AlphaFold2 and Protein Structure Prediction

• Demis Hassabis & John Jumper developed the AI Model, AlphaFold2
• AlphaFold2 is able to predict the structure of virtually all of the 200 million proteins identified by researchers
• It allows a better understanding of antibiotic resistance and the creation of images of enzymes that can decompose plastic
• In 2022, an image of a huge molecular structure in the human body was captured, where more than a thousand proteins form a pore through the membrane surrounding the cell nucleus
• In 2023, an image of a bacterial enzyme that causes antibiotic resistance was created, and its structure is important for discovering ways of preventing antibiotic resistance
• Natural enzymes that can decompose plastic was captured in 2022 with the aim to design proteins that can be used to recycle plastic

Microorganism “Learning”

• Microorganisms accumulate genetic mutations
• Detrimental mutations dies off quickly
• Neutral mutations slowly fades away with time
• Beneficial mutations have success of progeny increasing retention of the mutations

Replication Errors

• Replication an accurate process but between 10^-4 and 10^-12 errors may occur
• With 4 x 10^6 nucleotides per E. coli and many bacteria replicating, mistakes happen
• If DNApol makes a mistake, a single base mismatch occurs
• Replicating it again, match the "new" base

Base Pair Substitutions – Change of Genotype

• A change of "Genotype" always occurs, meaning the DNA sequence is altered
• However, a change in "Phenotype" is not necessary
• The enzyme might not change, or it might change very slightly
• First, it determined if the DNA has changed in a gene or not
• In Prokaryotes, about 50% of DNA is coding
• In humans, about 1% is coding, and 75-80% of that 1% is made up of introns

Outcomes in coding region of a gene

• Synonymous mutation: codes for same amino acid as original and can affect efficiency of translation
• Missense mutation: creates codon for different amino acids where the resulting protein often does not function normally
• Nonsense mutation: creates a stop codon and yields shorter, often non-functional proteins

Environmental Challenges

• Aerobic environments oxidizes guanine and incorrectly pairs with Adenine instead of cytosine
• Chemical mutagens, UV radiation, and other radiation also influence mutations

Nucleotides

• Adding or deleting a multiple of 3 nucleotides changes only one (or a few) amino acids
• If it is not a multiple of 3, the reading frame changes resulting in loss of all downstream function
• Adding random amino acids eventually terminates the process
• If a second mutation changes back to the original reading frame, it might only impact a few amino acids

Spontaneous Mutations

• Spontaneous Mutations are Rare, very seldom happen twice at the same time, like the story of HIV infections and ART therapy
• Bacteria have developed faster and riskier ways to change rather then the slow way to evolve new genes.

History

• 1920 – Frederick Griffith worked with S. pneumoniae in virulent and avirulent forms:
  • Dead virulent forms (encapsulated) could convert avirulent forms and bred true.
• 1940s – Avery, McCloud, and McCarty identified DNA as the carrier of that information, but the question remained how external DNA pieces change the chromosome

Bacterial Transformation

• Altered phenotype DNA was not within a cell or virus, but originated from cells that have burst or secreted it
• Addition of DNase prevents transformation, meaning the recipient cell must be competent
• Most take up DNA regardless of origin, but some only accept DNA from closely related bacteria
• Some species are always competent; others become so under certain conditions

Transformation Details

• A double-stranded donor DNA encoding streptomycin resistance (StrR) binds to a receptor on the surface of the competent cell
• One strand enters the cell while nucleases degrade the other strand
• New DNA integrates into the chromosome by homologous recombination
• One daughter cell will inherit donor DNA
• The transformed cell grows on a medium containing streptomycin
• Other donor genes are possible; the key is a STRONG selective agent

Homologous Recombination

• Donor DNA replaces the complementary region of the recipient cell's DNA
• If it is not integrated, the DNA will not replicate and will eventually be diluted from the population
• Stealing genes carries high risk, but also a high reward
• Microorganisms steal genes from others despite the occasional failures

Phage Transduction

• This is the transfer of bacterial genes by bacteriophages (phages)
• Phages infect bacterial cells by attaching to the cell and injecting its nucleic acid
• Phage enzymes then cut bacterial DNA into small pieces
• Bacterial cell enzymes produce phage nucleic acid and a phage coat the components of new phage particles
• Phage particles are released from the bacterial cell
• Generalized transduction results when a fragment of bacterial DNA enters the phage protein coat, producing a transducing particle

Transduction

• Still accidental, but a sufficiently strong selection process will see results

Conjugation

• This is a deliberate transfer of DNA through Plasmids
• Plasmids consist of a circular DNA, including oriT, meaning Plasmids don’t need to integrate into chromosome
• Plasmids Replicate within cells, some are single copy, replicate in unison with chromosome, others are multi-copy counting on enough to be in both halves of dividing cell and can have amplification effect of up to 1000’s per cell

Conjugative Plasmids

• These direct their own transfer between cooperating cells such as the F plasmid (fertility) of E. coli.
• F plasmid encodes the F pilus (sex pilus)
• F+ cells contain the plasmid, whereas F- cells do not and they copy from F+ to F-, resulting in two F+

Plasmid Encoded Traits

• Plasmids encode for many traits:
  • Antibiotic resistance - Many organisms
  • Antibiotic synthesis - Streptomyces species
  • Gas vacuole production - Halobacterium species
  • Increased virulence - Yersinia and Shigella species
  • Insect toxin synthesis - Bacillus thuringiensis
  • Nitrogen fixation - Rhizobium species
  • Oil degradation - Pseudomonas species
  • Pilus synthesis - E. coli, Pseudomonas species
  • Toxin production - Bacillus anthracis
  • Tumor formation in plants - Agrobacterium species

Antibiotic resistance major issue

• Original genes for Abx resistance evolved slowly as part of the bacterial wars and this takes a long time
• Stealing a readymade solution occurs rapidly
• Plasmids can contain multiple resistance genes for spread multi-resistance

The Downside of picking up DNA

• Transformation and other DNA stealing are rare events
• Viruses and viroids are real problems
• Bacteria must be ready to fight back against bad DNA

Restriction Enzymes

• Bacterial enzymes that cut up "foreign" DNA, while not cutting Chromosomal DNA
• Bacteria differentiates with the help of enzymes
• Double stranded DNA cuts at a particular sequence which is a sequences that different bacteria have with bacteria modifies own DNA sequences (methylates) to make resistant to enzyme
• Virus injects viral DNA, this starts to take over the cell, at the same time restriction enzymes try to cut up viral DNA, this renders it inactive
• Race, infect enough at times the virus wins

Ongoing War

• Host with mDNA can kill most non-methylated phages
• But if one phage succeeds all progeny have mDNA
• Subsequently, non-mDNA hosts have an advantage...

Restriction Enzymes in the Laboratory

• Commonly lused recognize a 4-6 bp sequence of double stranded DNA and most sequences are Palindromes which read the same on both strands, 5->3
• Named for bacteria isolated from; for example: Alul - Arthrobacter luteus (recognition sequence: 5' AGCT 3') BamHI - Bacillus amyloliquefaciens H (recognition sequence: 5' GGATCC 3') EcoRI - Escherichia coli RY13 (recognition Sequence: 5' GAATTC 3')
• Researchers use specific restriction enzymes to cut DNA in known places
• Some cut strands offset from center, known as sticky ends and this is useful in molecular cloning

Molecular Cloning

• Procedure involving the taking of DNA of Interest, inserting it into a high copy plasmid with a restriction enzyme, making DNA Probes, and making proteins (synthesized in Bacteria)

CRISPR System

• CRISPR systems include small segments of phage DNA that recognize the specific DNA if it invades the cell again
• First invasion: complex of Cas proteins cuts DNA into short fragments
• They are inserted into chromosome at CRISPR array where the integrated DNA fragment is called a spacer
• Subsequent invasion: transcription of CRISPR array generates crRNAs that direct DNA-cutting enzymes to invading DNA, similar to the antibody systems in animals
• This memory is embedded in the DNA and passed to progeny

CRISPR in the Laboratory

• You can make Guide RNA to target any sequence of interest
• Can inactivate a bad gene, or edit to restore correct function

New, Laboratory Variations

• dCas9 (“Dead”) is a new laboratory variation which does not cut DNA, but still uses RNA guide to bind to specific DNA sequence
• May block RNA polymerase from transcribing the gene and turn the gene off
• May deliver molecules to specific chromosome locations:
  • Carry an activator to turn the gene on
  • Fluorescent marker to locate a gene

PUBMED

• PUBMED is the Main Database of the National Library of Medicine
• PUBMED is reputable with Peer-Reviewed Journal Articles and Books and containing Search functionality with Full-Text available for free, online

PCR

• Principles:
  • DNA polymerase can copy ANY DNA, but must have a priming oligonucleotide; can make synthetic oligo as primer, but must know that sequence
• Process:
  • "melt" DNA with heat/buffer
  • "anneal" DNA primers with right conditions
  • Run polymerase reaction get twice as much DNA and is repeated

DNA sequencing

• Involves radioactive primer, DNA polymerase, dATP, dCTP, dGTP, dTTP in for different reaction tubes, each with one dideoxynucleoside triphosphate and the reaction products are separated by electrophoresis on polyacrylamide gel and identified by autoradiography

Microarray Probes

• Microarray Probes involves making "probe" a DNA oligo complementary to targeted sequence
• Labeled with radioactivity, fluorescence, enzyme) hybridized to separated DNA sequences on Gel, Plate, and Synthetic grid...