BIOL114 - Lecture 5 - Microbial Biotechnology

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  • Created on: 15-03-16 17:08
What is a recombinant protein?
A protein produced through the use of recombinant DNA techniques
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What is an example of a recombinant form of human insulin?
Humulin - used for type 1 diabetes
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What is the first step of generating recombinant proteins?
1.) cloning a cDNA copy of the gene - making a cDNA library and screening the library.
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What is cDNA?
Complementary DNA - a DNA version of the RNA molecule. It is initially a single strand but its copeid.
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Which enzyme copies the cDNA?
Reverse transcriptase
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Why do we want to clone a cDNA cope of a gene?
1.) Enrichment - eukaryotic genomes are very large and mostly non coding - cDNA represents only the protein coding part of the genome. 2.) To use in the production of recombinant proteins in E.coli. Introns in genomic DNA prevent its expression .
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What are the steps taken to construct double stranded cDNA.
F
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How do you clone your cDNA?
Ligation into plasmid. Transform this. Culture this. Plasmid isolation and purification to have cloned DNA.
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Which of the clones carries the sequence of interest?
You have to screen the cDNA libraries to find out. It is like finding a needle in the haystack
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How many choices are there to find this out?
3
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What are the 3 approaches to screeing?
Hybridization
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How can you detect clones by hybridization?
1
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What are the general principles of hybridization?
It is the most common approach to identifying a specific clone, using a hybrization probe.
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What is a hybridization probe?
It is a DNA (or RNA) fragment (usually 100-1000 bases) which is used to detect the presence of related nucleotide sequences in a biological sample
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What else is important about the probe?
It must be specific for the target sequence to allow base pairing. The probe must be labelled in some way that allows its location to be visualised
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What are the different types of hybridization probe?
Homology probe, degenerate oligonucleotide probe
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What does the homology probe depend on?
Using the corresponding gene sequence (or cDNA) from a related organism. E.g. if a cDNA clone for a specific mouse gene is available, it can be used as a probe to identify the corresponding human gene.
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What does the Degenerate oligonucleotide depend on?
Depends on knowing the protein sequence of the gene you wish to clone
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Which amino acids only have a single codon?
Tryptophan and Methionine
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In degenerate bases, which codon usually varies?
The last base
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if you're looking to work out the possible number of combinations that could encode a protein sequence, what do you do?
You multiply the number of codons that code for each amino acid all together for the sequence - e.g. 2x3x2x1x2x2x2
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What do you do once you have identified the least degenerate 20 base region?
You prepared 20more degenerate probes to screen library - 1 of the 96 oligos will be an exact match
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How many ways are there to label a probe?
2
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What are the ways of labelling a probe?
Radioactive and non-radioactive
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What appens in radioactive labelling?
DNA is labelled by incorporation of nucleotides containing radioactive 32P or 33P
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What are the disadvantages to using radioactivity to label?
Safety hazard, complicated handling, short half life, limited detection methods.
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What happens in non radioactive labelling?
DNA labelled by incorporation of nucleotides coupled to DIG
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What is DIG?
Digoxigenin
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What is DIG?
A highly immunogenic steroid that comes from foxgloves
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How does DIG work to labelling?
It is conjugateed to nucleotides
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Advantages to DIG labelling?
DIG labelled probes are safe and stable
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Disadvantages to DIG?
The detection method is more complex (multi-step_
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Wht is the detection process of DIG?
DIG label recognised by specific antibodies coupled to alkaline phosphatase enzyme. AMPPD is added - when cleaved by the enzyme it produces chemiluminescence
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Why is alkaline phosphatase used?
Because in the presence of the reagnt AMPPD, the alkaline phosphatase uses AMPPD in a reaction that produces light. (chemiluminescence)
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How many different ways are there to label things?
2
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What are the different ways of labelling probes?
Terminal transferase, and using Taq polymerase and PCR
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What is terminal transferase?
It is an unusual DNA polymerase that does not require a DNA template
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What does terminal transferase do?
It adds polynucleotide tails to the 3' end of DNA molecules
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When does terminal transferase work best?
When the 3' end is not recessed (e.g it can have a blunt end or 3' overhang)
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What does PCR do?
It provides a convenient way to synthesize large amounts of labelled DNA
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How does DNA help label?
The DNA fragment to be used as a probe is amplified by PCR in the presence of labelled dNTP
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Once you have labelled your probe, how does colony hybridization occur?
It is a way of making a DNA print from each colony on a membrane that can then be screened with a hybridization probe to detect the clones of interest. After denaturation of the DNA strands in the plasmid, the probe will bind to its complementary .
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What does denaturation of DNA mean?
Separating of the DNA strands.
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What will happen to the colonies carrying the targeted cDNA?
They will be radioactively tagged (detectable with an X ray film)
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What is another approach to screening cDNA libraries?
Immunodetection using antibodies
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What does immunodetection require?
Purifying the protein of interest, raising specific antibodies against purified protein, a cDNA expression library in bacteriophage lambda.
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If you want to use antibodies as a probe instead, you need a different type of library. What is this called?
A bacteriophage expression library - it is desirable because the bacteriophage lyses the E.coli cells and releases the proteins encoded by the cDNA.
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When the bacteriophage lyses the cells and releases the protein, what form are the proteins in?
Plaques - these contain the E.coli protein
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What is complementation cloning?
It uses the activity of the encoded protein to rescue a mutation in the host organism. If we wanted to clone a cDNA encoding enzyme required for histidine biosynthesis we could use a mutant E.coli lacking that enzyme (only able to grow with histidine
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What is the first step of complementation cloning?
You ligate plasmid and cDNA fragments
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What is the second step of complementation cloning?
Transform into His- mutant bacteria and culture in liquid medium (+histidine)
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What is the third step of complementation cloning?
Only cells transformed with cDNA encoded required enzyme for histidine biosynthesis are able to grow on madium lacking histidine
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What is an example of a recombinant form of human insulin?

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Humulin - used for type 1 diabetes

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What is the first step of generating recombinant proteins?

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Card 4

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What is cDNA?

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Card 5

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Which enzyme copies the cDNA?

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