Hypothesis free research Flashcards
you don’t always need a
tight hypothesis
drugs can be designed
without knowing much about the target
- we can combine several techniques to make try hi tech therapeutics
bacterial resistance
bacterial resistance is a growing problem and causes many deaths worldwide.
current option seems to make more antibiotics
- could use what we already know about bacteria and design some more
- or we could adapt old ones
rational drug design could be used to design
super anti bionics which are 25,000 times more potent than predecessor
another methods of hypothesis free design
analogy e.g. there is a man who wants to know which horse will win a race
- a physiologist, biochemist and physicist are asked for their opinions.
- the point is WE DONT NEED TO KNOW IN ADVANCE, JUST RUN THE RACE AND FIND OUT
what is an assay?
An assay is an investigative procedure in laboratory medicine
there are many microbes undiscovered in the ocean, therefore could have the potential
to have antimicrobial properties which could be used to design new antibiotics
how many bacteria in ml of seawater
1 million
an assay needs to be designed, to screen things
- what is the assay? does bacteria secretions cause death
- what sort of stuff are we going to screen? bacterial secretions
- what is our hypothesis? some soil bacterial will make anti-micorbial substance
we can culture less than ……% of bacteria
10
to access the rest we must
design high throughput technology
craig ventro
doesn’t need to culture bacteria
- sequenced Sargasso sea (sar17)
- he collects genes, not bothered whether the genes come from
what are scFv’s
A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids
when are scFv’s used
These molecules were created to facilitate phage display, where it is highly convenient to express the antigen-binding domain as a single peptide. As an alternative, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. ScFvs have many uses, e.g., flow cytometry, immunohistochemistry, and as antigen-binding domains of artificial T cell receptors.
production process of therapeutic antibodies
1) fuse the cDNA to a phage coat protein
2) genetically engineer a library of bacteriophages with many different cDNA sequences (more than 10^9)
3) fuse to cDNA for an Gab to a phage coat protein
4) genetically engineer a library of bacteriophages (M13) with many different cDNA sequences
5) expose hages to an immobilised target molecule - often coated on a piece of plastic
6) use high stringency washes to leave the strongest binders attached to the ligand- most will not attach and be washed away
7) strong bids are eluted- more stringent solution used to dislodge liana
8) stronger binders used to infect bacteria
9) natural mutations will occur when the bacteria grow and new phages are produced
10) these re used to infect fresh bacteria
11) as bacteria grow- more viruses produced- increase number of fab high affinity binders
12) mutations in bacteria will occur n the gene for Fab region
13) new variants of them are produced
14) repeat process
15) increase stringency
16) selective pressure- only the ones that stick get to divide and make progeny
17) repeat the cycle and ramp up salt conc to strip off high affinity binders
18) third generation phages produced
19) infect bacteria- more mutations
20) repeat cycle- higher affinity binders
21) production of therapeutic anybodies
