A human gene can be made to work inside a bacterium. Getting it there needs a copy of the gene, a carrier plasmid, a set of enzymes that cut and join DNA, a promoter the host recognises and a marker that shows which cells have taken it up.
Recombinant DNA is DNA made by joining together DNA from two different sources, often from different species. Genetic engineering is the deliberate manipulation of genetic material to modify specific characteristics of an organism; often a gene is transferred into another organism so that it is expressed there. The gene can be extracted from donor DNA with restriction endonucleases, made from mRNA with reverse transcriptase and DNA polymerase, or synthesised chemically from nucleotides. A plasmid cut with the same restriction endonuclease has complementary sticky ends; DNA ligase seals the gene into it. A promoter that the host's RNA polymerase recognises is transferred with the gene so that it is transcribed, and a marker gene coding for a fluorescent protein such as GFP shows which cells are expressing the new DNA.
Fig. 1: a gene and a plasmid cut with the same restriction endonuclease are joined by DNA ligase to form a recombinant plasmid carrying a promoter and a fluorescent marker gene
Section 01
Three ways to get the gene.
Pick each source in turn and press Run. From donor DNA, the restriction endonuclease drops onto the DNA and the yellow gene lifts out. From mRNA, reverse transcriptase builds an orange cDNA strand and then a blue second strand appears. Chemical synthesis adds nucleotides one at a time to a designed sequence.
Source of the gene
How it is done
Enzymes
Introns?
extracted from the DNA of a donor organism
DNA is cut at specific base sequences (restriction sites) on either side of the gene and the fragment is separated out
restriction endonuclease
yes, if the donor is eukaryotic
synthesised from the mRNA of a donor organism
mRNA is taken from cells that make the protein (for example β cells of the pancreas for insulin) and copied into complementary DNA (cDNA)
reverse transcriptase makes single-stranded cDNA; DNA polymerase makes the second strand
no: mRNA has already been spliced
synthesised chemically from nucleotides
if the base sequence is known, or worked out from the amino acid sequence, a machine joins nucleotides in order; short pieces are joined into the whole gene
none needed to build the strand
no; codons can also be chosen to suit the host
Restriction endonucleases come from bacteria, where they cut up the DNA of invading viruses. Each recognises a specific, usually palindromic, sequence of 4-8 base pairs. Many make staggered cuts that leave short single-stranded sticky ends; some make blunt ends.
Why cDNA is useful: bacteria cannot remove introns, so a eukaryotic gene cut straight from the chromosome would give a faulty protein in a bacterium. A gene made from mature mRNA has no introns.
Cells that make a lot of the protein contain many copies of its mRNA, which makes them a good starting point.
Examiner trap
Reverse transcriptase makes DNA from an RNA template (the reverse of transcription). It does not make mRNA from DNA, and it is not the enzyme that cuts DNA.
Section 02
Building and checking a recombinant plasmid.
Press Next step to work through all five stages. Watch the counter go from 1 of 5 to 5 of 5. At step 4 count how many of the 16 bacteria take up a plasmid, and at step 5 see which of them glow under ultraviolet light.
Plasmid: a small circle of double-stranded DNA found in bacteria, separate from the main chromosome and able to replicate on its own. It acts as a vector that carries the gene into the host cell.
Same restriction endonuclease: cutting the plasmid and the donor DNA with the same enzyme gives complementary sticky ends, which pair up by hydrogen bonding between bases.
DNA ligase forms phosphodiester bonds between nucleotides, sealing the sugar-phosphate backbone so the gene is permanently part of the plasmid. The result is a recombinant plasmid.
Promoter: a region of DNA next to a gene where RNA polymerase (with transcription factors) binds to start transcription. A gene taken from a eukaryote may lack a promoter that a bacterium's RNA polymerase can recognise, so a suitable promoter is inserted with the gene; without it the gene is present but not expressed.
Taking up the plasmid: bacteria are treated with calcium ions and a heat shock so that some take up plasmids. Only a small fraction do, so the cells that did must be identified.
Fluorescent marker genes: a gene for green fluorescent protein (GFP), from a jellyfish, is inserted next to the desired gene. Bacteria that glow green under ultraviolet light have taken up the recombinant plasmid and are expressing it, so they can be picked out and grown on. This is quicker and safer than older methods that relied on antibiotic resistance genes.
Examiner trap
Seeing the gene in the cell is not enough: the marker shows that the inserted DNA is being expressed (transcribed and translated), because the green colour only appears if a fluorescent protein has been made.
Stage 1 · Learn
Check what the sim just showed you
Four quick checks. Each correct answer earns XP and records this skill on your profile.
Quick check+10 XP
In the simulator's 'from mRNA' option, which enzyme builds the first (orange) strand of cDNA?
Quick check+10 XP
At step 2 of the recombinant DNA simulation, why is the plasmid cut with the same restriction endonuclease as the donor DNA?
Quick check+10 XP
At step 4, only 4 of the 16 bacteria take up a plasmid. What does the simulator use at step 5 to find them?
Quick check+10 XP
The simulator joins the gene to a promoter before inserting it. What does the promoter do?
Section 03
The toolkit in one table.
Every gene transfer uses the same small set of tools. Learn what each one does, because exam questions often give one step and ask for the enzyme.
Tool
Role in transferring a gene
restriction endonuclease
cuts DNA at a specific base sequence, cutting out the gene and opening the plasmid with matching sticky ends
reverse transcriptase
makes single-stranded cDNA using mRNA as a template
DNA polymerase
makes the second strand to give double-stranded cDNA (and copies DNA in PCR)
DNA ligase
joins the gene into the plasmid by forming phosphodiester bonds in the sugar-phosphate backbone
plasmid
vector that carries the gene into the host cell and is copied when the bacterium divides
promoter
allows the host's RNA polymerase to bind, so the gene is transcribed
marker gene (GFP)
codes for a fluorescent product that shows which cells contain and express the recombinant DNA
Stage 2 · Exam
Exam-style questions
Unlocks once the four checks above are done. Worth more XP, written to A Level Paper 4 standard.
Finish the four checks above to unlock the exam questions
Exam style+20 XP
Which statement defines recombinant DNA?
Exam style+20 XP
A human gene is cut directly from a chromosome and put into bacteria, but the protein made is not functional. What is the most likely reason?
Exam style+20 XP
Which enzyme forms phosphodiester bonds to seal a gene into a cut plasmid?
Exam style+20 XP
Why is a gene for a fluorescent protein used as a marker in genetic engineering?
Skill unlocked
Recombinant DNA, mastered.
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