rahma zakaria
how to grow almost anything

bio design: dna ladder

In this week's lab, we isolated pPSU1 and pPSU2 plasmids from cell culture, performed restriction digests to create DNA fragments for a ladder, and ran a gel to confirm the size of the fragments created.

Based on the resulting gel, it seems like our EcoRV digest did not work, but our PstI digests created a nice ladder with some clear bands!

Lanes:
A: NEB 100bp reference ladder
B: NEB 1kb reference ladder
C: Uncut plasmid pPSU1
D: Uncut plasmid pPSU2
E: pPSU1 and pPSU2 EcoRV digest
F: pPSU1 and pPSU2 PstI digest

Picture credits: Sara and Rae

discussion questions

1. What software are you using to read the sequence files?

o Benchling

2. What are the distances between the PstI (CTGCAG) sites in each plasmid?

o pPSU1: 494, 1994, 994, 694, 794, 894, 4094

o pPSU2: 1494, 44, 94, 194, 294, 394, 494, 594, 4094

3. What are the distances between the EcoRV (GATATC) sites in each plasmid?

o pPSU1: 494, 994, 1494, 1994, 4994.

o pPSU2: 744, 2994, 3994

o Add 6 bases to each distance (the size of the cut site)

4. Where do PstI and EcoRV cut within their recognition sequences?

o PstI: CTGCA|G

� G|ACGTC

o EcoRV: GAT|ATC

� CTA|TAG

o Add 6 bases to each distance (the size of the cut site)

5. What gene is encoded into the plasmids?

o AmpR, for ampicillin resistance

6. How many copies of the plasmid would you roughly expect in each cell?

o Since they are high copy number plasmids, you would expect thousands of copies of the plasmids in each cell.

7. How do the plasmids' replication relate to that of pBR322 (genbank)?

o pBR322 is a medium copy number plasmid, having about 10-1000 copies in each cell, so pPSU1 and pPSU2 replicates more in cells.

Pro Challenge

Add a restriction site into the E. coli genome through lambda-red recombination.

For this week, provide the oligo design that achieves your proposed genomic edit and describe what impact this change may have on the genetic code. In later weeks, you will carry out this recombination within the electroporator you build, and soon after measure your genome editing efficiency. Recall, George described lambda red recombination as being so precise that it can do what CRISPR can only wish to accomplish, for now.

I will try to introduce a restriction site for the enzyme EcoRV, with the cut site GATATC, in the lacZ gene of E. coli. There currently exists an EcoRV cut site at location 1126 of the lacZ gene. I would like to introduce a site at location 1492. Currently, the bases at location 1492-1497 are GATATT, so a point mutation of T1497C would turn this into an EcoRV cut site, and could be done with a ssDNA oligo. However, it would have the consequence of interrupting the lacZ gene and making it nonfunctional.

Forward primer:

GCCCGGTGCAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATCatgagtattcaacatttccg

Uppercase: lacZ sequence, ending at base 1496. Bold: point mutation. Lowercase: AmpR beginning.

Reverse primer:

CAGCCGGGAAGGGCTGGTCTTCATCCACGCGCGCGTACATCGGGCAAATAttaccaatgcttaatcagtg

Uppercase: lacZ sequence reverse complement, ending at base 1498. Lowercase: AmpR end reverse complement.