E. coli in-frame knockout primers. Church lab, Harvard Medical School

Go to the list of E. coli in-frame knockout primers

For background, refer to the lab pages about gene replacement using the pKO3 vector and E. coli in-frame gene deletion projects.

DETAILS

These primers were generated with PrimerFinder and data from Escherichia coli K-12 MG1655 M52, complete genome. They were designed for use with the pKO3 and pKO5 vectors (notes coming soon) designed in our lab. Two genes can only be knocked out with pKO5, and 27 only with pKO3. This is indicated in the list.

SELECTION OF RESTRICTION ENZYMES

Briefly, the pKO3 cloning site consists of:

5' - SmaI - NotI - BamHI - NotI - SalI - 3'

Since NotI removes the BamHI site, those two enzymes cannot be used together.

The pKO5 cloning site is:

5' - SmaI - NotI - stuffer - BamHI - SalI - 3'

where the stuffer is genomic sequence which must be removed, meaning that BamHI and SalI cannot be used together, and SmaI and NotI cannot be used together.

ALTERNATE ENZYMES

If the restriction sites above occur in the flanking regions of the target genes (i.e., the insert), alternate enzymes must be used. In these cases, the vector must be cut with the original enzyme, while the insert is cut with an enzyme that produces compatible ends.

BglII and BclI are compatible with BamHI. PmeI and SwaI are compatible with SmaI.

The order of preference is the selection of restriction enzymes was as follows:

order of preference	N enzyme	C enzyme	notes
1	NotI	SalI
2	BamHI	SalI	pKO3 only, since the stuffer must be removed in pKO5
3	PmeI	BamHI
4	PmeI	SalI
5	SwaI	SalI
6	SwaI	BamHI
7	BglII	SalI
8	PmeI	BglII
9	SwaI	BglII
10	BclI	SalI
11	PmeI	BclI
12	SwaI	BclI
13	NotI	BamHI	pKO5 only, since NotI removes the BamHI site in pKO3
14	SmaI	NotI	pKO3 only, since the stuffer must be removed in pKO5

The results were as follows:

N enzyme	C enzyme	# genes	notes
NotI	SalI	4001
PmeI	BamHI	332
PmeI	BglII	30
BamHI	SalI	26	pKO3 only
SwaI	BamHI	9
NotI	BamHI	2	pKO5 only
PmeI	BclI	2
SwaI	BglII	2
SmaI	NotI	1	pKO3 only
	total	4405

PRIMERFINDER PARAMETERS

The default parameters are conservative, but one can loosen them if PrimerFinder is unable to find primers. If you run PrimerFinder, be sure to specify the anchor sequences ("outer primer overhang sequence") for both the N and C restriction enzymes. The order of selection of parameters was as follows; this is useful if you try to generate primers for a difficult gene, but the order depends on the reason(s) for the rejection of primers (besides restriction sites).

1. Set primer length target to 30, primer length variance to 10, specify restriction enzymes and anchor sequences; ignore gene containment.

2. If unable to generate primers, turn off PCR genomic noise checking.

3. Next, turn on PCR genomic noise checking but turn off checking for uniqueness.

4. Turn off PCR genomic noise checking and checking for uniqueness.

5. Increase target Tm and variance by one degree each.

6. Increase max Tm for primer dimers and/or hairpins.

If you have questions or comments, please contact adnan@genetics.med.harvard.edu.

Updated 2/4/98.