Team:Penn State/Project

From 2012.igem.org

Revision as of 14:52, 13 July 2012

Objective

The focus of this years' projects is to question the central dogma of biology. This is the commonly held belief that information flows from the ordering of the bases within a cell's DNA to the ordering of the bases within RNA. This information can then be translated to the order of Amino Acids that make up proteins. We are currently working on projects that question aspects of this dogma.

Project 1: Codon Optimization

Project Overview

All of the proteins around us, with few exceptions, are made up of 20 fundamental building blocks of life - Amino Acids. Different arrangements and combinations of these basic building blocks give us the diversity of proteins that we see. Messenger RiboNucleic Acids (mRNA) is in essence a "photocopy" of DNA that codes for a gene. mRNA carry codons, which are groups of three bases that code for a single amino acid. There are 64 possible combinations of codons (4 x 4 x 4 = 64), but some of these combinations are degenerate, so there can be more than one codon that codes for the Amino Acid.

Proteins are assembled by ribosomes which read the mRNA and catalyze the binding between Amino Acids; however, they cannot read the codon code themselves and require the help of tRNA (transfer RNA). Each tRNA matches a single codon sequence and can only carry a single specific amino acid at a time. tRNA has complimentary bases to their respective codon sequences on the mRNA strand, and once the tRNA matches the sequence on the mRNA, the tRNA deposits the Amino Acids, which then bind together to form proteins.

We have mentioned how these can be a number of codons that can code for the same Amino Acid, and subsequently, a number of tRNA molecules that can carry a given Amino Acid. In nature, and in many organisms, only a select few of these tRNA and codon combinations are used instead of all sequences that code for the same amino acid. This is called codon bias.

Our goal for this project is to see which codons are preferred, or biased. We also want to investigate if this bias can change over time, different circumstances, or stresses.

Experiments

We have been using a piece (vector) designed that includes a promoter, leader sequence, repeat codon sequence, mCherry, and GFP all inserted into the sPB1C3 vector for our project. We will use this construct to digest out the existing repeat codon sequence and then ligate in our designed repeat pieces. We designed repeat sequences for 9, 6, and 3 repeats of the codon. These repeats coding for Threonine and Alanine will be designed and ligated into the vector construct. Once we have the replace the repeat sequences, we will transform them into chemically competent cells and plate them. With these, we will prepare them for testing on the flow cytometer in order to test for GFP and mCherry intensity.

Results

Coming soon…

Project 2: Multidirectional Promoters

Project Overview

Before we can make a protein, we need an mRNA to carry the information about the order of the amino acids. But before we can make mRNA we need to know where the genes are in the DNA of a cell. Before RNA polymerase can make a copy it needs to bind to the DNA. This is assisted by a variety of factors, other proteins, that look for a specific sequence in the DNA. This sequence is called a promoter because it promotes the transcription of the DNA into RNA by the RNA polymerase. These sequences are generally upstream, or ahead, of a gene's coding sequence.

However, not all promoters cause RNA polymerase to transcribe downstream in the expected "forward direction". Some promoters can cause RNA polymerase to go in the opposite direction from what is expected, or go in both directions. This is what we are trying to find out; do different promoters go in different directions, and what is the directional preference of different promoters.

Experiments

Coming soon…

Results

Coming soon…

Project 3: Multiple Start Codons

Project Overview

mRNA is the molecule that carries information about the sequence of amino acids in a protein. However, much like the lines on a sheet of paper, the protein coding sequence of an mRNA molecule does not start right at the beginning, or top of the page. Instead, once the mRNA is bound by a ribosome, a start codon must first be read before the protein can be translated. This start codon is generally AUG, or Methionine.

Once this start codon in read the ribosome will continue reading and building the polypeptide (protein) until a stop codon is reached. But what happens if you have two AUG codons close together? That is the question we are attempting to answer.

We are trying to understand what happens when there are two start codons very close together, but out of frame. Out of frame refers to how the ribosome reads the mRNA. Remember those codons and how they are groups of three bases on the mRNA? The reading frame refers to which group of three. If you start at one base and read the bases in groups of three from that point on, that is one frame of reference. If you then move your start point ahead one base, then you are reading in a new reading frame. If you advance you starting point one more base, that is the third reading frame. If you advance it again you are now back in your first reading frame, but you have skipped the first codon. We are looking into what happens when you have multiple start codons close together, but in different reading frames. Which frame will be preferred?

Experiments

Coming soon…

Results

Coming soon…