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.