***Don’t forget that on 03 Oct, he transitioned to “information covered on
Exam 3” ­­­ starting with “How to construct a plasmid” http://quizlet.com/54570964/flashcards

Steps to construct a plasmid
1. Amplify the gene of interest with PCR
2. Use Restriction Digest Enzymes to cut the gene and insert into plasmid 3. Insert plasmid into bacteria of interest
4. By using chemical transformation, electroporation (electroshock), conjugation, or bacteriophage
5. Grow bacteria on selective media
Also anyone have the iclicker answers from oct 10 and 13? iclicker answers oct 10:
1.a
2.c
3.b
<<<why is this answer b?
4.c
<<<<<why c???? iclicker answers from oct 13
1.a
2.b
3.b
<­­ Why is this the answer? It is asking about the best way to transfer antibiotic resistance from one strain of E. coli into another strain. This answer is Transduction.
Couldn’t Conjugation work just as well, it’s also an answer choice?
Its also asking for chromosomal transfer, not plasmid. Transduction is the best way for transfer of chromosomal DNA
4.

Answers to the questions from the 17th are
1) C

2)
3)
4)
5)

B
A
B why??? A

Slides 19­26 on Oct 1­3rd slides
NOT REQUIRED!

October 8th → Lecture 14 ● Utility of Plasmids
○ Foreign DNA­> gene of interest (PCR) ­> plasmid
○ Helpful video: https://www.youtube.com/watch?v=MIfDx417SDs
1. Promoters
a. How to study promoters
i.
Most promoters don’t have canonical ­10, ­35 sequences ii. Most of the time the ­35 is furthest from consensus iii. Most promoters are regulated by proteins called transcriptional regulators iv. Canonical promoter is constitutive (always finds RNA to transcribe at a constant rate)
1. This is not common.
Most promoters are regulated.
a. What they actually do is let the bacteria know where it is.
They look for cues (cues= signal specific sequences)
Study promoter activity
● Using reporter gene
­ Gene that produces a protein that is easily quantified
○ Plasmid that contains a reporter gene
■ Amount of protein = transcription from the promoter

1. B­galactosidase (LacZ alpha , LacZ omega )
2. Light production, (luxCDABE)
a. all wavelength
3. GFP Fluorescence

a. Looking at specific wavelengths

Two types of promoter­reporter fusions
1. Transcriptional
a. Shine Dalgarno(AGGAGG) is made up and optimal → it means that the SD is exactly consensus so that the maximum amount is made
→ the SD is the consensus AGGAGG
2. Translational
a. Shine Dalgarno(AGGAGG) is from the gene → the SD sequence is the original one in the gene and may not be consensus.

After we construct promoter­reporter fusions on a plasmid:
● Introduce the plasmid into the strain of interest
1.
Chemical transformation
a. Treat bacteria with solutions with high salt concentrations, making them randomly take up DNA including plasmids (ex.) E. coli in lab)
b. MgCl2 & CaCl2
c. Limited chemical transfer, works for few bacteria, but not most.

2.

Transformation using Electroshock (electrotransformation)

● Electrical current through the bacteria/plasmid
○ short duration 10 milliseconds 3.

Conjugation
● bacterium to bacterium transfer of plasmid

TN = Transposon

OCTOBER 10th (I think so?)
A donor (most likely to use E. Coli) creates a pilus that binds to recipient cell to bring them closer together 1. Donor creates, attaches to, and retracts pilus to bring the cells together
1. A pore between donor and recipient forms that the plasmid transfers through
○ Donor creates a copy of the plasmid that is then transferred over

Genes needed for Conjugation
Some genes are required for conjugation to occur
1. tra operon => encode proteins that make the pilus and retract the pilus; pilus ~ 10 nm in diameter, but can be micrometers long…. trans-acting gene
2. mob operon =>mobilization, prepares the plasmid for transfer and transfers the plasmid
○ Relaxase: makes a ss DNA cut in the plasmid that