The insertion of a human DNA fragment into a bacterial cell might make it possible for...... ?
1) The bacterial cell to produce a human protein. 2) The cloning of a human that donated the DNA fragment. 3) Human to become immune to an infection by this type of bacteria. 4) The cloning of this type of bacteria.
What are steps for inserting a gene into a plasmid from a cell of interest?
Here are the 7 steps for gene cloning using plasmids.... STEP 1. A gene of interest ( DNA fragment) is isolated from cells that have been grown in lab. culture ( you perhaps prepared it in the laboratory, have not you?); STEP 2. Both the human DNA and the plasmid are treated with the same RESTRICTION ENZYME to produce identical sticky ends; STEP 3. The restriction enzyme cuts the plasmid DNAat it's single recognitin sequence, disrupting the TETRACYCLINE resistance gene; STEP 4. The DNA fragment are mixed together and the complementary sticky ends are attached to each otherby base pairing. The enzyme DNA- LIGASE is added to bond the sticky ends; STEP 5. The recombitant plasmid , or molecular clone, is introduced into a bacterial cell by adding the DNA to a bacterial culture. Under the right condition some bacteria will take upthe plasmid from solution by the process TRANFORMATION; THIS IS THE PREPARATION OF CLONE upto this point. NOW, let's start CLONNING the gene...... STEP 6. The actual gene cloning process ( making multiple copies of human gene ) occurs when the bacterium with the recombinant plasmid is allowed to reproduce; STEP 7. Colonies of bacteria that carry the recombinant plasmid can be identified by the fact that they are resistant to ampicillin but sensitive to tetracycline. That's all. Are you happy of the answer? Or did you expect to know something more. E-mail me, then only I can help you. Best Wishes.
Plasmid gene orientation?
Firstly RNA polymerase reads the template strand in a 3'->5' Direction. Alternating transcription direction is because there is two strands. These plasmids are from bacteria not viruses so they don't need one continuous sense or antisense genomes, instead they have the sense and antisense strands interchanging depending on which transcription unit we are referring to. One operon may start with the sequence 3'-ATTATATAG-5' and RNA polymerase will come along and bind to this sequence specifically via its sigma subunit and start transcribing, now imagine it transcribed the same sequence into mRNA it would be 5'-UAAUAUAUC-3' - now the mRNA is always the sense and the strand that it was copied from is called the antisense. But imagine polymerase bound to the other strand which would be 5'-TAATATATC-3' this would mean that a different subunit could cause polymerase to recognise this sequence, this would mean that the mRNA will be transcribed as 3'-AUUAUAUAG-5' and this mRNA is always the sense and the sequence which is complementary to it is the antisense. So here you see that at one specific point in the plasmid polymerase can bind to either strand depending on things like sigma factors. This binding of polymerase to DNA will always orient polymerase so that it is reading in the 3'->5' direction, this means that the mRNA will always be transcribed in the 5'->3' direction. Here we see how we have transcribed two complementary sequences of DNA to produce two different mRNAs which are both sense and both strands are antisense - meaning that whether it is sense or antisense depends solely on the mRNA you get from it since it is always sense. So the ribosome will recognise the mRNA at the 5' and read along 5'->3' and produce a protein. That's about all I can say about that - 5' binds and is read along and produces an amino acid sequence dependent on its mRNA sequence. Using my example these two mRNAs will produce 2 different proteins even though the ribosome moved in two different directions - obviously at different times and locations along the plasmid in real life.
Biology- Bacterium genes?
Bacterial gene transfer is also used in the lab to introduce genes into organisms of different species, genera, phyla, or even kingdoms or domains. A major example is the use of a Ti plasmid of the soil bacterium Agrobacterium tumefaciens to transfer genes into dicotyledonous plants. The bacterium infects the plant root in the soil and transfers some of the Ti plasmid genes (T DNA) into the plant nucleus. Recombination may occur, and T DNA expression is controlled by plant compounds generated in response to wounding and infection. The T DNA is generated when there is a nick that creates a primer for replication, followed by transfer of a single-stranded DNA piece that converts to a double-stranded piece in the plant nucleus. Which of these processes is Ti plasmid transfer most like? A) conjugation B) generalized transduction C) specialized transduction D) transformation
Why can’t we put human DNA directly into bacteria and have it expressed (produce a protein)?
The question has some tricky generalisation, I assume the question implies about using bacteria as a factory to produce proteins normally generated by humans.First, there is a limit to the size of foreign DNA which can be inserted inside a bacteria. It generally correlates with the size of bacterial genome and method of introducing DNA (plasmid, chromosome integration) with no direct formula to calculate it. However, the limit is far below then the amount of genetic material carried by human genome. So you can’t put all it in or half or its half’s half’s half’s half’s half and probably some more half’s.As Mike Swanton in one of the answers mentioned that human DNA has intermediate non coding regions which will not be read as such by bacteria. The bacteria will read the sequence and translate into polypeptide of amino acids which will be different from proteins found in humans because there is an intermediate step called intron splicing. The strategy is as he mentions to reverse transcribe mRNA to DNA and then code.Certain proteins are polymers expressed from different genetic regions (Insulin was such an example) and while it is possible to express them individually, there is no guarantee that they will multimerize to form the final unit. Biochemical constraints are responsible for this. Even Insulin was developed in parts and then recombined downstream of bacterial expression.Polymers of amino acids translated by ribosomes do not mean they are expressed. Being functional is important to be considered expressed. However, proteins denature very often if they are perturbed from their natural environment even a little.Post translational modification like glycosylation cannot occur.Some proteins are coded in same regions but are splice variants of each other. None of such proteins can be produced.This is still a small list of possible reasons why they human DNA can’t be just put into bacteria and see the protein be expressed. However, it does not mean that no human protein can be artificially produced in bacteria. But, one do need to account into large number of variables that come into play and change their expression system from bacteria to yeast or mammalian cell lines as needed.
Describe the process involved in inserting the genes from one kind of organism into cells of another organism.
1. Isolate the genome of organism on which the desired gene sits. 2. Cut it with restriction enzyme with the desired recognition site. 3. Perform polymerase chain reaction procedure with oligomer primers which have the suspected complimentary sequence with your gene 4. Perform agarose gel electophoresis to isolate the multiplied sequences 5. Separate the duplex structure of primer:DNA in alkaline medium. 6. Perform Southern Blot Transfer (could be found in any molecular biology laboratory manual) 7. Transform the target organism with the resulting gene fragments from the southern blot. (transformation procedure vary with the varying target organism)
Why does a plasmid contain an antibiotics resistance gene?
The whole point of an artificial plasmid is to insert it into bacteria to change their gene expression. Say you have a plasmid that has a gene for making insulin. You inject it into a culture of bacteria and let them take it up.A few hours later, you get some bacteria producing insulin for you, but most of the bacteria in your culture haven’t taken up the plasmid, so they’re not doing anything useful. If anything, they’re competing with your insulin-producing bacteria for nutrients. So you decide to kill them with antibiotics.Well, it works. You’ve killed off the useless bacteria. Except oops, you’ve also killed the bacteria that were being useful.But if you include an antibiotic resistance gene in the plasmid, then any bacteria that take up the plasmid will produce insulin and survive when you kill off the useless ones, so that you’re only left with insulin-producing bacteria.