MAKING A RECOMBINANT PLASMID Transformation of Bacteria using plasmids & restriction enzymes By Kelly Riedell Brookings Biology Blue edged slides from slide show by Kim Foglia http://explorebiology.com Recombinant Plasmid activity modified from http://biologycorner.com/worksheets/DNA_analysis.html

Big Idea 3: Living systems store, retrieve, transmit and respond to information essential to life processes. Enduring understanding 3.A: Heritable information provides for continuity of life. Essential knowledge 3.A.1: DNA, and in some cases RNA, is the primary source of heritable information e. Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. To foster student understanding of this concept, instructors can choose an illustrative example such as: • Electrophoresis • Plasmid-based transformation • Restriction enzyme analysis of DNA • Polymerase Chain Reaction (PCR) f. Illustrative examples of products of genetic engineering include: • Genetically modified foods • Transgenic animals • Cloned animals • Pharmaceuticals, such as human insulin or factor

BACTERIA HAVE PLASMIDS Image from: https://pmgbiology.files.wordpress.com/2014/06/20_02a.jpg BACTERIA HAVE PLASMIDS Small, extra-chromosomal, self-replicating, rings of double stranded DNA that can carry genes for conjugation and antibiotic resistance Can be used in genetic engineering to transfer genes from other organisms into bacteria

GENETICALLY MODIFIED ORGANISMS SLIDE MODIFIED FROM KIM FOGLIA http://explore biology.com GENETICALLY MODIFIED ORGANISMS “Golden” rice (Vitamin A) Bt-corn-resists insect pests Frost resistant bacteria Toxic cleanup bacteria

Genetically modified organisms (GMO) SLIDE MODIFIED FROM KIM FOGLIA http://explore biology.com Genetically modified organisms (GMO) enabling plants to produce new proteins Protect crops from insects: BT corn corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn) Extend growing season: fishberries strawberries with an anti-freezing gene from flounder Improve quality of food: golden rice rice producing vitamin A improves nutritional value For example, a transgenic rice plant has been developed that produces yellow grains containing beta-carotene. Humans use beta-carotene to make vitamin A. Currently, 70% of children under the age of 5 in Southeast Asia are deficient in vitamin A, leading to vision impairment and increased disease rates.

How can plasmids help us? SLIDE FROM KIM FOGLIA http://explore biology.com How can plasmids help us? A way to get genes into bacteria easily insert new gene into plasmid insert plasmid into bacteria = vector bacteria now expresses new gene bacteria make new protein transformed bacteria gene from other organism recombinant plasmid vector plasmid cut DNA + glue DNA

 How do we cut DNA? Restriction enzymes restriction endonucleases SLIDE FROM KIM FOGLIA http://explore biology.com How do we cut DNA? Restriction enzymes restriction endonucleases discovered in 1960s evolved in bacteria to cut up foreign DNA “restrict” the action of the attacking organism protection against viruses & other bacteria bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA 

RESTRICTION ENDONUCLEASES ENZYMES cut DNA at a specific “palindrome” sequences = restriction site Produce sticky ends Different kinds of DNA cut with same enzyme will have the same “sticky ends” and can be joined Different enzymes recognize & cuts different sequences 100’s of different enzymes named after organism they are found in EcoRI, HindIII, BamHI, SmaI

http://www.slic2.wsu.edu:82/hurlbert/micro101/images/LigaseAnimation6.gif

Plasmid Image from: http://faculty. ccbcmd PLASMID DNA Cut the PLASMID DNA strips apart and tape them together in one long strip. Use the numbers to help you match ends in the correct sequence. Letters should all be facing in the same direction. Tape the two ends of the long strip together to form a circular plasmid with the letters FACING OUT Dark area represents ORIGIN OF REPLICATION

HUMAN INSULIN DNA Cut the HUMAN INSULIN DNA strips apart and tape them together in one long strip. Use the numbers to help you match ends in the correct sequence. Letters should all be facing in the same direction. Remember: Human chromosomes are LINEAR! Dark area represents INSULIN GENE http://nat5biopl.edubuzz.org/_/rsrc/1363098844922/unit-1-cell-biology/6-genetic-engineering/IMG_0226.JPG?height=204&width=400

Compare the sequences of base pairs on each of the RESTRICTION ENZYME SEQUENCES provided to the DNA sequences on the PLASMID DNA. If you find the same sequence, MARK THE LOCATION and the CUT PATTERN on BOTH THE ENZYME and the PLASMID DNA using the same color. Repeat comparison using RESTRICTION ENZYME SEQUENCES and HUMAN DNA sequence.

OBJECTIVE FIND a RESTRICTION ENZYME SEQUENCE that will: Cut the PLASMID DNA in ONLY ONE LOCATION NOT cut the ORIGIN OF REPLICATION SEQUENCE in the PLASMID DNA https://3c1703fe8d.site.internapcdn.net/newman/gfx/news/2015/bacteriaused.gif

IF CUT PLASMID IN MORE THAN ONE PLACE

IF CUT PLASMID IN MORE THAN ONE PLACE

IF CUT PLASMID IN MORE THAN ONE PLACE If plasmid has ORIGIN OF REPLICATION plasmid can copy itself when cell divides Can grow a whole vat of bacteria producing human insulin. Bacteria that have plasmids w/o ORIGIN OF REPLICATION can divide, but plasmid can’t copy itself. As cells divide, get more and more cells w/o human insulin gene.

OBJECTIVE ALSO FIND a RESTRICTION ENZYME SEQUENCE that will: NOT cut the INSULIN gene Make one cut in the HUMAN DNA on either side of AND as close as possible to the INSULIN GENE sequence

CREATE YOUR PLASMID After you have examined ALL of the enzyme sequences Provided, decide the best one to use. Make cuts on both the PLASMID and HUMAN DNA to create “sticky ends” as shown on the ENZYME sequence. USE LIGASE to join the “sticky ends” of the PLASMID and INSULIN gene together. CONGRATULATIONS! You have created a recombinant plasmid which can now be inserted into a bacterial cell to produce human insulin!

http://www.sliderbase.com/images/referats/694b/(6).PNG

Grow bacteria…make more SLIDE FROM KIM FOGLIA http://explore biology.com Grow bacteria…make more transformed bacteria gene from other organism + recombinant plasmid vector plasmid grow bacteria harvest (purify) protein

PROBLEM !!!!!!! https://wikispaces.psu.edu/download/thumbnails/42338209/image-1.jpg?version=2&modificationDate=1248490013000&api=v2 EUKARYOTIC mRNA is edited before it is translated into a protein NO POST TRANSCRIPTIONAL PROCESSING IN PROKARYOTES - NO INTRONS - NO EDITING ENZYMES CAN’T USE EUKARYOTIC DNA DIRECTLY NEED EDITED mRNA to make human proteins

reverse transcriptase “TRICKY” SOLUTION https://www.msu.edu/course/isb/202/ebertmay/images/HIV%20virus.png Don’t start with human DNA… Use mRNA for insulin to make cDNA(complementary DNA) copy of the gene without the introns How do you go from RNA  DNA? reverse transcriptase from RNA viruses retroviruses cDNA reverse transcriptase IMAGES FROM KIM FOGLIA

REVERSE TRANSCRIPTASE http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/AAAreverse.jpg

Plasmid- 1 cut NO cuts on human DNA Plasmid- 1 cut 3 cuts on human DNA Plasmid- 1 cut 2 close cuts on human DNA Plasmid- 2 cuts 2 cuts on human DNA Plasmid- 1 cut CUTS INSULIN gene