One very important ability of genes is their ability to regulate, i.e. to turn on and off as needed. Cancer is the result of a erroneous always on-regulation, Gregory (2013). The regulation happens by operators close to the promoter (the regulatory element briefly mentioned earlier) and occurs on basis of certain protein concentrations. For instance in E. coli enzymes for converting lactose to glucose are only being synthesised if there is not sufficient available glucose while lactose is present. The operator is located downstream of the promoter on the DNA.
Whenrepressor protein is bound to the operator, the operator effectively reg-ulates gene expression by physically denying RNA polymerase to bind to the promoter on the gene. There are two types of operators:
Inducible By default the repressor protein is bound to the operator, i.e. tran-scription is repressed. To unbind the repressor protein from the operator aninducer protein must bind to the repressor. See illustration in Fig. 2.8.
Repressible By default the repressor protein is not bound to the operator, i.e.
transcription is active. When a co-repressor is present it becomes active and binds the operator. See Fig. 2.9
Inducers and co-repressors are practically equivalent; it is the repressor protein that determines whether it is inducible or repressible. It is important to note
2.4 Gene regulation 17
that these repressor proteins are specific in the sense that they only bind to some specific operators and can only be repressed or induced by some specific inducer or co-repressors. This is illustrated in Fig. 2.8and2.9by their puzzle-shapes.
Figure 2.8: 1: RNA Polymerase,2: Inducible repressor,3: Promoter,4: Op-erator,5: Inducer,6,7,8: PCS.
Top)The repressor prevents the transcription process by blocking the promoter.
Bottom) An inducer becomes present, so the repressor unbinds from the operator to bind with the inducer. Transcription can now occur. Figure altered fromRAJU.
The time from releasing a regulatory protein, e.g. an inducer, to a complete switch can be observed, is typically in the order of minutes.
Some secondary regulation can occur outside the operator sequence due to ex-ternal changes. For instance the rate of transcription can also be influenced by other proteins and temperature changes but is usually not as determining as regulating the operator directly.
The regulation described above is usually referred to as transcriptional regula-tion, another type of regulation is referred to as translational regulation. One example of this is sRNA base-pairing with mRNA thus influencing translation or mRNA stability, effectively repressing gene expression,Shimoni et al.(2007).
As the behaviour of gene regulation is like that of an electric transistor, logic gates can also be created by genes hence, in theory, arbitrarily complex biological systems can be created. This leads us to the next chapter on engineering.
Figure 2.9: 1: RNA Polymerase, 2: Repressible repressor, 3: Promoter, 4:
Operator,5: Co-repressor,6,7,8: PCS.
Top)The repressor is inactive but present.
Middle)A co-repressor becomes present which binds to the repres-sor thus enables binding to the operator.
Bottom) The repressor binds to the operator and blocks further transcription. Figure altered fromRAJU.
Chapter 3
Engineering Biology
This chapter describes the foundational technologies for engineering DNA strands, how computer-tools can aid the design process, through which simplifying ab-stractions biological systems can be regarded and finally some of the difficulties in engineering biological systems will be discussed.
Several sources of literature have been used to compose this chapter: (Baldwin et al., 2012, Ch. 2-3,5), Beal et al. (2012), Beal et al. (2011), Densmore and Hassoun(2012) andPedersen and Phillips (2009).
3.1 Enabling technologies
The technologies in this section are the technologies that allow us to read, write and combine DNA fragments. Some of these are quite complex and therefore only briefly described while referring to more comprehensive literature on the matter.
3.1.1 DNA sequencing
Sequencing is the process of obtaining the base pair representation of a given DNA strand. Many DNA sequencing methods exist, each with varying accuracy, cost, speed and read length. Advances in DNA sequencing currently receive a lot of focus as it is believed to be the foundation of (near) future disease diagnosis by sequencing the entire genome and prescribe accordingly. The widely used Sanger sequencing method basically works as follows:
1. Split the double stranded DNA into a template strand and a complemen-tary strand by applying heat.
2. Put the template strand mixture in 4 different containers along with some polymerase.
3. Put some nucleotides (A, C, G and T) as well as one unique type of PCR terminating nucleotide (A, C, G or T) in each container.
4. Now the template strand will try to repair itself but will randomly get terminated by the PCR terminating nucleotide unique to that container.
5. By gel-electrolysis, which sorts the partial strands on their weight, it is now possible to identify the positions of the labeled nucleotides hence the location of all complementary nucleotides.
6. Merging the results from each of the containers now yields the complete sequence of DNA.
More details can be found in (Baldwin et al., 2012, Appendix 1) and detailed comparison of novel methods can be found in e.g. Liu et al.(2012).
3.1.2 DNA synthesis
Synthesis is the process of creating artificial DNA strands. Typically oligonu-cleotide synthesisbyMcBride and Caruthers(1983) is used which is a chemical process to produce short strands of 15-20 base pairs by treating nucleotides as building blocks that can be sequentially coupled in a growing order using four chemical processes for each addition. In general the majority of the synthesis methods are only able to produce small strands to avoid introducing errors why the assembly methods is of great importance to the synthesis of large strands.
3.1 Enabling technologies 21
3.1.3 DNA assembly
DNA assembly is the process of merging two strands. There are numerous ways to assemble DNA strands. One of the more intuitive, compelling methods is the standard assembly method which uses restriction enzymes to merge DNA strands as illustrated in Fig. 3.1. This method is cheap, but prone to errors, Densmore and Hassoun (2012). Some of the available assembly methods are compared inBaldwin et al.(2012).
Figure 3.1: Thestandard assembly method. EcoRi enzymes seek and remove theAATTC from 3’ strands and the complementaryTTAAG se-quence from 5’ strands. This forces the strands to combine. Fig-ure from Excellence.
BioBrickTM is an interface for painless assembly of biological parts. It works by having some universal defined DNA sequence appended to both ends of the part sequence with the ability of forming stable bonds with other parts that implement the same interface. The design along with the behavioural characteristics of these parts are stored and made available in rapidly increasing databases, e.g. the Registry of Standard Biological Parts1. A goal from the foundation behind the BioBrick, theBioBricks Foundation, is to make it possible to engineer entire organisms just from these parts.
1http://parts.igem.org