Part 2 Molecular Biology IGEM 20 June 2006

Part 2: Molecular Biology IGEM, 20 June 2006

Proteins • Structural and catalytic components of cells are mainly proteins. • Linear polymers of 20 different amino acids joined by amide linkages. • Spontaneously fold into 3 D shapes (tertiary structure). • Conventionally written from N to C terminus using three-letter or oneletter codes. • May have multiple identical or different subunits. Ribbon diagram of protein structure (H 2 N)-Met-Pro-Asp-Thr-Ser-Phe-Ser-Asn-Pro-Gly-Leu-(COO-) MPDTSFSNPGLFTPLQ…

Structure of DNA • • • Protein sequences are encoded in DNA. Linear polymer of four types of base: G, A, T, C. Strands directional: written from 5’ to 3’ end. Generally two antiparallel strands, hydrogen-bonded. G on one strand binds C on other; likewise A binds T. E. coli DNA forms a circle about 4. 5 million base pairs (Mbp) in length, encoding about 4000 proteins. 5’-AAGGAGGCAGAATGCCGGACACGTCCTTCTCCACCCCAGGCCTG-3’ 3’-TTCCTCCGTCTTACGGCCTGTGCAGGAAGAGGTGGGGTCCGGAC-5’

Encoding of proteins in DNA • • • Protein structure is determined by amino acid sequence. Encoded in DNA in ‘open reading frames’ (ORFs). Three DNA bases (one codon) specify an amino acid. There are 64 possible codons. Some amino acids are specified by a single codon, others by 2, 3, 4 or 6 different codons (degenerate). • The beginning of an ORF is indicated by the start codon ATG (rarely, GTG) which specifies N-formyl methionine. • The end of an ORF is indicated by a stop codon: TAA, TGA or TAG.

Transfer of sequence information from DNA to protein • TRANSCRIPTION: one strand of DNA (sense or coding strand) is copied as a single stranded messenger RNA (m. RNA). • TRANSLATION: the sequence information encoded in the m. RNA is used to generate a protein. DNA transcription m. RNA translation protein

Transcription • DNA sequence is copied to RNA by RNA Polymerase (RPol). • RPol initially binds to DNA at a promoter sequence, which specifies which strand is to be copied. • RPol binds to the promoter sequence and begins to make an RNA copy of the specified strand, from 5’ to 3’, using the other strand (template or antisense strand) as a template. • RNA structure is similar to DNA except that T (thymine) is replaced by U (uracil). • Transcription continues until a termination sequence is reached, at which point RPol dissociates from the DNA.

Translation • Sequence information in m. RNA is translated into protein sequence by ribosomes. • Ribosomes bind to m. RNA at ribosome binding sites (rbs), which partially match AAGGAGG. • If a start codon AUG (or GUG) is within 5 to 10 bp of a rbs, the ribosome will begin making a protein. • Translation continues until a stop codon is reached, at which point the ribosome dissociates from the protein and m. RNA. • The protein spontaneously folds into its final shape. • Multiple proteins can be encoded by one m. RNA if each has its own rbs. A group of ORFs expressed on a single m. RNA is an operon.

Control of expression • At any given time, only a subset of the 4000 ORFs are being expressed. • This is mainly controlled by switching promoters on and off in response to various stimuli. • Some promoters are controlled by activator proteins, which must bind near the promoter to recruit RPol. • Some promoters are controlled by repressor proteins, which can bind to the promoter to prevent RPol binding.

Positive Control • The promoter is switched on in response to the presence of a small molecule (inducer). • The inducer may bind to an activator protein, causing it to bind to the promoter, or to a repressor protein, causing it to dissociate from the promoter. RPol activator RPol repressor inducer RPol Inducer-activator complex binds promoter and recruits RPol inducer repressor Repressor binds promoter only when inducer is not present.

Negative control • A promoter is switched off in response to the presence of a small molecule (corepressor). • The corepressor may bind to an activator protein, causing it to dissociate from the promoter, or to a repressor protein, causing it to bind the promoter. activator RPol corepressor Corepressor-activator complex does not bind to promoter and recruit RPol. Repressor-corepressor complex binds promoter and blocks RPol.

Sequence of a gene/operon • • Promoter (eg TTGACA … TATA…). Operator site (binds activator or repressor protein). Ribosome binding site (eg AAGGAGG). Start codon (ATG). Open reading frame. Stop codon (TAG, TGA, TAA). May be other {rbs-start-ORF-stop} combinations. Transcription termination sequence. start codon promoter + operator site stop codon ORF termination sequence

Genetic Engineering • Techniques for transfer of genes from one organism to another. Considerations include: • Host (background) • Vector • Selectable marker • Restrictions sites for DNA insertion • Reporter genes

Host strains • • • Many specialized host strains of E. coli are available. Disabled: cannot colonise intestines. Mutation in rec. A: DNA cannot recombine. Other specialized mutations for particular purposes. Most common host strains (eg DH 5 a, JM 109) are derived from strain K 12.

Vectors • A way to carry DNA into the cell. • Usually plasmids: loops of DNA which are replicated independently of the main chromosome. • Require an origin of replication (ori). • Two plasmids with the same ori cannot be stably maintained in the same cell. • Usually carry one or more antibiotic resistance genes to select for cells which have the plasmid. Most commonly ampicillin resistance (ap. R, bla, beta lactamase).

Restriction sites • DNA can be cut at specific 6 bp sequences by restriction enzymes (type 2 restriction endonucleases). • May leave ‘sticky’ or ‘blunt’ ends. • Sites are usually palindromic (dyad symmetry). • Eg Eco. RI: GAATTC; Hind. III: AAGCTT 5’-NNNGAATTCNNN-3’ 3’-NNNCTTAAGNNN-5’ 5’-NNNG-3’ 5’-AATTCNNN-3’ 3’-NNNCTTAA-5’ 3’-GNNN-5’

Ligation • Compatible sticky ends tend to stick together by base pairing, especially at low temperatures. • Ends can be covalently joined using T 4 DNA ligase. 5’-NNNGAATT-3’ 5’-CNNN-3’ 3’-NNNC-5’ 3’-TTAAGNNN-5’ 5’-NNNGAATTCNNN-3’ 3’-NNNCTTAAGNNN-5’

Inserting a new gene into a plasmid • Digest plasmid and source DNA with the same pair of restriction enzymes. • Mix the DNA together and add DNA ligase. • A fraction of the resulting product molecules will contain the plasmid with the new gene inserted. Eco. RI Hind. III Eco. RI digest Hind. III Eco. RI mix and ligate Hind. III

Introducing a plasmid into a host • E. coli cells will take up DNA after being treated to make them competent. • Mix competent cells with plasmid or ligation to allow DNA uptake (transformation). • Spread cells on an agar plate containing the correct antibiotic (eg ampicillin). • Only cells with the plasmid will survive exposure to the antibiotic and grow to produce colonies. • Each colony is assumed to grow from a single transformed cell. Colonies on a plate

Selecting clones with the recombinant plasmid. • Many modern vectors use ‘blue-white’ selection. • Insertion of a new piece of DNA into the multi-cloning site disrupts a chromogenic gene (lac. Z’). • Addition of IPTG (inducer) and X-gal (chromogenic substrate) to the plates results in blue colonies if lac. Z’ is intact, white if it has been disrupted. • More generally, prepare plasmid DNA (miniprep) from each clone, digest with restriction enzymes, and analyse by agarose gel electrophoresis.

DNA synthesis • In contrast to RNA synthesis, DNA synthesis requires a primer: short piece of DNA (oligonucleotide, oligo) which binds to the template strand. • DNA Polymerase extends the primer, using the template strand as a basis for choosing which base to add. The new DNA has the same sequence as the other strand. 5’-GAATCCTGTAATCGGATACGGCGATTACGA T T primer new DNA G DNA pol C T 5’-GAATCCTGTAATCGGATACGGCGATTACGA AGTAGCCGGT-3’ 3’-CTTAGGACATTAGCCTATGCCGCTAATCGATCATCGGCCA-5’

DNA sequencing • Sanger dideoxynucleotide chain termination method. • A primer is extended in the presence of fluorescently labelled chain-terminating dd. ATP, dd. CTP, dd. GTP and dd. TTP substrates. • Prematurely terminated strands of defined lengths are detected by fluorescence following capillary electrophoresis and this information is used to assemble the sequence. • About 800 bases can be read from one primer new DNA pol Green product 34 bases long ending in A. 5’-GAATCCTGTAATCGGATACGGCGATTACGA 3’-CTTAGGACATTAGCCTATGCCGCTAATCGATCATCGGCCA-5’

Polymerase Chain Reaction • Can specifically amplify the region of DNA between two short stretches of known sequence. • Can modify the sequence at the ends. • Requires two primers binding opposite strands. • Exponential amplification process. Original DNA Primers New DNA (first round) New DNA (subsequent)

Reporter genes • • Genes whose expression can be easily monitored. lac. Z – produces blue colour with X-gal. ido. A: produces blue colour. xyl. E: produces yellow colour with catechol. lux. AB: glows if decanal added. lux. CDABE: glows. luc: glows brightly if luciferin added. gfp, rfp, cfp, yfp, ofp: cells fluorescent when viewed under UV. • None of these systems is easy to turn off.

Safety Considerations • No genetically modified organisms may be produced without a valid risk assessment approved in advance by SBS Genetic Modification and Biological Safety Committee. • Work using disabled E. coli hosts and harmless transgenes is category 1 and may be performed in category 1 laboratories. • Best practice must be followed to ensure that GMO do not leave the laboratory unless properly contained. • All potentially contaminated material must be sterilised prior to disposal. All working surfaces must be disinfected and proper protective equipment worn.

The End • This concludes your introduction to the wonderful world of Genetic Modification.