Team:Heidelberg/Templates/DelH week21

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Revision as of 21:11, 1 October 2013

16-09 - 22-09-13

Generation of DelH Plasmid pHM04 15-09

Colony-PCR Conditions CP.W21.A

Result

Expected band: 663 bp

Fig.21.1 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I(loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 1A-4A

Fig.21.2 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 4B-7B

Fig.21.3 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I(loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 7C-9H

Fig.21.4 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 10A-12H

The following colonies seem positive, because they show the expected band at 663 bp.

Test Restriction Digest

24 of these positive screened colonies were minipreped and digested with PvuI

Another restriction digest was performed. Colony 7D was digested with EcoRI-HF

Result

Expected bands: 11,621, 8,690 and 2,685 bp (PvuI) and 17,801 and 5,105 bp (EcoRI)

Fig.21.5 restriction digest of positive colonies with PvuI (loaded 10 µL of PCR)
l1:2 log ladder, l2-l13:colonies 1E-11E

None of the analyzed clones shows correct band pattern.

=> Clones are discarded.

Amplification of Backbone

New Template

Because the backbone amplification yield from pSB6A1 + BBa_J04450 from Spring distribution 2012 (plate 1, well K1) was so low and the screening did not result in positive restriction digests with PvuI or EcoRI, we go a step back and transform E.coli ToP10 with a new pSB6A1 part from the 2013 distribution. Therefore, we transform on the one hand with the part on plate 2 well 2L and on the other hand with the part from the plate 5 well 1K from the Spring 1023 diestricbution.
Aditionally we ran a PCR directly from the two wells with the backbone primers (HM20, HM17 & FS77).

PCR Conditions BB.W21.A

Amplification of pSB6A1 directly from the parts registry
1x PCR with HM_20 and 1x PCR with FS_77

Result

Expected band: 4. Kb

Fig.21.6 Amplification of backbone pSB6A1 directly from two wells (loaded 10 µL of PCR)
l1:2log ladder, l2HM20 aus 1K, l3FS77 aus 1K, l4HM20 aus 2L, l5FS77 aus 2L

Fig.21.7 Amplification of backbone pSB6A1 directly from two wells (loaded 10 µL of PCR)
l1:2log ladder, l2HM20 aus 1K, l3FS77 aus 1K, l4HM20 aus 2L, l5FS77 aus 2L - was cut out

Amplification worked with the template pSB6A1 from the biobrick distribution 2013 plate 2, well L2.

=> The fragment was gel extracted with QIAquick gel extraction kit, digested with DpnI, and gel extracted again, resulting in final concentrations of 10.7 ng/µl for FS_77 and 4.8 ng/µL for HM_20

=> PCR will be repeated with several samples from the colonies transformed with pSB6A1 from the biobrick distribution 2013 plate 2, well L2

PCR Conditions BB.W21.A

Amplification of backbone fragment pSB6A1 from colonies of DH10ß, transformed with pSB6A1 from the biobrick distribution 2013 plate 2, well L2
4x PCR with HM_20 and 4x PCR with FS_77

Result

Expected band: 4.4 Kb

Fig.21.8 Amplification of backbone pSB6A1 from colony transformed with pSB6A1 from biobrick distribution 2013 plate 2, well L2
l1:2log ladder, l2 FS_77, l3HM20 after DpnI digest

Fig.21.9 Amplification of backbone pSB6A1 from colony transformed with pSB6A1 from biobrick distribution 2013 plate 2, well L2
l1:2log ladder, l2 FS_77, l3HM20 after DpnI digest - was cut

Gel shows amplification of fragment.

=> Fragment was cut and gel extracted, then DpnI digested and gel purified again.

Generation of DelH Plasmid pHM04 18-09

Gibson Assembly

• Incubate for 1 h at 50°C

Electroporation

• Afterwards 3 µl of each Gibson assembly were taken and 2 µl ddH₂O were added
• With 17 µl of the Gibson assembly, isopropanol purification was performed, DNA was eluted in 20 µl ddH₂O
• 6 different electroporations in E.coli BL21 DE3 were performed and grown at RT (see scheme below)

Colony-PCR CP.W21.A

Result

Expected band: 663 bp

Fig.21.10 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I(loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies A1-C1

Fig.21.11 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies C2-E2

Fig.21.12 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I(loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies E3-G3

Fig.21.13 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies G4-H12

The following colonies seem positive, because they show the expected band at 663 bp

Colony-PCR CP.W21.B

Result

Expected band: 663 bp

Fig.21.14 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II(loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies A1-B12

Fig.21.15 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies C1-D12

Fig.21.16 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies E1-F12

Fig.21.17 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies G1-H12

The following colonies seem positive, because they show the expected band at 663 bp

Test Restriction Digest

The positive screened colonies were digested with PvuI.

Result

Expected bands: 11,621, 8,628 & 2,685 bp

Fig.21.18 Test digest with PvuI (loaded 10 µL of PCR)
l1: 2log ladder, l2: C3,l3: C5, l4: D1, l5: C7, l6: C11,l7: C12, l8: D3, l9: D5, l10: D7,l11: D8, l12:D9, l13:D12

Fig.21.19 Test digest with PvuI (loaded 10 µL of PCR)
l1: 2log ladder, l2: E2,l3: F4, l4: F9, l5: F11, l6: G6,l7: G11, l8: G12 l9: H6, l10: H8,l11: H11A, l12:H11B, l13:II B6

Fig.21.20 Test digest with PvuI (loaded 10 µL of PCR)
l1: 2log ladder, l2: II B7,l3: II G3, l4: II G10, l5:II H2

The following colonies show the correct pattern:

Sequencing

Clones C5, C7, C12, G11, II A6 and II D2 were send for sequencing with VF2.

Result

Amplification of Backbone

PCR Conditions BB.W21.B

Amplification of backbone parts registry well 2L, transformed 19-09 using HPLC purified HM11

Result

Expected band: 4.4 Kb

Fig.21.21 gel of amplified BB with HM11 (loaded 20 µL of PCR)
l1: BB amplified with HM11 HPLC-purified at 68°C td l2: 2log
no product

Gel does not show amplified backbone fragment.

=> Further optimize PCR conditions.

PCR Conditions BB.W21.C

Result

Expected band: ~ 4.4 Kb

Fig.21.22 Gel of BB amplified with HM11: l1:2 log ladder, l2: BB without DMSO, l3: BB with DMSO

Gel shows strong and specific amplification of backbone.

=> Fragments were cut and gel isolated.

Generation of DelH Plasmid pHM04 20-09

Gibson Assembly

• Incubate for 1 h at 50°C

Electroporation

• Afterwards 3 µl of each Gibson assembly were taken and 2 µl ddH₂O were added
• With 17 µl of the Gibson assembly, isopropanol purification was performed, DNA was eluted in 20 µl ddH₂O
• 6 different electroporations in E.coli DH10ß were performed and grown at RT (see scheme below)

New HPLC Purified Primers

As pointed out before, we suspect DelH to be toxic for E. coli and therefore, only colinies containing mutated constructs survive. To avoid this, we ordered new shorter and HPLC purified primers for the assembly of pHM04.

Mutagenesis of DelH clones I 6B and 15

Strategy

As our clones which have the DelH construct keep on having mutations and deletions we decided to fix this problem by carrying out site-directed mutagenesis. The targeted mutations are the deletion of parts of the ribsome binding site in clone I6B and the deletion at the beginning of DelH in clone 15. For this we make use of two restriction enzymes (MfeI & XbaI) which both only cut in the construct once. By this we excise the fragment with the mutation. The larger fragment (17.3 kbp) without mutation is kept.
Furthermore we ordered four new primers. The smaller fragment is then PCR amplified with those primers in two fragments. Primer HM22 and HM23 cure the mutations. With those primers also recognitions sites for the restriction enzyme BbsI are introduced, which is needed for religation of the two fragments. BbsI cuts two basepair after its recognition site.

HM21 and HM24 are the corresponding primer. They introduce XbaI and MfeI sites as well as in each on BbsI recognition site. Those are needed for religation.

In the end the two fragments are both digested with BbsI. In the adjacent ligation the two fragments can ligate together due to the BbsI recognition sites. Furthermore BbsI opens the introduced MfeI and XbaI sites through which the two fragments can ligate with the digested 17.3 kb fragment.

However the XbaI site is not introduced correctly again. Therefore we can test if the mutagenesis had been successful by digesting with XbaI. If the site is not present anymore we successfully got rid of the mutations.

New Primers

Purification of Midiprep of Clone I6B

Fig.21.23 Midipreps of colonies I6B and 15

As can be seen in figure 21.23, the midi-prep of colony I 6B is contaminated. Thus, we tried to purify only the largest band by gel extraction with the QIAEX II Gel Extraction Kit (Qiagen). The procedure was as following:

• Application of 2 µl of midiprep on gel (4596.7 ng/µl --> ~10 µg)
• Excision of band at about 17.4 kb
• Gel extraction of DNA with QIAEX II Gel Extraction Kit (Qiagen)

Result

• The concentration of the purified fragment was only 4.7 ng/µl after extraction.
  

=> We are trying to elute again.

• The second elution did not work either

Amplification of HM23 to HM24

1x 20 µl for each colony

Result

Gels for amplification of HM23 to HM24

Fig.21.24	Fig.21.25	Fig.21.26	Fig.21.27
Fig.21.28	Fig.21.29

Amplification of HM21 to HM22

Result

Gels for amplification of HM21 to HM22

Fig.21.30

Fig.21.31

Restriction Digest of pHM04 (I6B) with XbaI and MfeI

• Incubation for 2 h at 37°C

Result

• Expected band pattern: 17.3 kbp, 5.3 kbp

Gels for restriction Digest of pHM04 (I6B) with XbaI and MfeI

Fig.21.32

Fig.21.33

Fig.21.34

Fig.21.35

• The MfeI already expired in 2011

=> We will test digest the construct again with a new enzyme

Restriction Digest of pHM04 (I6B) with XbaI and MfeI

Result

Due to the contamination in DelH it was not possible to decide whether the fragment really had been digested. Thus we have to test whether MfeI Cutting site is really present.

Test for MfeI Cutting Site

Fig.21.36 Test if MfeI restriction site is present

Incubation at room temperature over night.
--> As the upper band is lower for the digest with both enzymes the MfeI restriction site is apparently present. Therefore the mutagenesis is tried.

Restriction Digest of Fragment HM21-HM22 (I6B) and HM23-HM24 (I6B) with BbsI

PCR fragments: HM21-HM22 (36.6 ng/µl) and HM23-HM24 (32.4 ng/µl)

Ligation of Digested pHM04 (I6B), HM21-HM22 and HM23-HM24

Transformation of Ligation

1 µl and 5 µl were transformed in 50 µl electrocompetent DH10β each by electroporation. The cells were recovered in 400 µl SOC media and plated on LB Amp.