From 0d93bbeb794a7e0e7954943eabc312fed79746b4 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Thu, 12 May 2022 11:54:37 -0500 Subject: [PATCH 01/24] Create Gene_Complementation_Klebsiella.md --- Gene_Complementation_Klebsiella.md | 37 ++++++++++++++++++++++++++++++ 1 file changed, 37 insertions(+) create mode 100644 Gene_Complementation_Klebsiella.md diff --git a/Gene_Complementation_Klebsiella.md b/Gene_Complementation_Klebsiella.md new file mode 100644 index 0000000..a45a30f --- /dev/null +++ b/Gene_Complementation_Klebsiella.md @@ -0,0 +1,37 @@ +# Gene Complementation in *Klebsiella Pneumoniae* + +This protocol describes complementation of a gene back into Klebsiella using Tn7. + +## Insert gene of interest into donor strain's vector backbone using Gibson Assembly + +Insert DNA cassette containing the gene of interest from KPPR1(MJM2383) into the pGP-Tn7-Gm backbone. + +1. Use Snapgene to make an *in silico* insert DNA cassette approximately 500 bp downstream and upstream of the gene of interest. +2. Make Primers on NEBuilder according to the [Gibson Assembly Protocol](https://github.com/mjmlab/protocols/blame/master/gibson-assembly.md). + * Use the [pGP-Tn7-Gm backbone](https://www.ncbi.nlm.nih.gov/nuccore/JQ429758.1?report=gbwithparts&log$=seqview) as the vector fragment, and amplify the vector from base pairs 1880 to 1810 (The MCS of the backbone). + * Add the *in silico* insert DNA cassette as a fragment to the vector. +3. Use the primers from NEBuilder to perform Gibson Assembly. +4. Using heat shock, transform the donor strain vector into chemically competent S17-1 λ pir *E. coli* cells (MJM661). + +## Transform recipient vector into recipient strain + +Using heat shock, transform pSTNSK into KPPR1. ???? + +## Deliver Tn7 into Klebsiella + +Using classical mating, integrate the Tn7 transposon into the KPPR1 chromosome. + +1. Start overnight cultures of the donor strain and the recipient strain. +2. Pellet 500 uL of donor and recipient strains in seperate Eppendorf tubes (5 min max speed, Klebsiella may need a longer spin since it doesn't form a tight pellet) +3. Remove all but 50 uL of LB from each tube and re-suspend each pellet in the remaining LB, then combine the resuspensions in a single tube. +4. Plate all 100 uL of mixed suspension on an LB plate and incubate at 30 degrees Celsius for 5 or 18 hrs. +5. Use a cotton swab dipped in PBS to scrape up the cells, agitate to dislodge the bacteria in 1 mL of PBS in a 2 mL tube. +6. Create serial dilutions out to 10^-8, and plate out onto LB-GM plates. + * Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. +7. Streak out single colonies onto LB-Gm, LB-Carb, and LB-Km plates. + * Look for resistance to Gm, sensitivity to Carb and Km. +8. Screen complementation candidates using PCR. +9. Freeze successful candidates and send to MIGS for sequencing. + + + From 670fd77a31fd073731fd0284b88f5b58dbed42f8 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Thu, 12 May 2022 16:30:13 -0500 Subject: [PATCH 02/24] Update Gene_Complementation_Klebsiella.md --- Gene_Complementation_Klebsiella.md | 10 +++++++++- 1 file changed, 9 insertions(+), 1 deletion(-) diff --git a/Gene_Complementation_Klebsiella.md b/Gene_Complementation_Klebsiella.md index a45a30f..c5dec36 100644 --- a/Gene_Complementation_Klebsiella.md +++ b/Gene_Complementation_Klebsiella.md @@ -15,7 +15,15 @@ Insert DNA cassette containing the gene of interest from KPPR1(MJM2383) into the ## Transform recipient vector into recipient strain -Using heat shock, transform pSTNSK into KPPR1. ???? +Introduce pSTNSK into KPPR1 via electroporation. + +1. Inoculate 100 mL LB with 1 mL of overnight culture. Incubate at 30C with stir bar at ~500 rpm until it reaches OD600 of 0.6 to 0.8 (approximately 4 hours). +2. Centrifuge cells at 6500 rpm for 5 min. Remove supernatant and wash twice with 50 mL of ice cold glycerol (resuspend with glycerol and then centrifuge it again). +3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA and electroporate 2500 mV. Add SOC medium immediately after electroporation. +4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto LB-Kan50 plates. +5. Incubate plates at 30 degrees C overnight and PCR Screen for pSTNSK in colonies. +6. Freeze successful colonies and use for complementation. + ## Deliver Tn7 into Klebsiella From 002ea30707a76cffe2bfdc4dc2e372d04004e36e Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Thu, 12 May 2022 16:32:29 -0500 Subject: [PATCH 03/24] Update Gene_Complementation_Klebsiella.md --- Gene_Complementation_Klebsiella.md | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/Gene_Complementation_Klebsiella.md b/Gene_Complementation_Klebsiella.md index c5dec36..9d80fe9 100644 --- a/Gene_Complementation_Klebsiella.md +++ b/Gene_Complementation_Klebsiella.md @@ -35,9 +35,9 @@ Using classical mating, integrate the Tn7 transposon into the KPPR1 chromosome. 4. Plate all 100 uL of mixed suspension on an LB plate and incubate at 30 degrees Celsius for 5 or 18 hrs. 5. Use a cotton swab dipped in PBS to scrape up the cells, agitate to dislodge the bacteria in 1 mL of PBS in a 2 mL tube. 6. Create serial dilutions out to 10^-8, and plate out onto LB-GM plates. - * Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. + * Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. 7. Streak out single colonies onto LB-Gm, LB-Carb, and LB-Km plates. - * Look for resistance to Gm, sensitivity to Carb and Km. + * Look for resistance to Gm, sensitivity to Carb and Km. 8. Screen complementation candidates using PCR. 9. Freeze successful candidates and send to MIGS for sequencing. From 3dd223861724c815157bee68a75682e280cfb457 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Thu, 12 May 2022 16:34:31 -0500 Subject: [PATCH 04/24] Update Gene_Complementation_Klebsiella.md --- Gene_Complementation_Klebsiella.md | 9 +++++---- 1 file changed, 5 insertions(+), 4 deletions(-) diff --git a/Gene_Complementation_Klebsiella.md b/Gene_Complementation_Klebsiella.md index 9d80fe9..cf2895d 100644 --- a/Gene_Complementation_Klebsiella.md +++ b/Gene_Complementation_Klebsiella.md @@ -12,6 +12,7 @@ Insert DNA cassette containing the gene of interest from KPPR1(MJM2383) into the * Add the *in silico* insert DNA cassette as a fragment to the vector. 3. Use the primers from NEBuilder to perform Gibson Assembly. 4. Using heat shock, transform the donor strain vector into chemically competent S17-1 λ pir *E. coli* cells (MJM661). +5. Use PCR screening to determine if the Gibson Assembly and transformation was successful. ## Transform recipient vector into recipient strain @@ -35,11 +36,11 @@ Using classical mating, integrate the Tn7 transposon into the KPPR1 chromosome. 4. Plate all 100 uL of mixed suspension on an LB plate and incubate at 30 degrees Celsius for 5 or 18 hrs. 5. Use a cotton swab dipped in PBS to scrape up the cells, agitate to dislodge the bacteria in 1 mL of PBS in a 2 mL tube. 6. Create serial dilutions out to 10^-8, and plate out onto LB-GM plates. - * Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. -7. Streak out single colonies onto LB-Gm, LB-Carb, and LB-Km plates. +7. Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. +8. Streak out single colonies onto LB-Gm, LB-Carb, and LB-Km plates. * Look for resistance to Gm, sensitivity to Carb and Km. -8. Screen complementation candidates using PCR. -9. Freeze successful candidates and send to MIGS for sequencing. +9. Screen complementation candidates using PCR. +10. Freeze successful candidates and send to MIGS for sequencing. From d214d65fd521bda9e96fea3fc7d95792f608289f Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Thu, 12 May 2022 16:39:07 -0500 Subject: [PATCH 05/24] Rename Gene_Complementation_Klebsiella.md to gene_complementation_klebsiella.md --- ...ementation_Klebsiella.md => gene_complementation_klebsiella.md | 0 1 file changed, 0 insertions(+), 0 deletions(-) rename Gene_Complementation_Klebsiella.md => gene_complementation_klebsiella.md (100%) diff --git a/Gene_Complementation_Klebsiella.md b/gene_complementation_klebsiella.md similarity index 100% rename from Gene_Complementation_Klebsiella.md rename to gene_complementation_klebsiella.md From 64f91ac2e1eea4ebc4472bf5070fb6f4a0787057 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Thu, 12 May 2022 16:39:21 -0500 Subject: [PATCH 06/24] Update README.md --- README.md | 1 + 1 file changed, 1 insertion(+) diff --git a/README.md b/README.md index 9ed3fd0..c7c0149 100644 --- a/README.md +++ b/README.md @@ -4,6 +4,7 @@ ### Molecular Biology - [Gene Deletion in *V. fischeri*](gene-deletion.md) +- [Gene Complementation in *K. Pneumoniae*](gene_complementation_klebsiella.md) - [Gibson Assembly](gibson-assembly.md) - [dNTP stocks for PCR](molecular-dntps.md) - [Semi-arbitrarily-primed PCR to identify transposon insertion sites](arbitrarily-primed-pcr.md) From 4bd32d5c396d971495fedd9d2bb1c4f12e925674 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Fri, 13 May 2022 11:03:26 -0500 Subject: [PATCH 07/24] add clarity --- gene_complementation_klebsiella.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index cf2895d..6f4f7b8 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -16,7 +16,7 @@ Insert DNA cassette containing the gene of interest from KPPR1(MJM2383) into the ## Transform recipient vector into recipient strain -Introduce pSTNSK into KPPR1 via electroporation. +Introduce pSTNSK into KPPR1 strain with respective deleted gene via electroporation. 1. Inoculate 100 mL LB with 1 mL of overnight culture. Incubate at 30C with stir bar at ~500 rpm until it reaches OD600 of 0.6 to 0.8 (approximately 4 hours). 2. Centrifuge cells at 6500 rpm for 5 min. Remove supernatant and wash twice with 50 mL of ice cold glycerol (resuspend with glycerol and then centrifuge it again). From 6e63f3db96fd16f460e3629346d3f951ffad74d2 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Mon, 16 May 2022 11:15:20 -0500 Subject: [PATCH 08/24] Edit primer creation in NEBuilder protocol --- gene_complementation_klebsiella.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index 6f4f7b8..c9d91e3 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -8,7 +8,7 @@ Insert DNA cassette containing the gene of interest from KPPR1(MJM2383) into the 1. Use Snapgene to make an *in silico* insert DNA cassette approximately 500 bp downstream and upstream of the gene of interest. 2. Make Primers on NEBuilder according to the [Gibson Assembly Protocol](https://github.com/mjmlab/protocols/blame/master/gibson-assembly.md). - * Use the [pGP-Tn7-Gm backbone](https://www.ncbi.nlm.nih.gov/nuccore/JQ429758.1?report=gbwithparts&log$=seqview) as the vector fragment, and amplify the vector from base pairs 1880 to 1810 (The MCS of the backbone). + * Use the [pGP-Tn7-Gm backbone](https://www.ncbi.nlm.nih.gov/nuccore/JQ429758.1?report=gbwithparts&log$=seqview) as the vector fragment, and amplify the vector from base pairs 1871 to 1824 (The MCS of the backbone). * Add the *in silico* insert DNA cassette as a fragment to the vector. 3. Use the primers from NEBuilder to perform Gibson Assembly. 4. Using heat shock, transform the donor strain vector into chemically competent S17-1 λ pir *E. coli* cells (MJM661). From 86917660adc89a3e8e7fb7b0e60bfc608b9f53e7 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Tue, 17 May 2022 10:50:28 -0500 Subject: [PATCH 09/24] edit protocol --- gene_complementation_klebsiella.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index c9d91e3..569fd44 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -21,7 +21,7 @@ Introduce pSTNSK into KPPR1 strain with respective deleted gene via electroporat 1. Inoculate 100 mL LB with 1 mL of overnight culture. Incubate at 30C with stir bar at ~500 rpm until it reaches OD600 of 0.6 to 0.8 (approximately 4 hours). 2. Centrifuge cells at 6500 rpm for 5 min. Remove supernatant and wash twice with 50 mL of ice cold glycerol (resuspend with glycerol and then centrifuge it again). 3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA and electroporate 2500 mV. Add SOC medium immediately after electroporation. -4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto LB-Kan50 plates. +4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto low salt LB-Kan50 plates. 5. Incubate plates at 30 degrees C overnight and PCR Screen for pSTNSK in colonies. 6. Freeze successful colonies and use for complementation. From 4401b5cdf0f4fb6eb696f0a7c04f8790ac177be0 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Tue, 21 Jun 2022 14:52:37 -0500 Subject: [PATCH 10/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index 569fd44..96175ba 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -20,7 +20,7 @@ Introduce pSTNSK into KPPR1 strain with respective deleted gene via electroporat 1. Inoculate 100 mL LB with 1 mL of overnight culture. Incubate at 30C with stir bar at ~500 rpm until it reaches OD600 of 0.6 to 0.8 (approximately 4 hours). 2. Centrifuge cells at 6500 rpm for 5 min. Remove supernatant and wash twice with 50 mL of ice cold glycerol (resuspend with glycerol and then centrifuge it again). -3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA and electroporate 2500 mV. Add SOC medium immediately after electroporation. +3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA and electroporate 2500 mV. Add 900 uL SOC medium immediately after electroporation. 4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto low salt LB-Kan50 plates. 5. Incubate plates at 30 degrees C overnight and PCR Screen for pSTNSK in colonies. 6. Freeze successful colonies and use for complementation. From 39f93b7503d1ef82d1011a9267dc1e03ff51872a Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Tue, 5 Jul 2022 12:55:10 -0500 Subject: [PATCH 11/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 10 ++++++---- 1 file changed, 6 insertions(+), 4 deletions(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index 96175ba..490ccf0 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -16,14 +16,16 @@ Insert DNA cassette containing the gene of interest from KPPR1(MJM2383) into the ## Transform recipient vector into recipient strain -Introduce pSTNSK into KPPR1 strain with respective deleted gene via electroporation. +Introduce pSTNSK-Cm into KPPR1 strain with respective deleted gene via electroporation. 1. Inoculate 100 mL LB with 1 mL of overnight culture. Incubate at 30C with stir bar at ~500 rpm until it reaches OD600 of 0.6 to 0.8 (approximately 4 hours). 2. Centrifuge cells at 6500 rpm for 5 min. Remove supernatant and wash twice with 50 mL of ice cold glycerol (resuspend with glycerol and then centrifuge it again). 3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA and electroporate 2500 mV. Add 900 uL SOC medium immediately after electroporation. -4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto low salt LB-Kan50 plates. -5. Incubate plates at 30 degrees C overnight and PCR Screen for pSTNSK in colonies. -6. Freeze successful colonies and use for complementation. +4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto low salt LB-Cam25 plates. +5. Incubate plates at 30 degrees C overnight. +6. Streak colonies onto low salt LB-Cam25 plates, and then again the next day. +7. PCR Screen for pSTNSK-Cm. +8. Freeze successful colonies and use for complementation. ## Deliver Tn7 into Klebsiella From 8f6a164f9a7a2b1a58c9a488c816b889a16781a1 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Fri, 29 Jul 2022 13:39:35 -0500 Subject: [PATCH 12/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index 490ccf0..b169328 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -20,7 +20,7 @@ Introduce pSTNSK-Cm into KPPR1 strain with respective deleted gene via electropo 1. Inoculate 100 mL LB with 1 mL of overnight culture. Incubate at 30C with stir bar at ~500 rpm until it reaches OD600 of 0.6 to 0.8 (approximately 4 hours). 2. Centrifuge cells at 6500 rpm for 5 min. Remove supernatant and wash twice with 50 mL of ice cold glycerol (resuspend with glycerol and then centrifuge it again). -3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA and electroporate 2500 mV. Add 900 uL SOC medium immediately after electroporation. +3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA (<= 6 uL) in an electroporation cuvette. Use the manual mode on the electroporation machine and use the arrows to increase the voltage to 2.5V. Make sure that the cuvette is perfectly straight before pressing the pulse button, or else there's a very large chance of arcing. Add 900 uL SOC medium immediately after electroporation. 4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto low salt LB-Cam25 plates. 5. Incubate plates at 30 degrees C overnight. 6. Streak colonies onto low salt LB-Cam25 plates, and then again the next day. @@ -37,7 +37,7 @@ Using classical mating, integrate the Tn7 transposon into the KPPR1 chromosome. 3. Remove all but 50 uL of LB from each tube and re-suspend each pellet in the remaining LB, then combine the resuspensions in a single tube. 4. Plate all 100 uL of mixed suspension on an LB plate and incubate at 30 degrees Celsius for 5 or 18 hrs. 5. Use a cotton swab dipped in PBS to scrape up the cells, agitate to dislodge the bacteria in 1 mL of PBS in a 2 mL tube. -6. Create serial dilutions out to 10^-8, and plate out onto LB-GM plates. +6. Create serial dilutions out to 10^-4, and plate out onto LB-GM plates. 7. Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. 8. Streak out single colonies onto LB-Gm, LB-Carb, and LB-Km plates. * Look for resistance to Gm, sensitivity to Carb and Km. From 3f664a7bc5c8250ba3af9dc955daa9529316d41d Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Fri, 29 Jul 2022 13:40:19 -0500 Subject: [PATCH 13/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index b169328..a33b58f 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -20,7 +20,7 @@ Introduce pSTNSK-Cm into KPPR1 strain with respective deleted gene via electropo 1. Inoculate 100 mL LB with 1 mL of overnight culture. Incubate at 30C with stir bar at ~500 rpm until it reaches OD600 of 0.6 to 0.8 (approximately 4 hours). 2. Centrifuge cells at 6500 rpm for 5 min. Remove supernatant and wash twice with 50 mL of ice cold glycerol (resuspend with glycerol and then centrifuge it again). -3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA (<= 6 uL) in an electroporation cuvette. Use the manual mode on the electroporation machine and use the arrows to increase the voltage to 2.5V. Make sure that the cuvette is perfectly straight before pressing the pulse button, or else there's a very large chance of arcing. Add 900 uL SOC medium immediately after electroporation. +3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA (<= 6 uL) in an electroporation cuvette. Use the manual mode on the electroporation machine and use the arrows to increase the voltage to 2.5V. Make sure that the cuvette is perfectly vertical before pressing the pulse button, or else there's a very large chance of arcing. Add 900 uL SOC medium immediately after electroporation. 4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto low salt LB-Cam25 plates. 5. Incubate plates at 30 degrees C overnight. 6. Streak colonies onto low salt LB-Cam25 plates, and then again the next day. From 240eaf38ef28b8fc33a153da435515ea568c465c Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Mon, 1 Aug 2022 14:31:23 -0500 Subject: [PATCH 14/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index a33b58f..1851f8e 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -39,7 +39,7 @@ Using classical mating, integrate the Tn7 transposon into the KPPR1 chromosome. 5. Use a cotton swab dipped in PBS to scrape up the cells, agitate to dislodge the bacteria in 1 mL of PBS in a 2 mL tube. 6. Create serial dilutions out to 10^-4, and plate out onto LB-GM plates. 7. Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. -8. Streak out single colonies onto LB-Gm, LB-Carb, and LB-Km plates. +8. Streak out single colonies onto LB-Gm, LB-Carb, and LB-Cam plates. * Look for resistance to Gm, sensitivity to Carb and Km. 9. Screen complementation candidates using PCR. 10. Freeze successful candidates and send to MIGS for sequencing. From f63e129b64020cdf0ce3d4844a1be8a9f664c4d2 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Mon, 1 Aug 2022 14:31:41 -0500 Subject: [PATCH 15/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index 1851f8e..498421e 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -40,7 +40,7 @@ Using classical mating, integrate the Tn7 transposon into the KPPR1 chromosome. 6. Create serial dilutions out to 10^-4, and plate out onto LB-GM plates. 7. Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. 8. Streak out single colonies onto LB-Gm, LB-Carb, and LB-Cam plates. - * Look for resistance to Gm, sensitivity to Carb and Km. + * Look for resistance to Gm, sensitivity to Carb and Cam. 9. Screen complementation candidates using PCR. 10. Freeze successful candidates and send to MIGS for sequencing. From cf09eb9e0fdfcfcc527a8d539abb2abcf48bf207 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Fri, 12 Aug 2022 13:46:25 -0500 Subject: [PATCH 16/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index 498421e..4a35126 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -35,12 +35,12 @@ Using classical mating, integrate the Tn7 transposon into the KPPR1 chromosome. 1. Start overnight cultures of the donor strain and the recipient strain. 2. Pellet 500 uL of donor and recipient strains in seperate Eppendorf tubes (5 min max speed, Klebsiella may need a longer spin since it doesn't form a tight pellet) 3. Remove all but 50 uL of LB from each tube and re-suspend each pellet in the remaining LB, then combine the resuspensions in a single tube. -4. Plate all 100 uL of mixed suspension on an LB plate and incubate at 30 degrees Celsius for 5 or 18 hrs. +4. Plate all 100 uL of mixed suspension on an LB-DAP plate and incubate at 30 degrees Celsius for 5 hrs. 5. Use a cotton swab dipped in PBS to scrape up the cells, agitate to dislodge the bacteria in 1 mL of PBS in a 2 mL tube. 6. Create serial dilutions out to 10^-4, and plate out onto LB-GM plates. 7. Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. -8. Streak out single colonies onto LB-Gm, LB-Carb, and LB-Cam plates. - * Look for resistance to Gm, sensitivity to Carb and Cam. +8. Streak out single colonies onto LB-Gm, LB-Carb, LB-Kan and LB-Cam plates. + * Look for resistance to Gm and Kan, sensitivity to Carb and Cam. 9. Screen complementation candidates using PCR. 10. Freeze successful candidates and send to MIGS for sequencing. From 7bff956ce7e64b451436cda3f6f5345ef827b1cc Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Fri, 12 Aug 2022 13:47:44 -0500 Subject: [PATCH 17/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index 4a35126..700da02 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -11,7 +11,7 @@ Insert DNA cassette containing the gene of interest from KPPR1(MJM2383) into the * Use the [pGP-Tn7-Gm backbone](https://www.ncbi.nlm.nih.gov/nuccore/JQ429758.1?report=gbwithparts&log$=seqview) as the vector fragment, and amplify the vector from base pairs 1871 to 1824 (The MCS of the backbone). * Add the *in silico* insert DNA cassette as a fragment to the vector. 3. Use the primers from NEBuilder to perform Gibson Assembly. -4. Using heat shock, transform the donor strain vector into chemically competent S17-1 λ pir *E. coli* cells (MJM661). +4. Using heat shock, transform the donor strain vector into chemically competent β3916 E. *coli* cells (MJM628). 5. Use PCR screening to determine if the Gibson Assembly and transformation was successful. ## Transform recipient vector into recipient strain From e10d1c67709ad9239c8ea9b48cbcc0f42c267f0a Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Mon, 15 Aug 2022 13:25:25 -0500 Subject: [PATCH 18/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 5 +++-- 1 file changed, 3 insertions(+), 2 deletions(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index 700da02..6d268cf 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -11,8 +11,9 @@ Insert DNA cassette containing the gene of interest from KPPR1(MJM2383) into the * Use the [pGP-Tn7-Gm backbone](https://www.ncbi.nlm.nih.gov/nuccore/JQ429758.1?report=gbwithparts&log$=seqview) as the vector fragment, and amplify the vector from base pairs 1871 to 1824 (The MCS of the backbone). * Add the *in silico* insert DNA cassette as a fragment to the vector. 3. Use the primers from NEBuilder to perform Gibson Assembly. -4. Using heat shock, transform the donor strain vector into chemically competent β3916 E. *coli* cells (MJM628). -5. Use PCR screening to determine if the Gibson Assembly and transformation was successful. +4. Using heat shock, transform the donor strain vector into chemically competent β3914 E. *coli* cells (MJM628). + * In the future, use MJM1424 +6. Use PCR screening to determine if the Gibson Assembly and transformation was successful. ## Transform recipient vector into recipient strain From f7aa943a2e32c302576a12dfe843148043fca027 Mon Sep 17 00:00:00 2001 From: adesikan1 <100712720+adesikan1@users.noreply.github.com> Date: Wed, 17 Aug 2022 14:44:25 -0500 Subject: [PATCH 19/24] Update gene_complementation_klebsiella.md --- gene_complementation_klebsiella.md | 6 ++++-- 1 file changed, 4 insertions(+), 2 deletions(-) diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md index 6d268cf..599e74d 100644 --- a/gene_complementation_klebsiella.md +++ b/gene_complementation_klebsiella.md @@ -25,7 +25,7 @@ Introduce pSTNSK-Cm into KPPR1 strain with respective deleted gene via electropo 4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto low salt LB-Cam25 plates. 5. Incubate plates at 30 degrees C overnight. 6. Streak colonies onto low salt LB-Cam25 plates, and then again the next day. -7. PCR Screen for pSTNSK-Cm. +7. PCR Screen for pSTNSK-Cm. 8. Freeze successful colonies and use for complementation. @@ -42,7 +42,9 @@ Using classical mating, integrate the Tn7 transposon into the KPPR1 chromosome. 7. Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. 8. Streak out single colonies onto LB-Gm, LB-Carb, LB-Kan and LB-Cam plates. * Look for resistance to Gm and Kan, sensitivity to Carb and Cam. -9. Screen complementation candidates using PCR. +9. Screen complementation candidates using PCR, looking for pGP-Gm, pSTNSK, and looking at the Tn7 site. + * If pSTNSK is still in the complemented strain, then streak onto LB-Gent10 and incubate at 42C for 5 hours again + * If pGP-Gm is still in the complemented strain, then streak onto LB, LB-Gent10, and LB-Carb100 until the strain is sensitive to Carb and resistant to Gent 10. Freeze successful candidates and send to MIGS for sequencing. From 8c212310581aca973ff0462f565f4a0ec6021f62 Mon Sep 17 00:00:00 2001 From: Ani Date: Mon, 10 Jul 2023 15:46:44 -0500 Subject: [PATCH 20/24] add kleb conjugation --- conjugation.md | 58 ++++++++++++++++++++++++++++++++++++++++++++++++++ 1 file changed, 58 insertions(+) diff --git a/conjugation.md b/conjugation.md index fdf3c12..4f137f2 100644 --- a/conjugation.md +++ b/conjugation.md @@ -35,3 +35,61 @@ Controls: Same as conjugation tube but excluding one of the constituent strains Plate freshness: Fresh LBS plates seem to help a lot. If possible, use them the same day you pour them; just make sure they’re not "sloppy wet." If you want to compare mating efficiencies of different constructs, be careful to use similar plates or multiple spots on the same plate. Citation: [Stabb EV, Ruby EG. RP4-based plasmids for conjugation between Escherichia coli and members of the Vibrionaceae. Meth Enzymol (2002) vol. 358 pp. 413-26](http://www.ncbi.nlm.nih.gov/pubmed/12474404) + +# Triparental Conjugation into Klebsiella + +This is a protocol describing the triparental conjugation of Tn7 transposon and Tn7 transposase plasmids into a deletion strain of KPPR1. The complementation of VK055_1398 into its deletion strain is used as an example. + +**Day 0** + +1. Make overnight cultures of each strain: + - Transposon Plasmid (pAD13, MJM5668): Grown in LB-Kan50-DAP liquid media at 37C, shaking + - Transposase Plasmid (pJMP1039, MJM4078): Grown in LB-Carb100-DAP liquid media at 37C, shaking + - Deletion Strain (∆1398, MJM): Grown in LB at 37C, shaking + - Positive control (Any previously complemented strain made) + + +**Day 1: Conjugation** +**Important: Do all steps in the BSC** + +1. Add 500 uL of each strain to a tube, mix well with a pipette + * At this point, make multiple *negative controls* using just the deletion strain, just the transposase plasmid, just the transposon plasmid, etc. + * Also make a *positive control* by using the previously complemented strain +2. For all samples: + * centrifuge for 3 min at max speed + * Resuspend pellet in 10 uL of LB-DAP + * Plate 10 uL spot onto LB-DAP plate + * Wait 10 min for spots to dry + * Parafilm plates and put into 30C incubator + * If putting in core room, make sure that you put all plates into box for Klebsiella plates + +**Day 2: Plate Klebsiella** +**Important: Do all steps in the BSC** + +1. Take spots out of the incubator +2. For each spot, do the following: + * Pick up spot using sterile cotton swab soaked in 1x PBS + * Swirl swab well (about 30 sec) in 1 mL of 1x PBS in 2 mL centrifuge tube + * Dilute to 10^-2 and 10^-4 using 1x PBS + * Plate onto LB-Kan50 plate *with no DAP* + * Parafilm plate +3. Put plates into 30C incubator overnight + +**Day 3-5: Kick out plasmids** + +1. Streak 4-8 colonies from yesterday onto 2 different plates: + * LB-Kan50: Shows that Kleb has the Tn7 site inserted + * LB-Carb100: If candidates grow on this plate, that means that plasmids haven't been kicked out of the Klebsiella. Restreak until candidate doesn't grow on this plate + +Usually it takes 1-2 days for the plasmids to be kicked out + +2. If candidates aren't growing on the LB-Carb100 plate, make overnight cultures in LB-Kan50 and grow overnight at 37C shaking + +**Day 6: PCR Screen** + +* Use AD_103 + 104 as the primers + * Sequences are in the lab database as well as in Pep Charusanti's paper +* For template DNA, use a 1:100 dilution of overnight culture +* After screen, run Q5 PCR and PCR purify the product +* Send the purified PCR product to Plasmidsuarus for linear amplicon sequencing + From c9ea7d4537fee1f9137e529d286e7e65adbc2ac5 Mon Sep 17 00:00:00 2001 From: Ani Date: Mon, 10 Jul 2023 15:47:49 -0500 Subject: [PATCH 21/24] change header --- conjugation.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/conjugation.md b/conjugation.md index 4f137f2..65c1ec2 100644 --- a/conjugation.md +++ b/conjugation.md @@ -36,7 +36,7 @@ Plate freshness: Fresh LBS plates seem to help a lot. If possible, use them the Citation: [Stabb EV, Ruby EG. RP4-based plasmids for conjugation between Escherichia coli and members of the Vibrionaceae. Meth Enzymol (2002) vol. 358 pp. 413-26](http://www.ncbi.nlm.nih.gov/pubmed/12474404) -# Triparental Conjugation into Klebsiella +## Triparental Conjugation into Klebsiella This is a protocol describing the triparental conjugation of Tn7 transposon and Tn7 transposase plasmids into a deletion strain of KPPR1. The complementation of VK055_1398 into its deletion strain is used as an example. From fff83cef54f41f7661128cce6de24475441c6b9a Mon Sep 17 00:00:00 2001 From: Ani Date: Mon, 10 Jul 2023 15:48:15 -0500 Subject: [PATCH 22/24] change spacing --- conjugation.md | 6 ++++++ 1 file changed, 6 insertions(+) diff --git a/conjugation.md b/conjugation.md index 65c1ec2..2cda54c 100644 --- a/conjugation.md +++ b/conjugation.md @@ -36,6 +36,12 @@ Plate freshness: Fresh LBS plates seem to help a lot. If possible, use them the Citation: [Stabb EV, Ruby EG. RP4-based plasmids for conjugation between Escherichia coli and members of the Vibrionaceae. Meth Enzymol (2002) vol. 358 pp. 413-26](http://www.ncbi.nlm.nih.gov/pubmed/12474404) + + + + + + ## Triparental Conjugation into Klebsiella This is a protocol describing the triparental conjugation of Tn7 transposon and Tn7 transposase plasmids into a deletion strain of KPPR1. The complementation of VK055_1398 into its deletion strain is used as an example. From 5a0cf20c5237d8179a593a350f39abbd3a5b6031 Mon Sep 17 00:00:00 2001 From: Ani Desikan Date: Fri, 21 Jul 2023 14:29:32 -0500 Subject: [PATCH 23/24] remove gene complementation --- gene_complementation_klebsiella.md | 51 ------------------------------ 1 file changed, 51 deletions(-) delete mode 100644 gene_complementation_klebsiella.md diff --git a/gene_complementation_klebsiella.md b/gene_complementation_klebsiella.md deleted file mode 100644 index 599e74d..0000000 --- a/gene_complementation_klebsiella.md +++ /dev/null @@ -1,51 +0,0 @@ -# Gene Complementation in *Klebsiella Pneumoniae* - -This protocol describes complementation of a gene back into Klebsiella using Tn7. - -## Insert gene of interest into donor strain's vector backbone using Gibson Assembly - -Insert DNA cassette containing the gene of interest from KPPR1(MJM2383) into the pGP-Tn7-Gm backbone. - -1. Use Snapgene to make an *in silico* insert DNA cassette approximately 500 bp downstream and upstream of the gene of interest. -2. Make Primers on NEBuilder according to the [Gibson Assembly Protocol](https://github.com/mjmlab/protocols/blame/master/gibson-assembly.md). - * Use the [pGP-Tn7-Gm backbone](https://www.ncbi.nlm.nih.gov/nuccore/JQ429758.1?report=gbwithparts&log$=seqview) as the vector fragment, and amplify the vector from base pairs 1871 to 1824 (The MCS of the backbone). - * Add the *in silico* insert DNA cassette as a fragment to the vector. -3. Use the primers from NEBuilder to perform Gibson Assembly. -4. Using heat shock, transform the donor strain vector into chemically competent β3914 E. *coli* cells (MJM628). - * In the future, use MJM1424 -6. Use PCR screening to determine if the Gibson Assembly and transformation was successful. - -## Transform recipient vector into recipient strain - -Introduce pSTNSK-Cm into KPPR1 strain with respective deleted gene via electroporation. - -1. Inoculate 100 mL LB with 1 mL of overnight culture. Incubate at 30C with stir bar at ~500 rpm until it reaches OD600 of 0.6 to 0.8 (approximately 4 hours). -2. Centrifuge cells at 6500 rpm for 5 min. Remove supernatant and wash twice with 50 mL of ice cold glycerol (resuspend with glycerol and then centrifuge it again). -3. Resuspend cells in residual glycerol solution after final wash. Mix 50 uL of dense suspended cells with 1 ug of plasmid DNA (<= 6 uL) in an electroporation cuvette. Use the manual mode on the electroporation machine and use the arrows to increase the voltage to 2.5V. Make sure that the cuvette is perfectly vertical before pressing the pulse button, or else there's a very large chance of arcing. Add 900 uL SOC medium immediately after electroporation. -4. Incubate sample for 1 hour at 30 degrees C on a rotary shaker at 150 rpm and plate onto low salt LB-Cam25 plates. -5. Incubate plates at 30 degrees C overnight. -6. Streak colonies onto low salt LB-Cam25 plates, and then again the next day. -7. PCR Screen for pSTNSK-Cm. -8. Freeze successful colonies and use for complementation. - - -## Deliver Tn7 into Klebsiella - -Using classical mating, integrate the Tn7 transposon into the KPPR1 chromosome. - -1. Start overnight cultures of the donor strain and the recipient strain. -2. Pellet 500 uL of donor and recipient strains in seperate Eppendorf tubes (5 min max speed, Klebsiella may need a longer spin since it doesn't form a tight pellet) -3. Remove all but 50 uL of LB from each tube and re-suspend each pellet in the remaining LB, then combine the resuspensions in a single tube. -4. Plate all 100 uL of mixed suspension on an LB-DAP plate and incubate at 30 degrees Celsius for 5 hrs. -5. Use a cotton swab dipped in PBS to scrape up the cells, agitate to dislodge the bacteria in 1 mL of PBS in a 2 mL tube. -6. Create serial dilutions out to 10^-4, and plate out onto LB-GM plates. -7. Incubate plates at 42 C for 4 hours, then 37 C for 18 hrs. -8. Streak out single colonies onto LB-Gm, LB-Carb, LB-Kan and LB-Cam plates. - * Look for resistance to Gm and Kan, sensitivity to Carb and Cam. -9. Screen complementation candidates using PCR, looking for pGP-Gm, pSTNSK, and looking at the Tn7 site. - * If pSTNSK is still in the complemented strain, then streak onto LB-Gent10 and incubate at 42C for 5 hours again - * If pGP-Gm is still in the complemented strain, then streak onto LB, LB-Gent10, and LB-Carb100 until the strain is sensitive to Carb and resistant to Gent -10. Freeze successful candidates and send to MIGS for sequencing. - - - From e9b4de9e467bb857be08726b5590fe1b775aefd5 Mon Sep 17 00:00:00 2001 From: Ani Desikan Date: Fri, 21 Jul 2023 14:30:29 -0500 Subject: [PATCH 24/24] remove gene complementation --- README.md | 1 - 1 file changed, 1 deletion(-) diff --git a/README.md b/README.md index c7c0149..9ed3fd0 100644 --- a/README.md +++ b/README.md @@ -4,7 +4,6 @@ ### Molecular Biology - [Gene Deletion in *V. fischeri*](gene-deletion.md) -- [Gene Complementation in *K. Pneumoniae*](gene_complementation_klebsiella.md) - [Gibson Assembly](gibson-assembly.md) - [dNTP stocks for PCR](molecular-dntps.md) - [Semi-arbitrarily-primed PCR to identify transposon insertion sites](arbitrarily-primed-pcr.md)