1. Bacterial Transformations and PCR (Part I of II)
Exercises 5 and 6
Bacterial Transformations and PCR (Part I of II)

2. Learning Outcomes Describe the structure and function of plasmids
Summarize the steps and requirements for bacterial transformations
Perform bacterial transformation
Isolate genomic DNA from check swab
Summarize the steps and requirements for PCR
Prepare a PCR sample

3. Procedures PLASMID STUFF
5.1: Digest plasmid with restriction enzymes: SKIP
5.2: Transform Bacteria with pGLO plasmid
5.3: Bacterial Growth and Selection
GENOMIC STUFF
5.4: Isolate your DNA
5.5: Prepare your DNA for PCR

4. Plasmids Some bacteria have smaller rings of DNA called plasmids
Plasmids replicate separately
from the bacterial chromosome
Bacterium with a plasmid
plasmid
Engineered plasmid
Antibiotic resistance gene

5. Transforming Bacteria (5.2)
Bacteria can be made ‘chemically competent’… meaning they will take up plasmids from the environment
Begin by treating E. coli with a high concentration of calcium ions
Makes their phospholipid bilayer ‘leaky’’
Are now ‘chemically competent’
We will provide you with chemically competent bacteria!
We will provide you with the pGLO plasmid

6. We will give you chemically competent E. coli
We will give you the pGLO plasmid
You will transform bacteria with the pGLO plasmid: Heat shock followed by recovery
Only a few cells will successfully incorporate a plasmid.

7. Your plasmid: pGLO Components of your pGLO plasmid
ori (origin for DNA replication)
bla (ampicillin resistance)
Multiple Cloning Site (MCS)
GFP gene
araC gene
bla gene: codes for the enzyme beta-lactamase: gives transformed bacteria resistance to the beta-lactam family of antibiotics (such as ampicillin)
A bacterium that incorporates the pGLO plasmid (is transformed) has what ‘new’ features?

8. GFP Gene
GPF gene codes for the green fluorescent protein. It is composed of 238 amino acids and fluoresces bright green when exposed UV light.
Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein isolated from the jellyfish Aequorea victoria
No UV UV

9. araC protein (produced by the araC gene)
In the absence of the sugar arabinose, the araC protein prevents transcription of the GFP gene.
In the presence of arabinose, the araC protein no longer prevents transcription of the GFP gene.
The result…. RNA polymerase transcribes the GFP gene and the fluorescent protein is produced.

10. Bacteria Require a growth medium (LB media)
10 g tryptone (a.a.)
5 g yeast extract (nutrients)
10 g NaCl (osmotic balance)
Elective growth compounds may also be added to the media, such as antibiotics.
Pour the media into a petri dish
Individual microorganisms added using a technique known as "streaking”
Loop

11. Bacterial lawn vs colonies
A bacterial lawn: forms when all the individual colonies merge to form a field or mat of bacteria.
A colony of bacteria: all bacteria in the colony are descendants of a single mother cell (bacteria all genetically identical in a colony)

12. 5.3 Bacterial Growth + LB +LB +LB + AMP +AMP – AMP + ARA -ARA – ARA
You will be given three different plates:
+ LB
+ AMP
+ ARA
+LB
+AMP
-ARA
+LB
– AMP
– ARA 1 2 3
On which plate(s) do you expect to see a lawn?
On which plates(s) do you expect to see colonies?
On which plate(s) do you expect transformed bacteria to grow? (genotype +plasmid)
On which plate(s) do you expect GFP to be produced?
Will transformed bacteria always fluoresce?
On which plate(s) do you expect the bacteria to fluoresce? (phenotype +fluoresce)

13. WHAT DETERMINES PHENOTYPE?
1) Only those bacteria that have been transformed will survive on plates with ampicillin.
2) Arabinose allows for the transcription of the gene that codes for GFP.
3) Fluoresce ONLY in UV light
With the right genotype AND environmental conditions….you get glowing bacteria!
Genotype + Environment = PHENOTYPE

14. Genomic DNA Isolation 5.4.3: Collect Cells (swab)
5.4.5: Disrupt Cell membranes (Genomic Lysis Buffer)
5.4.9: Spin to discard cell debris, DNA Binds to matrix
and : Removing impurities (Wash buffer)
5.4.16: DNA released from matrix, collected

15. Isolate Human DNA from Buccal Samples
You will isolate all 46 of your chromosomes during lab from cheek cells using a kit (5.4)
However, you will study only one small region (locus) on chromosome 1 (D1S80 locus).
D1S80 locus located on distal end of short arm ‘p’ (1p36)
What is at this locus?
Chromosome 1 is the designation for the largest human chromosome. Humans have two copies of chromosome 1, as they do with all of the autosomes, which are the non-sex chromosomes. Chromosome 1 spans about 249 million nucleotide base pairs, which are the basic units of information for DNA.[2] It represents about 9% of the total DNA in human cells.[3]
Identifying genes on each chromosome is an active area of genetic research. Chromosome 1 is currently thought to have 4,316 genes, exceeding previous predictions based on its size.[2] It was the last completed chromosome, sequenced two decades after the beginning of the Human Genome Project.

16. The Human Genome https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1186966/
A variable number tandem repeat (or VNTR) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length (number of repeats) among individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting.

17. D1S80 Minisatellite or VNTR’s (variable number tandem repeats)
16 bp repeat unit
[GAGGACCACCGGCAAG]
16 bp repeat unit repeats 20 times
Sam
16 bp repeat unit repeats 15 times
16 bp repeat unit repeats 20 times
Beth
16 bp repeat unit repeats 20 times

18. D1S80 28 known alleles in human population 14 - 41 repeats
16(r) +146 = 370 to 802 bp
Individuals can be homozygous or heterozygous
80% of population is heterozygous

19. D1S80
How will you figure out the size of your alleles and number of repeats?
How will you figure out whether you are homozygous or heterozygous?
PCR and Gel Electrophoresis!

20. The Steps of Polymerase Chain Reaction
1. Denaturation – heating the mixture to 95°C separates the two strands of the DNA.
2. D1S80 Primer annealing – cooling the mixture allows the D1S80 primers to bond, or anneal, to complementary sections of single-stranded target DNA flanking D1S80 locus.
3. Extension – heating the mixture to 72°C causes the Taq polymerase to synthesize the complementary DNA strand from the dNTPs, starting at the primer.
Steps 1–3 are continually repeated to yield the necessary number of copies.

21. Polymerase Chain Reaction (PCR): making copies of targeted regions of DNA
Ingredients
Your DNA (all 46 chromosomes). Each student will make their own tube.
D1S80 Primers
MasterMix: Taq polymerase, MgCl2, DNA building-blocks, deoxyribonucleoside triphosphates (dNTPs)
Thermus aquaticus
dGTP
Taq polymerase

22. Forward: 5'-GAAACTGGCCTCCAAACACTGCCCGCCG-3'
Forward and Reverse D1S80 primers correspond to the sequences on either side of the D1S80 locus to be copied! We buy them for you!
Forward: 5'-GAAACTGGCCTCCAAACACTGCCCGCCG-3'
Reverse: 5'-GTCTTGTTGGAGATGCACGTGCCCCTTGC-3'
DNA polymerase
D1S80 LOCUS
DNA polymerase

23. Positive control (prep staff)
5.5: PCR Setup
Tube letter
Sample NAME
Master mix
DNA template
Primer Mix
D1S80
Total Volume A
Student #1 genomic DNA
12.5 ul
10.0 ul
D1S80: 2.5 ul
25.0 ul B
Student #2 genomic DNA C
Student #3 genomic DNA D
Student #4 genomic DNA
D1S80: 2.5 u E
Negative control
10.0 ul dH2O
Positive control (prep staff)
Master Mix: Taq polymerase, MgCl2, dNTPs and buffer
Remember to keep your samples on ice and ALWAYS use a new sterile pipette tip with aerosol-barrier (labeled for PCR-Only) for each transfer to avoid contaminating the stock reagents as well as your samples!
How is the negative control different? What is its purpose?
How is the positive control different? What is its purpose?

24. Place the tubes in the PCR machine
Thermocycler

25. What will be amplified. All 46 chromosomes
What will be amplified? All 46 chromosomes? The entire length of Chromosome 1? A small segment or locus on Chromosome 1?