![]() ![]() With this in mind, it is necessary to change the codon to alter the restriction site whilst at the same time ensuring that the same amino acid is coded for (a different amino acid means an incorrect protein). ![]() This sequence is present in the DNA sequence for the protein Dispersin B, meaning that EcoRI would cut within the coding sequence, leading to a shortened amino acid sequence and a nonfunctional protein. Before using restriction enzymes, it is therefore crucial to check all of the DNA sequences for any disallowed restriction enzyme sites.įor example, the DNA sequence where the EcoRI restriction enzyme cuts is GAATTC. If the restriction enzyme sites were present within any of the DNA sequences (and not only within the prefixes and suffixes) then the restriction enzymes would cut within and mix up all the sequences, rendering them useless. iGEM specifies certain restriction sites at the start and end of DNA sequences so that any sequences can be cut and stuck together using only a few restriction enzymes. When you have the codon optimised DNA sequences, restriction enzymes are then used to cut and stick the sequences and plasmids. These enzymes cut at specific sequences of DNA called restriction sites and allow us to stick our pieces of DNA together. This is important to avoid very slow or incorrect protein synthesis. coli, the codon optimization software gives the DNA sequence using the codons that are most commonly used by E. When taking the amino acid sequence of the protein you want to express in E. For example, AGA is the most commonly used codon for serine in E. The relative frequency of codon use varies widely depending on the organism and organelle. For example, AGA, AGG, AGT and ACG all code for the amino acid serine. With only 20 amino acids, this means that some amino acids are coded for by more than one codon. As there are 4 different nucleotides (A,T,C,G), there are 64 different possible codons. There are 20 amino acids and each is coded for by three DNA bases, which are called codons. This is an important step, for which I will now describe the biochemistry.Īmino acids are the building blocks of proteins. coli (or whichever bacteria species you are wanting to express your proteins of interest in). This programme also optimizes the DNA sequence for expression in E. This software gives the DNA sequence when you enter the amino acid. We used IDT codon optimization to get the DNA sequence. The paper from which we sourced the sequence of Art-175 only gave the amino acid sequence. For us, this was the case with the protein Art-175. In other cases, you may have the amino acid sequence of a protein you want to express, but not the DNA sequence that you need to insert into the plasmid. If the sequence you need is not a biobrick, you can find it on online databases such as GenBank. This includes the promoter regions, which are regions of DNA that initiate the expression of a particular gene (in effect an on/off switch), and the ribosome binding sites that facilitate the initiation of protein synthesis. Many of the parts we need to use for our project are already biobricks. For this component of our project, we used the iGEM parts registry to locate the DNA sequence. The DNA sequences that you want to add to a plasmid can come from a variety of sources. In the simplest case, the sequence you are looking for is already a biobrick, such as the protein DNase. These days, we can put the gene for human insulin into a plasmid, put this plasmid into bacteria and have the bacteria make insulin for us. It used to be that the only way to obtain insulin was purifying it from the pancreas of cows and pigs that had been slaughtered for food. For example, human insulin is made using bacteria that have been genetically-engineered in this way. The DNA that codes for the new proteins can be added into a plasmid, which can then be put into bacteria (through a process called transfection) and, providing the plasmid contains the all necessary extra features for gene expression, the bacteria will be able to make the brand new proteins. A plasmid containing 1) promoter 2) coding sequence 3) origin of replication 4) antibiotic resistance gene to select only those bacteria that have successfully taken up the plasmidĪ plasmid is a circular piece of DNA. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |