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Biological Dynamics Group
Central Dogma:
DNA->RNA->Protein
Genes I
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Genes consist of DNA that contains a
promoter, a DNA sequence that enables a
gene to be transcribed , and a coding
sequence, which determines what the gene
produces.
In our case, the riHC (nucleoside hydrolase)
enzyme is produced during translation.
DNA
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DNA is a long polymer of simple units called
nucleotides.
DNA bonding
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Phosphodiester bonds (covalent)
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Complementary (noncovalent) bonding between
bases
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Between 3’OH and 5’phosphate on the adjacent
nucleotide
A hydrogen bonds with T (or U in RNA).
G hydrogen bonds with C.
Forming a double helix with antiparallel strands
We use restriction enzymes to cut the riHC DNA.
Genetic (DNA) Elements
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Genome
Chromosome
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E. coli has one.
Extrachromosomal genetic elements
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Plasmids
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Usually small, circular dsDNA (10kbp)
We can cut a plasmid and insert the rihC
gene by a ligation reaction.
On the way to protein!
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The plasmid must have a promoter sequence
recognized by RNA polymerase, which then initiates
transcription.
DNA->RNA=transcription
The coding sequence is copied in a process called
transcription, producing a RNA copy of the gene's
information.
This RNA can then direct the synthesis of proteins or
translation via the genetic code.
RNA->Protein=Translation
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RNA that will be translated into protein is
called messenger RNA (mRNA).
There are two other types of RNA that are
important to translation.
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tRNA transfers the next amino acid (aa) to the
growing peptide chain.
rRNA is part of the ribosome complex.
Translation and Mutations
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The ribosomes “read” triplets of the mRNA.
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The genetic code is a triplet code.
If a change (mutation) occurs in the DNA
code, then a different aa may be
incorporated into the protein chain.
To change the active site of rihC we have
engineered specific point mutations.
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A point mutation is a single base change.
PCR
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We engineer point mutations using
the polymerase chain reaction
(PCR)
PCR is DNA replication in vitro.
dsDNA is “melted”
ssDNA primers bind to C’ regions
DNA polymerase extends the
template
Repeated through 30 cycles
Site-directed mutagenesis
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The primers containing the desired mutation
are utilized.
A mis-match during the first cycle in binding
the template DNA strand, after that first round
occurs.
After successive cycles, the mutated strand
would exponentially grow, and after 25
cycles, would outnumber the original 8
million : 1.
Ligation and Transformation
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The mutated PCR product is then ligated into
a linearized plasmid with C’ ends via the
enzyme DNA ligase that forms
phosphodiester bonds.
The ligated DNA is then transformed into a
special strain of E. coli that will express the
mutated rihC gene.
rihC Nucleic Acid sequence 27293..28207
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27241 taagttatgc gaaaatgccg gtcttgttac cggcattttt tatggagaaa acatgcgttt
27301 acctatcttc ctcgatactg accccggcat tgacgatgcc gtcgccattg ccgccgcgat
27361 ttttgcaccc gaactcgacc tgcaactgat gaccaccgtc gcgggtaatg tctcggttga
27421 gaaaactacc cgcaatgccc tgcaactgct gcatttctgg aatgcggaga ttccgctcgc
27481 ccaaggggcc gctgtgccac tggtacgcgc accgcgtgat gcggcatctg tgcacggcga
27541 atcgggaatg gctggctacg actttgttga gcacaaccga aagccgctcg ggataccggc
27601 gtttctggcg attcgggatg ccctgatgcg tgcaccagag cctgttaccc tggtggccat
27661 cggcccgtta accaatattg cgctgttact ttcacaatgc ccggaatgca agccgtatat
27721 tcgccgtctg gtgatcatgg gtggttctgc cggacgcggc aactgtacgc caaacgccga
27781 gtttaatatt gctgccgatc cagaagctgc tgcctgtgtc ttccgcagtg gtattgaaat
27841 cgtcatgtgc ggtttggatg tcaccaatca ggcaatatta actcctgact atctctctac
27901 actgccgcag ttaaaccgta ccgggaaaat gcttcacgcc ctgtttagcc actaccgtag
27961 cggcagtatg caaagcggct tgcgaatgca cgatctctgc gccatcgcct ggctggtgcg
28021 cccggacctg ttcactctca aaccctgttt tgtggcagtg gaaactcagg gcgaatttac
28081 ctcaggcacg acggtggttg atatcgacgg ttgcctgggc aagccagcca atgtacaggt
28141 ggcattggat ctggatgtga aaggcttcca gcagtgggtg gctgaggtgc tggctctggc
28201 gtcgtaacct
Primers for site-directed mutagenesis
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14 -rihC F-ATA CTG ACC CCG GAA TTG CCG ATG
rihC R-CAT CGG CAA TTC CGG GGT CAG TAT
15 -rihC F-ATA CTG ACC CCG GAA TTG CCG ATG
rihC R-GGC GGC GTC AAT TCC GGG GTC AGT AT
164-rihC F-CGC CGA GGC TAA TAT TGC TGC
rihC R-GCA GCA ATA TTA GCC TCG GCG
222-rihC F-CAC TAC GCT AGC GGC AGT ATG
rihC R-CAT ACT GCC GCT AGC GTA GTG
233-rihC F-TGC GAA TGG CCG ATC TCT GCG
rihC R-CGC AGA GAT CGG CCA TTC GCA
234-rihC F-TGC GAA TGC ACG CCC TCT GCG
-rihC R-CGC AGA GGG CGT GCA TTC GCA
241-rihC F-ATC GCC TGG GCG GTG CGC CCG GA
-rihC R-TCC GGG CGC ACC GCC CAG GCG AT
242-rihC F-ATC GCC TGG CTG GCG CGC CCG GA
-rihC R-TCC GGG CGC GCC AGC CAG GCG AT
Genetic code -> AA code
One letter code
rihC Amino Acid sequence
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0
MRLPIFLDTD PGIDDAVAIA AAIFAPELDL
QLMTTVAGNV SVEKTTRNAL QLLHFWNAEI
61 PLAQGAAVPL VRAPRDAASV HGESGMAGYD
FVEHNRKPLG IPAFLAIRDA LMRAPEPVTL
121 VAIGPLTNIA LLLSQCPECK PYIRRLVIMG
GSAGRGNCTP NAEFNIAADP EAAACVFRSG
181 IEIVMCGLDV TNQAILTPDY LSTLPQLNRT
GKMLHALFSH YRSGSMQSGL RMHDLCAIAW
241 LVRPDLFTLK PCFVAVETQG EFTSGTTVVD
IDGCLGKPAN VQVALDLDVK GFQQWVAEVL
301 ALAS