Permeability and transformation of cells

Abstract

A method of inducing competence in cells, the method comprising contacting the cell with a composition comprising a quaternary ammonium compound including a silicon-containing functional group and a hydrocarbyl-saccharide compound.

Claims

1. A method of inducing competence in cells, the method comprising contacting the cell with a composition comprising a quaternary ammonium compound including a silicon-containing functional group and a hydrocarbyl-saccharide compound of general formula (III): ##STR00004## wherein n is from 5 to 12, preferably from 6 to 10, more preferably from 7 to 9 and m is from 1 to 6, preferably from 2 to 5, more preferably 3 or 4.

2. A method according to claim 1 wherein the cells are bacteria cells.

3. A method according to claim 2 wherein the cells are gram negative bacteria cells.

4. A method according to claim 1 wherein the cells are yeast cells.

5. A method according to claim 1 wherein the cells are prokaryotic cells.

6. A method according to claim 1 wherein the cells are eukaryotic cells.

7. A method according to claim 1 wherein the quaternary ammonium compound including a silicon-containing functional group is a compound of general formula (I): ##STR00005## or a derivative salt thereof wherein L is a linking group; each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 is independently selected from H or an optionally substituted alkyl, alkenyl, aryl or alkoxy group; and n is 0 or 1.

8. A method according to claim 7 wherein the quaternary ammonium compound including silcon-containing functional group is a compound of formula II: ##STR00006##

9. A method of transformation of cells, the method comprising the steps of: (i) contacting the cells with a composition comprising a quaternary ammonium compound including a silicon-containing functional group and a hydrocarbyl-saccharide compound of general formula (III): ##STR00007## wherein n is from 5 to 12, preferably from 6 to 10, more preferably from 7 to 9 and m is from 1 to 6, preferably from 2 to 5, more preferably 3 or 4; (ii) adding a source of exogenous DNA to the cells; and (iii) thermally shocking the cells.

10. A method according to claim 9 wherein the source of exogenous DNA is a plasmid carrying exogenous DNA.

11. A kit for transformation of cells, the kit comprising: a composition comprising a quaternary ammonium compound including a silicon-containing functional group and a hydrocarbyl-saccharide compound of general formula (III): ##STR00008## wherein n is from 5 to 12, preferably from 6 to 10, more preferably from 7 to 9 and m is from 1 to 6, preferably from 2 to 5, more preferably 3 or 4; and a source of exogenous DNA.

Description

EXAMPLE 1

(1) An E. coli propriety strain was treated with an aqueous test composition comprising 0.08 wt % of the quaternary ammonium compound of formula (II) described in the specification and 0.08 wt % of the monoalkyl polyglucoside compound of formula (III) described in the specification.

(2) Commercially available InvitroGen competent cells were tested as a comparative composition.

(3) Treatment was performed over an ON culture diluted to an OD.sub.330 of 0.01 and grown again to an OD.sub.330 of 0.11

(4) The resulting exponentially growing cells were thoroughly mixed with the test compositions and the control composition and kept on ice for 15 minutes, before performing the thermal shock as described by Maniatis et al. in Current Protocols in Molecular Biology. An analogous procedure was carried out using the control samples.

(5) A commercially available vector, pVC19 was used and a sample of cloned DNA. These were added to the cooled composition prior to the thermal shock. In each case the DNA sample was added 5 minutes after the cells were put on ice, allowing the DNA to interact with the cells for 10 minutes prior to the thermal shock.

(6) pUC19 with no cloned insert is used as a positive control. The clean plasmid with no cloned DNA is the default choice for its reduced toxicity and impairment in terms of cell metabolism and recombination machinery.

(7) In treatment A, 480 L of cells were mixed with 20 L test composition.

(8) In treatment B, 470 L of cells were mixed with 30 L of test composition.

(9) TABLE-US-00001 Number of Treatment transformants InvitroGen competent cells/pUC19 control 78 InvitroGen competent cells/no DNA (ve control) 0 Treatment A 3 Treatment B 16

EXAMPLE 2

(10) A further E. coli strain (DH5 F) was treated using the same test composition and procedure as example 1 and compared with commercially available InvitroGen competent cells.

(11) Again, the treatment was performed over an ON culture diluted to an OD.sub.330 of 0.01 and grown again to an OD.sub.330 of 0.11

(12) The resulting exponentially growing cells were thoroughly mixed with the composition and kept on ice for 15 minutes, before adding the cloned DNA and performing the thermal shock as described by Maniatis et al. in Current Protocols in Molecular Biology. The cloned DNA was added 5 minutes after the cells were put on ice, allowing the DNA to interact with the cells for 10 minutes prior to the thermal shock.

(13) TABLE-US-00002 Number of Treatment transformants InvitroGen competent cells/pUC18 control 80 InvitroGen competent cells/no DNA (ve control) 0 Treatment A 9 Treatment B 29

EXAMPLE 3

(14) A proprietary E. coli strain was treated with the same test composition as example 1 and compared with commercially available InvitroGen competent cells.

(15) Treatment was performed over an ON culture diluted to an OD.sub.330 of 0.01 and grown again to an OD.sub.330 of 0.11

(16) The resulting exponentially growing cells were thoroughly mixed with the test composition and kept on ice for 15 minutes, before adding the cloned DNA and performing the thermal shock as described by Maniatis et al. in Current Protocols in Molecular Biology. The cloned DNA was added 5 minutes after the cells were put on ice, allowing the DNA to interact with the cells for 10 minutes prior to the thermal shock.

(17) TABLE-US-00003 Number of Treatment transformants InvitroGen competent cells/+pUC19 control >50 InvitroGen competent cells/no DNA (ve control) 0 Treatment B 7

EXAMPLE 4

(18) E. coli standard strain DH5 F was treated with the same composition test and procedure as example 1 and compared with commercially available InvitroGen competent cells.

(19) Treatment was performed over an ON culture diluted to an OD.sub.330 of 0.01 and grown again to an OD.sub.330 of 0.11.

(20) The resulting exponentially growing cells were thoroughly mixed with the ARCIS sample(s) and kept on ice for 15 minutes, before adding the cloned DNA and performing the thermal shock as described by Maniatis et al. in Current Protocols in Molecular Biology. The cloned DNA was added 5 minutes after the cells were put on ice, allowing the DNA to interact with the cells for 10 minutes prior to the thermal shock.

(21) TABLE-US-00004 Number of Treatment transformants InvitroGen competent cells + pUC18 control 70 InvitroGen competent cells no DNA (ve control) 0 Treatment A 2 Treatment B 12