosequencing platform and a robotic system images and picks individual beads corresponding to accurate sequence. In another approach, a complex oligonucleotide library is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by dial-out PCR.

Increasingly, genes are ordered in sets including functionally related genes or multiple sequence variants on a single gene. Virtually all of the therapeutic proteins in development, such as monoclonal antibodies, are optimised by testing many gene variants for improved function or expression.

Unnatural base pairs

Main article: Unnatural base pair

While traditional nucleic acid synthesis only uses 4 base pairs - adenine, thymine, guanine and cytosine, oligonucleotide synthesis in the future could incorporate the use of unnatural base pairs, which are artificially designed and synthesized nucleobases that do not occur in nature.

In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team designed an unnatural base pair (UBP). The two new artificial nucleotides or Unnatural Base Pair (UBP) were named d5SICS and dNaM. More technically, these artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA. In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a plasmid containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed, and inserted it into cells of the common bacterium E. coli that successfully replicated the unnatural base pairs through multiple generations. This is the first known example of a living organism passing along an expanded genetic code to subsequent generations. This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into E. coli bacteria. Then, the natural bacterial replication pathways use them to accurately replicate the plasmid containing d5SICS–dNaM.

The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids which can be encoded by DNA, from the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel proteins. In the future, these unnatural base pairs could be synthesised and incorporated into oligonucleotides via DNA printing methods.

DNA assembly

DNA printing can thus be used to produce DNA parts, which are defined as sequences of DNA that encode a specific biological function (for example, promoters, transcription regulatory sequences or open reading frames). However, because oligonucleotide synthesis typically cannot accurately produce oligonucleotides sequences longer than a few hundred base pairs, DNA assembly methods have to be employed to assemble these parts together to create functional genes, multi-gene circuits or even entire synthetic chromosomes or genomes. Some DNA assembly techniques only define protocols for joining DNA parts, while other techniques also define the rules for the format of DNA parts that are compatible with them. These processes can be scaled up to enable the assembly of entire chromosomes or genomes. In recent years, there has been proliferation in the number of different DNA assembly standards with 14 different assembly standards developed as of 2015, each with their pros and cons. Overall, the development of DNA assembly standards has greatly facilitated the workflow of synthetic biology, aided the exchange of material between research groups and also allowed for the creation of modular and reusable DNA parts.

The various DNA assembly methods can be classified into three main categories – endonuclease-mediated assembly, site-specific recombination, and long-overlap-based assembly. Each group of methods has its distinct characteristics and their own advantages and limitations.

Endonuclease-mediated assembly

Endonucleases are enzymes that recognise and cleave nucleic acid segments and they can be used to direct DNA assembly. Of the different types of restriction enzymes, the type II restriction enzymes are the most commonly available and used because their cleavage sites are located near or in their recognition sites. Hence, endonuclease-mediated assembly methods make use of this property to define DNA parts and assembly protocols.

BioBricks

Main article: BioBrick

BBF RFC 10 assembly of two BioBricks compatible part. Treating the upstream fragment with EcoRI and SpeI, and the downstream fragment with EcoRI and XbaI allows for the assembly in the desired sequence. Because SpeI and XbaI produce complementary overhangs, they help link the two DNA fragments together, producing a scar sequence. All the original restriction sites are maintained in the final construct, which can then be used for further BioBricks reactions.

The BioBricks assembly standard was described and introduced by Tom Knight in 2003 and it has been constantly updated since then. Currently, the most commonly used BioBricks standard is the assembly standard 10, or BBF RFC 10. BioBricks defines the prefix and suffix sequences required for a DNA part to be compatible with the BioBricks assembly method, allowing the joining together of all DNA parts which are in the BioBricks format.

The prefix contains the restriction sites for EcoRI, NotI and XBaI, while the suffix contains the SpeI, NotI and PstI restriction sites. Outside of the prefix and suffix regions, the DNA part must not contain these restriction sites. To join two BioBrick parts together, one of the plasmids is digested with EcoRI and SpeI while the second plasmid is digested with EcoRI and XbaI. The two EcoRI overhangs are complementary and will thus anneal together, while SpeI and XbaI also produce complementary overhangs which can also be ligated together. As the resulting plasmid contains the original prefix and suffix sequences, it can be used to join with more BioBricks parts. Because of this property, the BioBricks assembly standard is said to be idempotent in nature. However, there will also be a "scar" sequence (either TACTAG or TACTAGAG) formed between the two fused BioBricks. This prevents BioBricks from being used to create fusion proteins, as the 6bp scar sequence codes for a tyrosine and a stop codon, causing translation to be terminated after the first domain is expressed, while the 8bp scar sequence causes a frameshift, preventing continuous readthrough of the codons. To offer alternative scar sequences that for example give a 6bp scar, or scar sequences that do not contain stop codons, other assembly standards such as the BB-2 Assembly, BglBricks Assembly, Silver Assembly and the Freiburg Assembly were designed.

While the easiest method to assemble BioBrick parts is described above, there also exist several other commonly used assembly methods that offer several advantages over the standard assembly. The 3 antibiotic (3A) assembly allows for the correct assembly to be selected via antibiotic selection, while the amplified insert assembly seeks to overcome the low transformation efficiency seen in 3A assembly.

The BioBrick assembly standard has also served as inspiration for using other types of endonucleases for DNA assembly. For example, both the iBrick standard and the HomeRun vector assembly standards employ homing endonucleases instead of type II restriction enzymes.

Type IIs restriction endonuclease assembly

Some assembly methods also make use of type IIs restriction endonucleases. These differ from other type II endonucleases as they cut several base pairs away from the recognition site. As a result, the overhang sequence can be modified to contain the desired sequence. This provides Type IIs assembly methods with two advantages – it enables "scar-less" assembly, and allows for one-pot, multi-part assembly. Assembly methods that use type IIs endonucleases include Golden Gate and its associated variants.

Golden Gate cloning

Main article: Golden Gate Cloning

The sequence of DNA parts for the Golden Gate assembly can be directed by defining unique complementary overhangs for each part. Thus, to assemble gene 1 in order of fragment A, B and C, the 3' overhang for fragment A is complementary to the 5' overhang for fragment B, and similarly for fragment B and fragment C. For the destination plasmid, the selectable marker is flanked by outward-cutting BsaI restriction sites. This excises the selectable marker, allowing the insertion of the final construct. T4 ligase is used to ligate the fragments together and to the destination plasmid.

The Golden Gate assembly protocol was defined by Engler et al. 2008 to define a DNA assembly method that would give a final construct without a scar sequence, while also lacking the original restriction sites. This allows the protein to be expressed without containing unwanted protein sequences which could negatively affect protein folding or expression. By using the BsaI restriction enzyme that produces a 4 base pair overhang, up to 240 unique, non-palindromic sequences can be used for assembly.

Plasmid design and assembly

In Golden Gate cloning, each DNA fragment to be assembled is placed in a plasmid, flanked by inward facing BsaI restriction sites containing the programmed overhang sequences. For each DNA fragment, the 3' overhang sequence is complementary to the 5' overhang of the next downstream DNA fragment. For the first fragment, the 5' overhang is complementary to the 5' overhang of the destination plasmid, while the 3' overhang of the final fragment is complementary to the 3' overhang of the destination plasmid. Such a design allows for all DNA fragments to be assembled in a one-pot reaction (where all reactants are mixed together), with all fragments arranged in the correct sequence. Successfully assembled constructs are selected by detecting the loss of function of a screening cassette that was originally in the destination plasmid.

MoClo and Golden Braid

The original Golden Gate Assembly only allows for a single construct to be made in the destination vector . To enable this construct to be used in a subsequent reaction as an entry vector, the MoClo and Golden Braid standards were designed.

The MoClo standard involves defining multiple tiers of DNA assembly:

• 

  The MoClo assembly standard allows for Golden Gate constructs to be further assembled in subsequent tiers. In the example here, four genes assembled via tier 1 Golden Gate assembly are assembled into a multi-gene construct in a tier 2 assembly.

  

  The Golden Braid assembly standard also builds on the first tier of Golden Gate assembly and assembles further tiers via a pairwise protocol. Four tier one destination vectors (assembled via Golden Gate assembly) are assembled into two tier 2 destination vectors, which are then used as tier 3 entry vectors for the tier 3 destination vector. Alternating restriction enzymes (BpiI for tier 2 and BsaI for tier 3) are used.

  The MoClo and Golden Braid assembly standards are derivatives of the original Golden Gate assembly standard.
  Tier 1: Tier 1 assembly is the standard Golden Gate assembly, and genes are assembled from their components parts (DNA parts coding for genetic elements like UTRs, promoters, ribosome binding sites or terminator sequences). Flanking the insertion site of the tier 1 destination vectors are a pair of inward cutting BpiI restriction sites. This allows these plasmids to be used as entry vectors for tier two destination vectors.
• Tier 2: Tier 2 assembly involves further assembling the genes assembled in tier 1 assembly into multi-gene constructs. If there is a need for further, higher tier assembly, inward cutting BsaI restriction sites can be added to flank the insertion sites. These vectors can then be used as entry vectors for higher tier constructs.

Each assembly tier alternates the use of BsaI and BpiI restriction sites to minimise the number of forbidden sites, and sequential assembly for each tier is achieved by following the Golden Gate plasmid design. Overall, the MoClo standard allows for the assembly of a construct that contains multiple transcription units, all assembled from different DNA parts, by a series of one-pot Golden Gate reactions. However, one drawback of the MoClo standard is that it requires the use of 'dummy parts' with no biological function, if the final construct requires less than four component parts. The Golden Braid standard on the other hand introduced a pairwise Golden Gate assembly standard.

The Golden Braid standard uses the same tiered assembly as MoClo, but each tier only involves the assembly of two DNA fragments, i.e. a pairwise approach. Hence in each tier, pairs of genes are cloned into a destination fragment in the desired sequence, and these are subsequently assembled two at a time in successive tiers. Like MoClo, the Golden Braid standard alternates the BsaI and BpiI restriction enzymes between each tier.

The development of the Golden Gate assembly methods and its variants has allowed researchers to design tool-kits to speed up the synthetic biology workflow. For example EcoFlex was developed as a toolkit for E. Coli that uses the MoClo standard for its DNA parts, while a similar toolkit has also been developed for engineering the Chlamydomonas reinhardtii mircoalgae.

Site-specific recombination

Main article: Gateway Technology

The Gateway Cloning entry vectors must first be produced using a synthesised DNA fragment containing the required attB sites. Recombination with the donor vector is catalysed by the BP clonase mix and produces the desired entry vector with attL sites.

The desired construct is obtained by recombining the entry vector with the destination vector. In this case, the final construct involves the assembly of two DNA fragments of interest. The attL sites on the entry vector recombine with the attR sites on the destination vector. The lethal gene ccdB is lost from the destination vector, and any bacteria that uptakes the unwanted vector will die, allowing selection of the desired vector.

The discovery of orthogonal att sites that are specific (i.e. each orthogonal attL only reacts with its partner attR) allowed for the developed of Multisite Gateway Cloning technology. This allows for multiple DNA fragments to be assembled, with the order of assembly directed by the att sites.

Summary of the key Gateway Cloning technologies.

Site-specific recombination makes use of phage integrases instead of restriction enzymes, eliminating the need for having restriction sites in the DNA fragments. Instead, integrases make use of unique attachment (att) sites, and catalyse DNA rearrangement between the target fragment and the destination vector. The Invitrogen Gateway cloning system was invented in the late 1990s and uses two proprietary enzyme mixtures, BP clonase and LR clonase. The BP clonase mix catalyses the recombination between attB and attP sites, generating hybrid attL and attR sites, while the LR clonase mix catalyse the recombination of attL and attR sites to give attB and attP sites. As each enzyme mix recognises only specific att sites, recombination is highly specific and the fragments can be assembled in the desired sequence.

Vector design and assembly

Because Gateway cloning is a proprietary technology, all Gateway reactions must be carried out with the Gateway kit that is provided by the manufacturer. The reaction can be summarised into two steps. The first step involves assembling the entry clones containing the DNA fragment of interest, while the second step involves inserting this fragment of interest into the destination clone.

1. Entry clones must be made using the supplied "Donor" vectors containing a Gateway cassette flanked by attP sites. The Gateway cassette contains a bacterial suicide gene (e.g. ccdB) that will allow for survival and selection of successfully recombined entry clones. A pair of attB sites are added to flank the DNA fragment of interest, and this will allow recombination with the attP sites when the BP clonase mix is added. Entry clones are produced, and the fragment of interest is flanked by attL sites.
2. The destination vector also comes with a Gateway cassette, but is instead flanked by a pair of attR sites. Mixing this destination plasmid with the entry clones and the LR clonase mix will allow for recombination to occur between the attR and attL sites. A destination clone is produced, with the fragment of interest successfully inserted. The lethal gene is inserted into the original vector, and bacteria transformed with this plasmid will die. The desired vector can thus be easily selected.

The earliest iterations of the Gateway cloning method only allowed for only one entry clone to be used for each destination clone produced. However, further research revealed that four more orthogonal att sequences could be generated, allowing for the assembly of up to four different DNA fragments, and this process is now known as the Multisite Gateway technology.

Besides Gateway cloning, non-commercial methods using other integrases have also been developed. For example, the Serine Integrase Recombinational Assembly (SIRA) method uses the ϕC31 integrase, while the Site-Specific Recombination-based Tandem Assembly (SSRTA) method uses the Streptomyces phage φBT1 integrase. Other methods, like the HomeRun Vector Assembly System (HVAS), build on the Gateway cloning system and further incorporate homing endoucleases to design a protocol that could potentially support the industrial synthesis of synthetic DNA constructs.

Long-overlap-based assembly methods require the presence of long overlap regions on the DNA parts that are to be assembled. This enables the construction of complementary overhangs that can anneal via complementary base pairing. There exist a variety of methods, e.g. Gibson assembly, CPEC, MODAL that make use of this concept to assemble DNA.

Long-overlap-based assembly

There have been a variety of long-overlap-based assembly methods developed in recent years. One of the most commonly used methods, the Gibson assembly method, was developed in 2009, and provides a one-pot DNA assembly method that does not require the use of restriction enzymes or integrases. Other similar overlap-based assembly methods include Circular Polymerase Extension Cloning (CPEC), Sequence and Ligase Independent Cloning (SLIC) and Seamless Ligation Cloning Extract (SLiCE). Despite the presence of many overlap assembly methods, the Gibson assembly method is still the most popular. Besides the methods listed above, other researchers have built on the concepts used in Gibson assembly and other assembly methods to develop new assembly strategies like the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy, or the Biopart Assembly Standard for Idempotent Cloning (BASIC) method.

Gibson assembly

Main article: Gibson assembly

The Gibson assembly method is a relatively straightforward DNA assembly method, requiring only a few additional reagents: the 5' T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase. The DNA fragments to be assembled are synthesised to have overlapping 5' and 3' ends in the order that they are to be assembled in. These reagents are mixed together with the DNA fragments to be assembled at 50 °C and the following reactions occur:

1. The T5 exonuclease chews back DNA from the 5' end of each fragment, exposing 3' overhangs on each DNA fragment.
2. The complementary overhangs on adjacent DNA fragments anneal via complementary base pairing.
3. The Phusion DNA polymerase fills in any gaps where the fragments anneal.
4. Taq DNA ligase repairs the nicks on both DNA strands.

Because the T5 exonuclease is heat labile, it is inactivated at 50 °C after the initial chew back step. The product is thus stable, and the fragments assembled in the desired order. This one-pot protocol can assemble up to 5 different fragments accurately, while several commercial providers have kits to accurately assemble up to 15 different fragments in a two-step reaction. However, while the Gibson assembly protocol is fast and uses relatively few reagents, it requires bespoke DNA synthesis as each fragment has to be designed to contain overlapping sequences with the adjacent fragments and amplified via PCR. This reliance on PCR may also affect the fidelity of the reaction when long fragments, fragments with high GC content or repeat sequences are used.

 

The MODAL standard provides a common format to allow any DNA part to be made compatible with Gibson assembly or other overlap assembly methods. The DNA fragment of interest undergoes two rounds of PCR, first to attach the adaptor prefix and suffixes, and next to attach the predefined linker sequences. Once the parts are in the required format, assembly methods like Gibson assembly can carried out. The order of the parts is directed by the linkers, i.e the same linker sequence is attached to the 3' end of the upstream part and the 5' end of the downstream part.

MODAL

The MODAL strategy defines overlap sequences known as "linkers" to reduce the amount of customisation that needs to be done with each DNA fragment. The linkers were designed using the R2oDNA Designer software and the overlap regions were designed to be 45 bp long to be compatible with Gibson assembly and other overlap assembly methods. To attach these linkers to the parts to be assembled, PCR is carried using part-specific primers containing 15 bp prefix and suffix adaptor sequences. The linkers are then attached to the adaptor sequences via a second PCR reaction. To position the DNA fragments, the same linker will be attached to the suffix of the desired upstream fragment and the prefix of the desired downstream fragments. Once the linkers are attached, Gibson assembly, CPEC, or the other overlap assembly methods can all be used to assemble the DNA fragments in the desired order.

BASIC

The BASIC assembly strategy was developed in 2015 and sought to address the limitations of previous assembly techniques, incorporating six key concepts from them: standard reusable parts; single-tier format (all parts are in the same format and are assembled using the same process); idempotent cloning; parallel (multipart) DNA assembly; size independence; automatability.

DNA parts and linker design

The DNA parts are designed and cloned into storage plasmids, with the part flanked by an integrated prefix (iP) and an integrated suffix (iS) sequence. The iP and iS sequences contain inward facing BsaI restriction sites, which contain overhangs complementary to the BASIC linkers. Like in MODAL, the 7 standard linkers used in BASIC were designed with the R2oDNA Designer software, and screened to ensure that they do not contain sequences with homology to chassis genomes, and that they do not contain unwanted sequences like secondary structure sequences, restriction sites or ribosomal binding sites. Each linker sequence is split into two halves, each with a 4 bp overhang complementary to the BsaI restriction site, a 12 bp double stranded sequence and sharing a 21 bp overlap sequence with the other half. The half that is will bind to the upstream DNA part is known as the suffix linker part (e.g. L1S) and the half that binds to the downstream part is known as the prefix linker part (e.g. L1P). These linkers form the basis of assembling the DNA parts together.

Besides directing the order of assembly, the standard BASIC linkers can also be modified to carry out other functions. To allow for idempotent assembly, linkers were also designed with additional methylated iP and iS sequences inserted to protect them from being recognised by BsaI. This methylation is lost following transformation and in vivo plasmid replication, and the plasmids can be extracted, purified, and used for further reactions.

Because the linker sequence are relatively long (45bp for a standard linker), there is an opportunity to incorporate functional DNA sequences to reduce the number of DNA parts needed during assembly. The BASIC assembly standard provides several linkers embedded with RBS of different strengths. Similarly to facilitate the construction of fusion proteins containing multiple protein domains, several fusion linkers were also designed to allow for full read-through of the DNA construct. These fusion linkers code for a 15 amino acid glycine and serine polypeptide, which is an ideal linker peptide for fusion proteins with multiple domains.

DNA parts to be used for BASIC assembly need to contain integrated prefix and suffix sequences (iP and iS). These contain BsaI restriction sites that will allow for the iP and iS linkers (which contain complementary overhang sequences) to be attached to the DNA part. Once the linkers are attached, the part is ready for assembly.

The sequence of assembly is directed by the positioning of the linkers. The suffix linker is ligated to the 3' end of the upstream fragment, while the prefix linker is ligated to the 5' end of the downstream fragment. Shown here is an example of a BASIC assembly construct using four DNA parts.

Assembly

There are three main steps in the assembly of the final construct.

1. First, the DNA parts are excised from the storage plasmid, giving a DNA fragment with BsaI overhangs on the 3' and 5' end.
2. Next, each linker part is attached to its respective DNA part by incubating with T4 DNA ligase. Each DNA part will have a suffix and prefix linker part from two different linkers to direct the order of assembly. For example, the first part in the sequence will have L1P and L2S, while the second part will have L2P and L3S attached. The linker parts can be changed to change the sequence of assembly.
3. Finally, the parts with the attached linkers are assembled into a plasmid by incubating at 50 °C. The 21 bp overhangs of the P and S linkers anneal and the final construct can be transformed into bacteria cells for cloning. The single stranded nicks are repaired in vivo following transformation, producing a stable final construct cloned into plasmids.

Applications

As DNA printing and DNA assembly methods have allowed commercial gene synthesis to become progressively and exponentially cheaper over the past years, artificial gene synthesis represents a powerful and flexible engineering tool for creating and designing new DNA sequences and protein functions. Besides synthetic biology, various research areas like those involving heterologous gene expression, vaccine development, gene therapy and molecular engineering, would benefit greatly from having fast and cheap methods to synthesise DNA to code for proteins and peptides. The methods used for DNA printing and assembly have even enabled the use of DNA as an information storage medium.

Synthesising bacterial genomes

Synthia and Mycoplasma laboratorium

Main article: Mycoplasma laboratorium

On June 28, 2007, a team at the J. Craig Venter Institute published an article in Science Express, saying that they had successfully transplanted the natural DNA from a Mycoplasma mycoides bacterium into a Mycoplasma capricolum cell, creating a bacterium which behaved like a M. mycoides.

On Oct 6, 2007, Craig Venter announced in an interview with UK's The Guardian newspaper that the same team had synthesized a modified version of the single chromosome of Mycoplasma genitalium artificially. The chromosome was modified to eliminate all genes which tests in live bacteria had shown to be unnecessary. The next planned step in this minimal genome project is to transplant the synthesized minimal genome into a bacterial cell with its old DNA removed; the resulting bacterium will be called Mycoplasma laboratorium. The next day the Canadian bioethics group, ETC Group issued a statement through their representative, Pat Mooney, saying Venter's "creation" was "a chassis on which you could build almost anything". The synthesized genome had not yet been transplanted into a working cell.

On May 21, 2010, Science reported that the Venter group had successfully synthesized the genome of the bacterium Mycoplasma mycoides from a computer record, and transplanted the synthesized genome into the existing cell of a Mycoplasma capricolum bacterium that had its DNA removed. The "synthetic" bacterium was viable, i.e. capable of replicating billions of times. The team had originally planned to use the M. genitalium bacterium they had previously been working with, but switched to M. mycoides because the latter bacterium grows much faster, which translated into quicker experiments. Venter describes it as "the first species.... to have its parents be a computer". The transformed bacterium is dubbed "Synthia" by ETC. A Venter spokesperson has declined to confirm any breakthrough at the time of this writing.

Synthetic Yeast 2.0

As part of the Synthetic Yeast 2.0 project, various research groups around the world have participated in a project to synthesise synthetic yeast genomes, and through this process, optimise the genome of the model organism Saccharomyces cerevisae. The Yeast 2.0 project applied various DNA assembly methods that have been discussed above, and in March 2014, Jef Boeke of the Langone Medical Centre at New York University, revealed that his team had synthesized chromosome III of S. cerevisae. The procedure involved replacing the genes in the original chromosome with synthetic versions and the finished synthetic chromosome was then integrated into a yeast cell. It required designing and creating 273,871 base pairs of DNA – fewer than the 316,667 pairs in the original chromosome. In March 2017, the synthesis of 6 of the 16 chromosomes had been completed, with synthesis of the others still ongoing.

Biomolecular engineering

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Biomolecular_engineering 

Biomolecular engineering is the application of engineering principles and practices to the purposeful manipulation of molecules of biological origin. Biomolecular engineers integrate knowledge of biological processes with the core knowledge of chemical engineering in order to focus on molecular level solutions to issues and problems in the life sciences related to the environment, agriculture, energy, industry, food production, biotechnology and medicine.

Biomolecular engineers purposefully manipulate carbohydrates, proteins, nucleic acids and lipids within the framework of the relation between their structure (see: nucleic acid structure, carbohydrate chemistry, protein structure,), function (see: protein function) and properties and in relation to applicability to such areas as environmental remediation, crop and livestock production, biofuel cells and biomolecular diagnostics. The thermodynamics and kinetics of molecular recognition in enzymes, antibodies, DNA hybridization, bio-conjugation/bio-immobilization and bioseparations are studied. Attention is also given to the rudiments of engineered biomolecules in cell signaling, cell growth kinetics, biochemical pathway engineering and bioreactor engineering.

Timeline

History

During World War II, the need for large quantities of penicillin of acceptable quality brought together chemical engineers and microbiologists to focus on penicillin production. This created the right conditions to start a chain of reactions that lead to the creation of the field of biomolecular engineering. Biomolecular engineering was first defined in 1992 by the U.S. National Institutes of Health as research at the interface of chemical engineering and biology with an emphasis at the molecular level". Although first defined as research, biomolecular engineering has since become an academic discipline and a field of engineering practice. Herceptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the U.S. FDA. Also, Biomolecular Engineering was a former name of the journal New Biotechnology.

Future

Bio-inspired technologies of the future can help explain biomolecular engineering. Looking at the Moore's law "Prediction", in the future quantum and biology-based processors are "big" technologies. With the use of biomolecular engineering, the way our processors work can be manipulated in order to function in the same sense a biological cell work. Biomolecular engineering has the potential to become one of the most important scientific disciplines because of its advancements in the analyses of gene expression patterns as well as the purposeful manipulation of many important biomolecules to improve functionality. Research in this field may lead to new drug discoveries, improved therapies, and advancement in new bioprocess technology. With the increasing knowledge of biomolecules, the rate of finding new high-value molecules including but not limited to antibodies, enzymes, vaccines, and therapeutic peptides will continue to accelerate. Biomolecular engineering will produce new designs for therapeutic drugs and high-value biomolecules for treatment or prevention of cancers, genetic diseases, and other types of metabolic diseases. Also, there is anticipation of industrial enzymes that are engineered to have desirable properties for process improvement as well the manufacturing of high-value biomolecular products at a much lower production cost. Using recombinant technology, new antibiotics that are active against resistant strains will also be produced.

Basic biomolecules

Biomolecular engineering deals with the manipulation of many key biomolecules. These include, but are not limited to, proteins, carbohydrates, nucleic acids, and lipids. These molecules are the basic building blocks of life and by controlling, creating, and manipulating their form and function there are many new avenues and advantages available to society. Since every biomolecule is different, there are a number of techniques used to manipulate each one respectively.

Proteins

Main article: Protein engineering

Proteins are polymers that are made up of amino acid chains linked with peptide bonds. They have four distinct levels of structure: primary, secondary, tertiary, and quaternary. Primary structure refers to the amino acid backbone sequence. Secondary structure focuses on minor conformations that develop as a result of the hydrogen bonding between the amino acid chain. If most of the protein contains intermolecular hydrogen bonds it is said to be fibrillar, and the majority of its secondary structure will be beta sheets. However, if the majority of the orientation contains intramolecular hydrogen bonds, then the protein is referred to as globular and mostly consists of alpha helices. There are also conformations that consist of a mix of alpha helices and beta sheets as well as a beta helixes with an alpha sheets.

The tertiary structure of proteins deal with their folding process and how the overall molecule is arranged. Finally, a quaternary structure is a group of tertiary proteins coming together and binding. With all of these levels, proteins have a wide variety of places in which they can be manipulated and adjusted. Techniques are used to affect the amino acid sequence of the protein (site-directed mutagenesis), the folding and conformation of the protein, or the folding of a single tertiary protein within a quaternary protein matrix. Proteins that are the main focus of manipulation are typically enzymes. These are proteins that act as catalysts for biochemical reactions. By manipulating these catalysts, the reaction rates, products, and effects can be controlled. Enzymes and proteins are important to the biological field and research that there are specific divisions of engineering focusing only on proteins and enzymes.

Carbohydrates

Carbohydrates are another important biomolecule. These are polymers, called polysaccharides, which are made up of chains of simple sugars connected via glycosidic bonds. These monosaccharides consist of a five to six carbon ring that contains carbon, hydrogen, and oxygen - typically in a 1:2:1 ratio, respectively. Common monosaccharides are glucose, fructose, and ribose. When linked together monosaccharides can form disaccharides, oligosaccharides, and polysaccharides: the nomenclature is dependent on the number of monosaccharides linked together. Common dissacharides, two monosaccharides joined together, are sucrose, maltose, and lactose. Important polysaccharides, links of many monosaccharides, are cellulose, starch, and chitin.

Cellulose is a polysaccharide made up of beta 1-4 linkages between repeat glucose monomers. It is the most abundant source of sugar in nature and is a major part of the paper industry. Starch is also a polysaccharide made up of glucose monomers; however, they are connected via an alpha 1-4 linkage instead of beta. Starches, particularly amylase, are important in many industries, including the paper, cosmetic, and food. Chitin is a derivation of cellulose, possessing an acetamide group instead of an –OH on one of its carbons. Acetimide group is deacetylated the polymer chain is then called chitosan. Both of these cellulose derivatives are a major source of research for the biomedical and food industries. They have been shown to assist with blood clotting, have antimicrobial properties, and dietary applications. A lot of engineering and research is focusing on the degree of deacetylation that provides the most effective result for specific applications.

Nucleic acids

Nucleic acids are macromolecules that consist of DNA and RNA which are biopolymers consisting of chains of biomolecules. These two molecules are the genetic code and template that make life possible. Manipulation of these molecules and structures causes major changes in function and expression of other macromolecules. Nucleosides are glycosylamines containing a nucleobase bound to either ribose or deoxyribose sugar via a beta-glycosidic linkage. The sequence of the bases determine the genetic code. Nucleotides are nucleosides that are phosphorylated by specific kinases via a phosphodiester bond. Nucleotides are the repeating structural units of nucleic acids. The nucleotides are made of a nitrogenous base, a pentose (ribose for RNA or deoxyribose for DNA), and three phosphate groups. See, Site-directed mutagenesis, recombinant DNA, and ELISAs.

Lipids

Lipids are biomolecules that are made up of glycerol derivatives bonded with fatty acid chains. Glycerol is a simple polyol that has a formula of C3H5(OH)3. Fatty acids are long carbon chains that have a carboxylic acid group at the end. The carbon chains can be either saturated with hydrogen; every carbon bond is occupied by a hydrogen atom or a single bond to another carbon in the carbon chain, or they can be unsaturated; namely, there are double bonds between the carbon atoms in the chain. Common fatty acids include lauric acid, stearic acid, and oleic acid. The study and engineering of lipids typically focuses on the manipulation of lipid membranes and encapsulation. Cellular membranes and other biological membranes typically consist of a phospholipid bilayer membrane, or a derivative thereof. Along with the study of cellular membranes, lipids are also important molecules for energy storage. By utilizing encapsulation properties and thermodynamic characteristics, lipids become significant assets in structure and energy control when engineering molecules.

Of molecules

Recombinant DNA

Main article: Recombinant DNA

Recombinant DNA are DNA biomolecules that contain genetic sequences that are not native to the organism's genome. Using recombinant techniques, it is possible to insert, delete, or alter a DNA sequence precisely without depending on the location of restriction sites. Recombinant DNA is used for a wide range of applications.

Method

Creating recombinant DNA. After the plasmid is cleaved by restriction enzymes, ligases insert the foreign DNA fragments into the plasmid.

The traditional method for creating recombinant DNA typically involves the use of plasmids in the host bacteria. The plasmid contains a genetic sequence corresponding to the recognition site of a restriction endonuclease, such as EcoR1. After foreign DNA fragments, which have also been cut with the same restriction endonuclease, have been inserted into host cell, the restriction endonuclease gene is expressed by applying heat, or by introducing a biomolecule, such as arabinose. Upon expression, the enzyme will cleave the plasmid at its corresponding recognition site creating sticky ends on the plasmid. Ligases then joins the sticky ends to the corresponding sticky ends of the foreign DNA fragments creating a recombinant DNA plasmid.

Advances in genetic engineering have made the modification of genes in microbes quite efficient allowing constructs to be made in about a weeks worth of time. It has also made it possible to modify the organism's genome itself. Specifically, use of the genes from the bacteriophage lambda are used in recombination. This mechanism, known as recombineering, utilizes the three proteins Exo, Beta, and Gam, which are created by the genes exo, bet, and gam respectively. Exo is a double stranded DNA exonuclease with 5’ to 3’ activity. It cuts the double stranded DNA leaving 3’ overhangs. Beta is a protein that binds to single stranded DNA and assists homologous recombination by promoting annealing between the homology regions of the inserted DNA and the chromosomal DNA. Gam functions to protect the DNA insert from being destroyed by native nucleases within the cell.

Applications

Recombinant DNA can be engineered for a wide variety of purposes. The techniques utilized allow for specific modification of genes making it possible to modify any biomolecule. It can be engineered for laboratory purposes, where it can be used to analyze genes in a given organism. In the pharmaceutical industry, proteins can be modified using recombination techniques. Some of these proteins include human insulin. Recombinant insulin is synthesized by inserting the human insulin gene into E. coli, which then produces insulin for human use. Other proteins, such as human growth hormone, factor VIII, and hepatitis B vaccine are produced using similar means. Recombinant DNA can also be used for diagnostic methods involving the use of the ELISA method. This makes it possible to engineer antigens, as well as the enzymes attached, to recognize different substrates or be modified for bioimmobilization. Recombinant DNA is also responsible for many products found in the agricultural industry. Genetically modified food, such as golden rice, has been engineered to have increased production of vitamin A for use in societies and cultures where dietary vitamin A is scarce. Other properties that have been engineered into crops include herbicide-resistance and insect-resistance.

Site-directed mutagenesis

Site-directed mutagenesis is a technique that has been around since the 1970s. The early days of research in this field yielded discoveries about the potential of certain chemicals such as bisulfite and aminopurine to change certain bases in a gene. This research continued, and other processes were developed to create certain nucleotide sequences on a gene, such as the use of restriction enzymes to fragment certain viral strands and use them as primers for bacterial plasmids. The modern method, developed by Michael Smith in 1978, uses an oligonucleotide that is complementary to a bacterial plasmid with a single base pair mismatch or a series of mismatches.

General procedure

Site directed mutagenesis is a valuable technique that allows for the replacement of a single base in an oligonucleotide or gene. The basics of this technique involve the preparation of a primer that will be a complementary strand to a wild type bacterial plasmid. This primer will have a base pair mismatch at the site where the replacement is desired. The primer must also be long enough such that the primer will anneal to the wild type plasmid. After the primer anneals, a DNA polymerase will complete the primer. When the bacterial plasmid is replicated, the mutated strand will be replicated as well. The same technique can be used to create a gene insertion or deletion. Often, an antibiotic resistant gene is inserted along with the modification of interest and the bacteria are cultured on an antibiotic medium. The bacteria that were not successfully mutated will not survive on this medium, and the mutated bacteria can easily be cultured.

This animation shows the basic steps of site directed mutagenesis, where X-Y is the desired base pair replacement of T-A.

Applications

Site-directed mutagenesis can be useful for many different reasons. A single base pair replacement, could change a codon, and thus replace an amino acid in a protein. This is useful for studying the way certain proteins behave. It is also useful because enzymes can be purposefully manipulated by changing certain amino acids. If an amino acid is changed that is in close proximity to the active site, the kinetic parameters may change drastically, or the enzyme might behave in a different way. Another application of site directed mutagenesis is exchanging an amino acid residue far from the active site with a lysine residue or cysteine residue. These amino acids make it easier to covalently bond the enzyme to a solid surface, which allows for enzyme re-use and use of enzymes in continuous processes. Sometimes, amino acids with non-natural functional groups (such as ketones and azides) are added to proteins These additions may be for ease of bioconjugation, or to study the effects of amino acid changes on the form and function of the proteins. The coupling of site directed mutagenesis and PCR are being utilized to reduce interleukin-6 activity in cancerous cells. The bacteria bacillus subtilis is often used in site directed mutagenesis. The bacteria secretes an enzyme called subtilisin through the cell wall. Biomolecular engineers can purposely manipulate this gene to essentially make the cell a factory for producing whatever protein the insertion in the gene codes.

Bio-immobilization and bio-conjugation

Bio-immobilization and bio-conjugation is the purposeful manipulation of a biomolecule's mobility by chemical or physical means to obtain a desired property. Immobilization of biomolecules allows exploiting characteristics of the molecule under controlled environments. For example, the immobilization of glucose oxidase on calcium alginate gel beads can be used in a bioreactor. The resulting product will not need purification to remove the enzyme because it will remain linked to the beads in the column. Examples of types of biomolecules that are immobilized are enzymes, organelles, and complete cells. Biomolecules can be immobilized using a range of techniques. The most popular are physical entrapment, adsorption, and covalent modification.

• Physical entrapment - the use of a polymer to contain the biomolecule in a matrix without chemical modification. Entrapment can be between lattices of polymer, known as gel entrapment, or within micro-cavities of synthetic fibers, known as fiber entrapment. Examples include entrapment of enzymes such as glucose oxidase in gel column for use as a bioreactor. Important characteristic with entrapment is biocatalyst remains structurally unchanged, but creates large diffusion barriers for substrates.
• Adsorption- immobilization of biomolecules due to interaction between the biomolecule and groups on support. Can be physical adsorption, ionic bonding, or metal binding chelation. Such techniques can be performed under mild conditions and relatively simple, although the linkages are highly dependent upon pH, solvent and temperature. Examples include enzyme-linked immunosorbent assays.
• Covalent modification- involves chemical reactions between certain functional groups and matrix. This method forms stable complex between biomolecule and matrix and is suited for mass production. Due to the formation of chemical bond to functional groups, loss of activity can occur. Examples of chemistries used are DCC coupling PDC coupling and EDC/NHS coupling, all of which take advantage of the reactive amines on the biomolecule's surface.

Because immobilization restricts the biomolecule, care must be given to ensure that functionality is not entirely lost. Variables to consider are pH, temperature, solvent choice, ionic strength, orientation of active sites due to conjugation. For enzymes, the conjugation will lower the kinetic rate due to a change in the 3-dimensional structure, so care must be taken to ensure functionality is not lost. Bio-immobilization is used in technologies such as diagnostic bioassays, biosensors, ELISA, and bioseparations. Interleukin (IL-6) can also be bioimmobilized on biosensors. The ability to observe these changes in IL-6 levels is important in diagnosing an illness. A cancer patient will have elevated IL-6 level and monitoring those levels will allow the physician to watch the disease progress. A direct immobilization of IL-6 on the surface of a biosensor offers a fast alternative to ELISA.

Polymerase chain reaction

Main article: Polymerase chain reaction

Polymerase chain reaction. There are three main steps involved in PCR. In the first step, the double stranded DNA strands are "melted" or denatured forming single stranded DNA. Next, primers, which have been designed to target a specific gene sequence on the DNA, anneal to the single stranded DNA. Lastly, DNA polymerase synthesizes a new DNA strand complementary to the original DNA. These three steps are repeated multiple times until the desired number of copies are made.

The polymerase chain reaction (PCR) is a scientific technique that is used to replicate a piece of a DNA molecule by several orders of magnitude. PCR implements a cycle of repeated heated and cooling known as thermal cycling along with the addition of DNA primers and DNA polymerases to selectively replicate the DNA fragment of interest. The technique was developed by Kary Mullis in 1983 while working for the Cetus Corporation. Mullis would go on to win the Nobel Prize in Chemistry in 1993 as a result of the impact that PCR had in many areas such as DNA cloning, DNA sequencing, and gene analysis.

Biomolecular engineering techniques involved in PCR

A number of biomolecular engineering strategies have played a very important role in the development and practice of PCR. For instance a crucial step in ensuring the accurate replication of the desired DNA fragment is the creation of the correct DNA primer. The most common method of primer synthesis is by the phosphoramidite method. This method includes the biomolecular engineering of a number of molecules to attain the desired primer sequence. The most prominent biomolecular engineering technique seen in this primer design method is the initial bioimmobilization of a nucleotide to a solid support. This step is commonly done via the formation of a covalent bond between the 3’-hydroxy group of the first nucleotide of the primer and the solid support material.

Furthermore, as the DNA primer is created certain functional groups of nucleotides to be added to the growing primer require blocking to prevent undesired side reactions. This blocking of functional groups as well as the subsequent de-blocking of the groups, coupling of subsequent nucleotides, and eventual cleaving from the solid support are all methods of manipulation of biomolecules that can be attributed to biomolecular engineering. The increase in interleukin levels is directly proportional to the increased death rate in breast cancer patients. PCR paired with Western blotting and ELISA help define the relationship between cancer cells and IL-6.

Enzyme-linked immunosorbent assay (ELISA)

Main article: ELISA

Enzyme-linked immunosorbent assay is an assay that utilizes the principle of antibody-antigen recognition to test for the presence of certain substances. The three main types of ELISA tests which are indirect ELISA, sandwich ELISA, and competitive ELISA all rely on the fact that antibodies have an affinity for only one specific antigen. Furthermore, these antigens or antibodies can be attached to enzymes which can react to create a colorimetric result indicating the presence of the antibody or antigen of interest. Enzyme linked immunosorbent assays are used most commonly as diagnostic tests to detect HIV antibodies in blood samples to test for HIV, human chorionic gonadotropin molecules in urine to indicate pregnancy, and Mycobacterium tuberculosis antibodies in blood to test patients for tuberculosis. Furthermore, ELISA is also widely used as a toxicology screen to test people's serum for the presence of illegal drugs.

Techniques involved in ELISA

Although there are three different types of solid state enzyme-linked immunosorbent assays, all three types begin with the bioimmobilization of either an antibody or antigen to a surface. This bioimmobilization is the first instance of biomolecular engineering that can be seen in ELISA implementation. This step can be performed in a number of ways including a covalent linkage to a surface which may be coated with protein or another substance. The bioimmobilization can also be performed via hydrophobic interactions between the molecule and the surface. Because there are many different types of ELISAs used for many different purposes the biomolecular engineering that this step requires varies depending on the specific purpose of the ELISA.

Another biomolecular engineering technique that is used in ELISA development is the bioconjugation of an enzyme to either an antibody or antigen depending on the type of ELISA. There is much to consider in this enzyme bioconjugation such as avoiding interference with the active site of the enzyme as well as the antibody binding site in the case that the antibody is conjugated with enzyme. This bioconjugation is commonly performed by creating crosslinks between the two molecules of interest and can require a wide variety of different reagents depending on the nature of the specific molecules.

Interleukin (IL-6) is a signaling protein that has been known to be present during an immune response. The use of the sandwich type ELISA quantifies the presence of this cytokine within spinal fluid or bone marrow samples.

Applications and fields

In industry

Graph showing number of biotech companies per country

 

Graph showing percentages of biotech firms by application

Biomolecular engineering is an extensive discipline with applications in many different industries and fields. As such, it is difficult to pinpoint a general perspective on the Biomolecular engineering profession. The biotechnology industry, however, provides an adequate representation. The biotechnology industry, or biotech industry, encompasses all firms that use biotechnology to produce goods or services or to perform biotechnology research and development. In this way, it encompasses many of the industrial applications of the biomolecular engineering discipline. By examination of the biotech industry, it can be gathered that the principal leader of the industry is the United States, followed by France and Spain. It is also true that the focus of the biotechnology industry and the application of biomolecular engineering is primarily clinical and medical. People are willing to pay for good health, so most of the money directed towards the biotech industry stays in health-related ventures.

Scale-up

Scaling up a process involves using data from an experimental-scale operation (model or pilot plant) for the design of a large (scaled-up) unit, of commercial size. Scaling up is a crucial part of commercializing a process. For example, insulin produced by genetically modified Escherichia coli bacteria was initialized on a lab-scale, but to be made commercially viable had to be scaled up to an industrial level. In order to achieve this scale-up a lot of lab data had to be used to design commercial sized units. For example, one of the steps in insulin production involves the crystallization of high purity glargin insulin. In order to achieve this process on a large scale we want to keep the Power/Volume ratio of both the lab-scale and large-scale crystallizers the same in order to achieve homogeneous mixing. We also assume the lab-scale crystallizer has geometric similarity to the large-scale crystallizer. Therefore,

P/V α N_i³d_i³
where d_i= crystallizer impeller diameter
N_i= impeller rotation rate

Related industries

Bioengineering

Main article: Biological engineering

A broad term encompassing all engineering applied to the life sciences. This field of study utilizes the principles of biology along with engineering principles to create marketable products. Some bioengineering applications include:

• Biomimetics - The study and development of synthetic systems that mimic the form and function of natural biologically produced substances and processes.
• Bioprocess engineering - The study and development of process equipment and optimization that aids in the production of many products such as food and pharmaceuticals.
• Industrial microbiology - The implementation of microorganisms in the production of industrial products such as food and antibiotics. Another common application of industrial microbiology is the treatment of wastewater in chemical plants via utilization of certain microorganisms.

Biochemistry

Main article: Biochemistry

Biochemistry is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemical processes govern all living organisms and living processes and the field of biochemistry seeks to understand and manipulate these processes.

Biochemical engineering

Main article: Biochemical engineering

• Biocatalysis – Chemical transformations using enzymes.
• Bioseparations – Separation of biologically active molecules.
• Thermodynamics and Kinetics (chemistry) – Analysis of reactions involving cell growth and biochemicals.
• Bioreactor design and analysis – Design of reactors for performing biochemical transformations.

Biotechnology

Main article: Biotechnology

• Biomaterials – Design, synthesis and production of new materials to support cells and tissues.
• Genetic engineering – Purposeful manipulation of the genomes of organisms to produce new phenotypic traits.
• Bioelectronics, Biosensor and Biochip – Engineered devices and systems to measure, monitor and control biological processes.
• Bioprocess engineering – Design and maintenance of cell-based and enzyme-based processes for the production of fine chemicals and pharmaceuticals.

Bioelectrical engineering

Main article: Bioelectric

Bioelectrical engineering involves the electrical fields generated by living cells or organisms. Examples include the electric potential developed between muscles or nerves of the body. This discipline requires knowledge in the fields of electricity and biology to understand and utilize these concepts to improve or better current bioprocesses or technology.

• Bioelectrochemistry - Chemistry concerned with electron/proton transport throughout the cell
• Bioelectronics - Field of research coupling biology and electronics

Biomedical engineering

Main article: Biomedical engineering

Biomedical engineering is a sub category of bioengineering that uses many of the same principles but focuses more on the medical applications of the various engineering developments. Some applications of biomedical engineering include:

• Biomaterials - Design of new materials for implantation in the human body and analysis of their effect on the body.
• Cellular engineering – Design of new cells using recombinant DNA and development of procedures to allow normal cells to adhere to artificial implanted biomaterials
• Tissue engineering – Design of new tissues from the basic biological building blocks to form new tissues
• Artificial organs – Application of tissue engineering to whole organs
• Medical imaging – Imaging of tissues using CAT scan, MRI, ultrasound, x-ray or other technologies
• Medical Optics and Lasers – Application of lasers to medical diagnosis and treatment
• Rehabilitation engineering – Design of devices and systems used to aid the disabled
• Man-machine interfacing - Control of surgical robots and remote diagnostic and therapeutic systems using eye tracking, voice recognition and muscle and brain wave controls
• Human factors and ergonomics – Design of systems to improve human performance in a wide range of applications

Chemical engineering

Main article: Chemical engineering

Chemical engineering is the processing of raw materials into chemical products. It involves preparation of raw materials to produce reactants, the chemical reaction of these reactants under controlled conditions, the separation of products, the recycle of byproducts, and the disposal of wastes. Each step involves certain basic building blocks called “unit operations,” such as extraction, filtration, and distillation. These unit operations are found in all chemical processes. Biomolecular engineering is a subset of Chemical Engineering that applies these same principles to the processing of chemical substances made by living organisms.

Education and programs

Newly developed and offered undergraduate programs across the United States, often coupled to the chemical engineering program, allow students to achieve a B.S. degree. According to ABET (Accreditation Board for Engineering and Technology), biomolecular engineering curricula "must provide thorough grounding in the basic sciences including chemistry, physics, and biology, with some content at an advanced level… [and] engineering application of these basic sciences to design, analysis, and control, of chemical, physical, and/or biological processes." Common curricula consist of major engineering courses including transport, thermodynamics, separations, and kinetics, with additions of life sciences courses including biology and biochemistry, and including specialized biomolecular courses focusing on cell biology, nano- and biotechnology, biopolymers, etc.