Standard Biological Parts

Today, I began looking at the standard biological parts used in the process of synthesising DNA. Here are their definitions:


Promoters
A promoter deals with preparing the DNA for transcription and assisting with the process. Transcription is the process of turning DNA into RNA (which is itself later turned into proteins).

In the DNA sequence, the Promoter is typically located near the gene it regulates. The enzyme that is responsible for synthesizing RNA from the DNA, Ribonucleic Polymerase (RNAP) needs somewhere on the DNA strand to bind to so that it can perform its job. This is where the Promoter comes in, providing a secure binding site for the RNAP as well as proteins called transcription factors that help recruit RNAP.

Once bound, a complicated process of synthesis begins, resulting in different types of RNA, dependent on what the gene encodes for (mRNA for genes that encode proteins).



Ribosome Binding Site
The Ribosome Binding Site (RBS) is a sequence found in mRNA that is bound by the ribosome when initiating protein translation.

The mRNA is produced from DNA in the process known as transcription, described above. The Ribosome Binding Site is then bound by a Ribosome. A Ribosome is a component of cells that uses the mRNA as a template for the correct sequence of amino acids. Transfer RNA (tRNA) molecules which are RNA molecules which are bound to a specific amino acid are brought in and mapped to the mRNA sequence. The Ribosome then adds the tRNA's amino acid to the growing peptide chain which is a chain of amino acids.

The polypeptide is later folded into a 3D structure to form a protein.

Protein Domains
Protein domains are logical sections of a protein sequence. Each Protein domain can evolve, function and exist, separate from the rest of the protein chain.

The Protein Domain is a 3D structure and can be thought of as a functioning protein that can join with other Protein Domains to form a larger Protein, with a new function. Because they are self-stable, domains can be swapped by Genetic Engineering between one protein and another.

Because of their nature, Protein Domains play an important role in evolution.

Pyruvate Kinase, a protein from 3 domains

Protein Coding Sequences
Protein coding sequences are DNA sequences that are transcribed into mRNA and in which the corresponding mRNA molecules are translated into a polypeptide chain. Every three nucleotides, termed a codon, in a protein coding sequence encodes 1 amino acid in the polypeptide chain.

Translational Units
Translational units are composed of a ribosome binding site and a protein coding sequence.

Since Translational Units are composed of an RBS and a Protein Coding Sequence, they can be thought of as composite components. Translational units begin with the RBS, the site of ribosome binding and translational initiation, and end with a stop codon, the site of translational termination.


Terminators
A Terminator is a DNA sequence that causes RNA Polymerase to cease Transcription. This sequence marks the end of the gene and signals to the RNAP to release the newly made RNA molecule.

Plasmids
A plasmid is a DNA molecule that is separate from and can replicate independently of the Chromosomal DNA. They are double stranded and typically circular. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state.

Plasmids are imporant in Genetic Engineering where they are called 'Vectors'. They are used to multiply or to express a gene within a host cell. Plasmids are inserted into the host cell typically via transformation which are then cloned to proliferate the cells with the added genes.



Primer
A Primer is a short, single strand of DNA that serves as the starting point for DNA replication. A Primer is required because the enzymes that catalyze replication, called DNA polymerases, can only add new nucleotides to an existing strand of DNA.

Parts Database

In order to form a synthetic cell, certain biological parts are required. I will research what these parts are and what their purpose is in a later post.

For now, I wanted to check out the 'Registry of Standard Biological Parts'. This is an online compendium of known biological parts and is a colloborative effort to index all such parts. Sara had shown me this website and for now, I was mainly concerned to see what sort of options were available for getting hold of the parts data.

It seems the ROSBP has set-up something called a 'DAS' - a Distributed Annotation System, described as being:

The distributed annotation system (DAS) is a client-server system in which a single client integrates information from multiple servers. It allows a single machine to gather up genome annotation information from multiple distant web sites, collate the information, and display it to the user in a single view. Little coordination is needed among the various information providers.

From initial inspection, the DAS can provide me with XML output of all the parts and specifics on each part. This would allow me to populate a Database of my own with Parts data, however it seems - and is noted on the website - that the data is sparse and a work in progress.

The following link provides the URLs for accessing the DAS: http://partsregistry.org/DAS_-_Distributed_Annotation_System

Plasmids and Transformation

The method we are mainly concerned with for the project is biological transformation, using 'plasmids' or 'vectors' as they are known in Genetic Engineering.

Plasmids
"Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes.[2] Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics.

However, a plasmid can only contain inserts of about 1–10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used."

source: http://en.wikipedia.org/wiki/Plasmid

Transformation
Transformation is the process of inserting exogenous DNA material into a cell. For our purposes, this means the uptake of the plasmid - containing the desired foreign DNA - into the host cell.

The following diagram illustrates the whole process.

source: http://www.accessexcellence.org/RC/VL/GG/plasmid.php

Here, we see that the desired gene is cut from the foreign DNA using a restriction enyzme. The plasmid is similarly cut. This produces 'Sticky Ends', with the ends of the genes not having matching bases. A DNA Ligase is then used to join the ends of the gene of interest with the plasmid. This produces the recombinant DNA.

The plasmids are then inserted into the host cells. To make sure we only have cells that contain the gene of interest, we introduce an anti-bacterial mixture. The plasmids with the foreign DNA have been given a marker that protects against the anti-bacterial, preserving all the cells which contain the foreign gene.

Preliminary Research: Recombinant DNA Basics

Recombinant DNA

Recombinant DNA involves taking a piece of DNA and combining it with another strand of DNA.

There are three methods of doing this: Transformation, Non-Bacterial Transformation and Phase Introduction.

Transformation

1. Select a piece of DNA to be inserted into a vector (Plasmid).
2. Cut the selected DNA using a restriction enzyme and then insert the DNA into the vector with DNA Ligase.
3. Add markers to the insert.
4. Insert the vector into a host cell e.g. E. Coli.

Markers that can be added include a marker for identifying recombinant molecules and an antibiotic marker. An antibiotic marker is typically used so that the host cell doesn't die when exposed to an antibiotic. This is used to kill cells with no recombinant DNA.

Non-Bacterial Transformation

Same as above except that a bacterial host is not used (Perhaps eukaryotic cell?).

Typically the DNA is inserted in one of two ways:

Microinjection - DNA is inserted directly into the nucleus of the cell being transformed.
Biolistics - The host cells are bombarded with high velocity microprojectiles, such as particles of gold or tungsten that have been coated with DNA.

Phage Introduction

The process of 'transfection' which is equivalent to transformation except a phage (virus that infects bacteria) is used instead of bacteria.

How rDNA works

The host cell expressed protein from the recombinant genes. A significant amount of recombinant protein will not be produced by the host unless expression factors are added. Protein expression depends upon the gene being surrounded by a collection of signals which provide instructions for the expression and translation of the gene by the cell. These signals include the promoter, the ribosome binding site and the terminator. Expression vectors contain these signals. Signals are species specific e.g. E. Coli signals must be used with E.Coli.

Problems occur if the gene contains introns or signals which act as signals to the bacterial host. This results in premature termination and the protein will be severely affected.

Source: http://rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html

Preliminary Research: DNA Basics

DNA - Deoxyribonucleic Acid. A nucleic acid that contains the genetic instructions used in the development and functioning of all known organism.

All DNA is made up of a base consisting of sugar (deoxyribose) and phosphate/ There is also one Nitrogen base of four possible bases which are:

• Adenine (A)
• Thymine (T)
• Guanine (G)
• Cytosine (C)

Nitrogen bases are found in pairs, with A binding to T and G binding to C.
All these come together to form the famous 'Double Helix'

The sequence and number of bases is what creates diversity in organisms.

DNA is transcribed into mRNA (Messenger Ribonucleic Acid) which is then translated into a protein.

Source: http://rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html

First Meeting

So after exchanging a few emails with Sara and being somewhat confused as to what the project would require, I thought it was time to pop onto Campus.

I'd spent the Bank Holiday weekend back in Yorkshire with the family so thought I'd pass through Coventry on the way back to Southampton and kill two birds with one stone! After having lunch and catching up with my good friend James, I had my meeting with Sara at 14:00.

After explaining Synthetic Biology basics, Sara showed me some documentation sent to her by her Biologist colleagues in Nottingham. It described the process of forming a plasmid for insertion into a bacteria. The plasmid has to be made up of certain genetic parts, however, finding which ones are best and checking that they are compatible is hard. Therefore, the project Sara was proposing is to find a combination of genetic parts that would create the desired genetic code, and then to attempt to resolve any DNA code conflicts. These conflicts occur when a sequence of ATCG modules that "end" a genetic part, appear before the genetic sequence for that part is complete. Due to the fact that amino acids can be coded for using many different combinations, it is probably possible to permute these ATCG sequences, until all such conflicts are resolved.

I told Sara that I thought the project sounded very interesting and was more than happy to take it on! After a little head scratching, we came up with a title of 'DNA Synthesis Tool with Conflict Resolution". While not the jazziest title, it certainly seemed to summarise what we had discussed.

Before leaving, I asked her what resources I could consult to get a decent enough grounding for the biology part of the project. My first job is to Google and maybe find a book or two covering, 'Recombinent DNA'.

After that, I packed up, wished Sara a good day and travelled back to Southampton feeling good that I'd got the Third Year Project stuff sorted out. I had however, managed to leave behind the documentation describing the process! Can't win them all >.<.

It Lives!

Taking the advice of Sara, my supervisor, I have setup a blog to record the progress of my Third Year Project at the Department of Computer Science, University Warwick. I predict tears, laughter, success, failure and moments of pure incoherence.