Team:Heidelberg/Templates/DelH week15

From 2013.igem.org

Contents

05-08 - 13-08-13

Generation of DelH Plasmid 01-08

Colony-PCR Conditions CP.W15.A

From colonies inoculated on 04-08

Reagent 3c 3c 3c 3c 3c
Expected length [kb] 663 663 663 663 663
Named 3c 1 3c 2 3c 3 3c 4 3c 5
Template (1 µl) 1 colonies of E3c 1 colonies of E3c 1 colonies of E3c 1 colonies of E3c 1 colonies of E3c
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 5 µl 5 µl 5µl 5 µl 5 µl
Reagent 4a 4a 4a 4a 4a
Expected length [kb] 663 663 663 663 663
Named 4a 1 4a 2 4a 3 4a 4 4a 5
Template (1 µl) 1 colonies of E4a 1 colonies of E4a 1 colonies of E4a 1 colonies of E4a 1 colonies of E4a
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 5 µl 5 µl 5 µl 5 µl 5 µl
Reagent 6b 6b 6b 6b 6b
Expected length [kb] 663 663 663 663 663
Named 6b 1 6b 2 6b 3 6b 4 6b 5
Template (1 µl) 1 colonies of E6b 1 colonies of E6b 1 colonies of E6b 1 colonies of E6b 1 colonies of E6b
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 5 µl 5 µl 5 µl 5 µl 5 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 (touchdown -0.5°C) 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.15.2 Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR)
l1-5: 5 seperate colonies which were together in 3a PCR earlier, l6: 2log
no bands = no colony positive
Fig.15.1 Gel of picked colonies with insert DelH-BB (loaded 20 µL of PCR)
l1-5: 5 seperate colonies which were together in 3a PCR earlier, l6: 2log, l7-11: 5 seperate colonies which were together in 3a PCR earlier,
no bands = no colony positive


None of the gels shows expected band.

=> None of the colonies is positive.


Miniprep

Of colonies 6b(1)-6b(5), 6a(1), 6a(2), 3c(3), a miniprep was performed.

Sample Concentration [ng/µl] µl avaiblable
6b(1) 33.8 18
6b(2) 33.4 18
6b(3) 36.9 18
6b(4) 59.6 18
6b(5) 18 18
6a(1) 60 18
6a(2) 62.3 18
3c(3) 40.9 18

Test Restriction Digest

Sample Miniprep [µl] CutSmart Buffer [µl] NotI-HF [µl] H2O [µl] Total [µl]
6b(1) 15 2 1 3 20
6b(2) 15 2 1 3 20
6b(3) 15 2 1 3 20
6b(4) 10 2 1 8 20
6b(5) 15 2 1 3 20
6a(1) 10 2 1 8 20
6a(2) 10 2 1 8 20
3c(3) 15 2 1 3 20

Result

Expected bands: 11,079 bp, 6,201 bp, 3,998 bp, 2,371 bp

Fig.15.3 test digest of listed colonies (loaded 20 µL of PCR)
l1-5: 5 seperate colonies of 6b, l6: 2log, l7 6a(1), l8 6a(2), l9 3c(3)
results are shown in table below
Expected bands Present bands
11.079 bp  ?
6.201 bp no
3.998 bp yes
2.371 bp no

in silico PCR of BB shows the following result:

2 fragments generated:
1: 3.998 bp - From NotI[9] To NotI[4007]
2: 1.093 bp - From NotI[4007] To NotI[9]

But an additional band between 1-2 Kb is present!

=> Talk to Advisors, because it isn't what we expected. What could it be?

Possible next steps:

a) Wait for the new strain of D. acidovorans SHP1
b) Perform a re-PCR of minipreps (one with BB and one with DelH G0 fragment)


Generation of Backbone pSB6A1-lacI-mRFP

Restriction Digest with DpnI

After consulting the advisors, we decided not to continue the strategy with the old construct and wait with DelH until the new strain of D. acidovorans arrives. Until then, we digest the backbone (which is used also in following experiments) with DpnI to get rid of any remaining template.

Reagent Amount [µl]
Gel extracted lineralized by PCR (template=10.4 Kb) 17
DpnI 1
CutSmart buffer 2
Total 20
  • 1 h incubation at 37°C
  • 20 min inactivation of enzyme by heat shock at 80°C for 20 min


Purification

  • As template, the PCR of 10,4 PCR, which was digested with DpnI on 06-08, was used.
  • Purification was performed with nucleotide removal kit (wrong column was used, but there was some yield at the nanodrop)


=> Again, checking on gel (2 µl + 8 µl 1xloading dye)

Fig.15.4 checking concentration of purified BB (PSB6A1-lacI-mRFP) (loaded 2 µL of PCR)
l1 2log ladder l2: pSB6A1-lacI-mRFP
l2 show expected band at 5 Kb
=> Concentration at nanodrop was 27.2 ng/µl


Amplification of DelH G0

PCR Conditions G0.W15.A

Reagent Amount [µl]
Expected length [Kb] 18.521
Named G0
Template 1 µl New strain culture SHP1 (resuspended pellet)
Primer fw 10 µM 1 µl short2
Primer rev 10 µM 1 µl HM08
Phusion Flash Ready Mix 10
ddH2O 5
DMSO 1
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 18.521 Kb

Fig.15.5 amplified fragment DelH-complete (loaded 20 µL of PCR)
l1: G0, l21Kb+ ladder
no band at 18 Kb only a specific band at 5 Kb, but that is not useful

Gel does not show the expected band.

=> Further improve PCR conditions.


PCR Conditions G0.W15.B

Reagent Amount [µl]
Expected length [Kb] 18.521
Named G0
Template Picked colony
Primer fw 10 µM 1 µl short2
Primer rev 10 µM 1 µl HM08
Phusion Flash Ready Mix 10
ddH2O 5
DMSO 1
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start
=> Becaus it worked well we amplified 3x G0 with the same conditions.

PCR Conditions G0.W15.C

3x 20 µl PCR reactions were prepared

Reagent Amount [µl]
Expected length [Kb] 18.521
Named G0
Template Picked colony
Primer fw 10 µM 1 µl short2
Primer rev 10 µM 1 µl HM08
Phusion Flash Ready Mix 10
ddH2O 5
DMSO 1
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 18 Kb

Fig.15.7 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: band at 18 Kb was cut out l5-6: band at 13 Kb was cut out
Fig.15.6 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: show the expected band at 18 Kb and l5-6: show the specific band at 13 Kb


Gel shows the expected bands.

=> Fragments were cut and gel extracted.


Amplification of DelH G1/2a

PCR Conditions G1/2a.W15.A

Reagent Amount [µl] Amount [µl]
Expected length [Kb] 13.083 13.083
Named G1/2a G1/2a
Template Picked colony Picked colony
Primer fw 10 µM 1 µl short2 1 µl short2
Primer rev 10 µM 1 µl HM06 1 µl HM06
Phusion Flash Ready Mix 10 10
ddH2O 5 4
DMSO 1 0
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Fig.15.7 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: band at 18 Kb was cut out l5-6: band at 13 Kb was cut out
Fig.15.6 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: show the expected band at 18 Kb and l5-6: show the specific band at 13 Kb


Gel shows the expected bands.

=> Fragments were cut and gel extracted.


PCR Conditions G1/2a.W15.B

Reagent Amount [µl]
Expected length [Kb] 13.083
Named G1/2a
Template Picked colony
Primer fw 10 µM 1 µl short2
Primer rev 10 µM 1 µl HM06
Phusion Flash Ready Mix 10
ddH2O 5
DMSO 1
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 13 Kb

Fig.15.9 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: band at 18 Kb was cut out l5-6: band at 13 Kb was cut out
Fig.15.8 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1-3:3x G0 (complete fragment amplified with DN11 & HM08), l4: 1kb+ ladder, l5-6:2x G1/2a (complete fragment amplified with DN11 & HM06)
l1-3: show the expected band at 18 Kb and l5-6: show the specific band at 13 Kb


Gel shows the expected bands.

=> Fragments were cut and gel extracted.


Amplification of DelH G2b

PCR Conditions G2b.W15.A

Reagent Amount [µl] Amount [µl]
Expected length [Kb] 5.472 5.472
Named G2b G2b
Template Picked colony Picked colony
Primer fw 10 µM 1 µl HM07 1 µl HM07
Primer rev 10 µM 1 µl HM08 1 µl HM08
Phusion Flash Ready Mix 10 10
ddH2O 5 4
DMSO 1 0
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
68 5
72 120
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Fig. 15.10: Gel of amplified DelH G2b fragment l1:2log, l2-3:G2b amplified


Summary of Gel Extracted Fragments

Concentrations (by nanodrop) are summarized in the following table and gel picture.

Fragment Concentration [ng/µl]
G0 3.5
G1/2a 4.9
G2b 18.6
Fig.15.11 Analyse-gel of DelH-fragments (loaded 5 µL of G0 and G1/2a and 1 µl of G2b)
lane 1 1kb+ ladder, lane 2 G0 (complete DelH with DN11 & HM08), lane 3 G1/2a (part of DelH with DN11 & HM06), lane 4 G2b (part of DelH with HM07 & HM08)


Generation of DelH Plasmid 09-08

Gibson Assembly

We decided two assemble 2 different constructs and one control (only the backbone).

Construct G0 [µl] G1/2a [µl] G2b [µl] BB [µl] 2x Gibson Master Mix [µl] Final amount [µl]
1 9 0 0 1 10 20
2 0 8 1 1 10 20
Control (C) 0 0 0 1 (+9 ddH2O ) 10 20
  • Incubated 1 h at 50°C

Electroporation

  • Of each Gibson assembly, 5 µl are diluted in 10 µl ddH2O
  • The rest of the Gibson assembly is stored at -20°C


Conoly-PCR Conditions CP.W15.A

Picked 8 colonies from plates with 10µl and 100 µl construct 1, and plate with 100 µl of construct 2. 1 additional colony was picked from plate with 10 µl of construct 2 (because there were no more cells) and 1 colony of the control(100µl), and one colony of the control (100µl) with new electrocompetent cells.

  • In the following table construct 1 is named C1, and construct 2 = C2 and Control = Co
Reagent C1 (10 µl) C1 (10 µl) C1 (10 µl) C1 (10 µl) C1 (10 µl) C1 (10 µl) C1 (10 µl) C1 (10 µl)
Expected length [bp] 663 663 663 663 663 663 663 663
Named 1 2 3 4 5 6 7 8
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6µl 6 µl 6 µl 6 µl 6 µl 6 µl
Reagent C1 (100 µl) C1 (100 µl) C1 (100 µl) C1 (100 µl) C1 (100 µl) C1 (100 µl) C1 (100 µl) C1 (100 µl)
Expected length [bp] 663 663 663 663 663 663 663 663
Named 9 10 11 12 13 14 15 16
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl 6µl 6 µl 6 µl 6 µl
Reagent C2 (10 µl) C2 (10 µl) C2 (10 µl) C2 (10 µl) C2 (10 µl) C2 (10 µl) C2 (10 µl) C2 (10 µl))
Expected length [bp] 663 663 663 663 663 663 663 663
Named 17 18 19 20 21 22 23 24
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl
Reagent C2 (10 µl) C2 (100 µl) Co (100 µl) Co(100 µl)
Expected length [bp] 663 663 no product no product
Named 24 25 26 27
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 (touchdown -0.5°C) 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.15.12 screening PCR with DN07 & VF2 (loaded 1 µL of PCR)
l1:2log ladder,l2-17: colonies C1, l18-24: colonies C2, l25:2log ladder
None of the colonies showed expected band, instead, all have band at ~6 kb

Gel shows no banda at 663 bp. Instead, they show an unexpected band at ~6 Kb.

=> None of the colonies is positive.


Conoly-PCR Conditions CP.W15.B

Picked 20 colonies from plates with 10 µl and 100 µl construct 1 and construct 2 (80 colonies). 4 colonies were picked from the control (10 µl) and control (100 µl).

  • In the following table construct 1 is named C1, and construct 2 = C2 and Control = Co
Reagent C1 (10 µl) C1 (10 µl) C1 (10 µl) C1 (10 µl) C1 (100 µl) C1 (100 µl) C1 (100 µl) C1 (100 µl)
Expected length [bp] 663 663 663 663 663 663 663 663
Named A B C D E F G H
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl
Reagent C2 (10µl) C2 (10µl) C2 (10µl) C2 (10µl) C2 (100µl) C2 (100µl) C2 (100µl) C2 (100µl)
Expected length [bp] 663 663 663 663 663 663 663 663
Named I J K L M N O P
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl
Reagent Co (10µl) Co (10µl) Co (10µl) Co (10µl) Co(100µl) Co(100µl) Co(100µl) Co(100µl)
Expected length [bp] 663 663 663 663 663 663 663 663
Named C1 C2 C3 C4 C5 C6 C7 C8
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl 6 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 (touchdown -0.5°C) 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.15.13 screening PCR with DN07 & VF2 (loaded 1 µL of PCR)
l1-9: colonies A-I, l10:2log ladder, l11-17: colonies J-P, l12:2log ladder, l13-18: PCR performed with 6 colonies of the control plate
Neither the colonies A-P show the expected band nor the control shows any band
=> New Gibson Assembly will be performed

Gel shows no banda at 663 bp.

=> Neither the colonies A-P show the expected band, nor the control shows any band.


Generation of DelH Plasmid 11-08

Gibson Assembly

Because of the past results, we assume that the colonies can grow because the backbone religates. We also found out that the first overhang of the backbone has some homologity with a sequence at the end and we can assume that with Gibson, it primes itself.
So in the following step we will use less backbone. We prepared an 1:10 dilution of the backbone construct.

Reagent Concentration [ng/µl] Amount [µl] Mix A Amount [µl] Mix B Amount [µl] Mix C
G0 3.5 9 0 0
BB 1.8 1 1 1
Gibson Assembly Master Mix 2x 10 10 0

The time constant for the electroporation was unexpectedly low, hopefully high enough...

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