Validation of the strain from Marahiel by Colony PCR

Primers used

TycA_A-domain: IK09, IK10
TycB1_A-domain: IK11, PW01
TycB1_E-domain: PW02, PW03

Conditions

Colony-PCR of B. brevis. Lane 4: NEB 2-log; lane 1: primers IK09+IK10; lane 2: primers IK11+PW01;lane 3: primers PW02+PW03

Handling of the strain

• received Brevibacillus parabrevis ATCC 8185 from Marahiel lab
• plate stood 1 week at RT in Marburg => wet, stinks
• plate cells on LB, inoculate 2x YT medium
• scratch everything from plate, centrifuge, wash with H₂O, centrifuge, resuspend in H₂O

Result

Three bands were visible in the UV-chamber. The three respective lanes implicate a fragment size of 1.5kb, 1.5kb and 1.2kb. This matches well the expected sizes. Therefore we can conclude that our colony PCRs have been successful!

Date: 19-07-2013

Evaluation of the strain

Result

Colonies grew over night -> as colony PCRs were positive and the strain was cultivatable, the plate of marburg seemed to be intact (despite long storage under bad conditions).

Amplifications

Amplification of fragment 1

A

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5

Result band in gel--> cut and extraction

B

all constructs extracted by gel extraction; fragment 15 missing -> Optimization

Amplification of fragment 2

A

Lane 1: NEB 2-log; lane 2: pJM5 with primers IK21+IK22; lane 3: B. parabrevis with primers IK13+IK14; lane 4: B. parabrevis with primers IK15+IK12

Result Successful

B

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction

PCR redone over night as gel was clumpy and did not show the expected fragments.

Amplification of fragment 3

A

Lane 1: NEB 2-log; lane 2: pJM5 with primers IK21+IK22; lane 3: B. parabrevis with primers IK13+IK14; lane 4: B. parabrevis with primers IK15+IK12

Result no band; new PCR settings

B

Lane 1: NEB 2-log; lane 2: B. parabrevis with fragment 3

Result xxxxxxxx; new: touchdown PCR with Phusion Flash

C

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5

Result Just a light band, have to improve conditions.

D

PCR Results of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and extracted with Qiagen Gel Extraction Kit

Amplification with Q5

E

Amplification with Phusion Flash

F

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction

PCR redone over night as gel was clumpy and did not show the expected fragments.

Amplification of fragment 4

A

Lane 1: NEB 2-log; lane 2: pJM5 with primers IK21+IK22; lane 3: B. parabrevis with primers IK13+IK14; lane 4: B. parabrevis with primers IK15+IK12

Result Successful

B

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction

Result PCR redone over night as gel was clumpy and did not show the expected fragments.

C

all constructs extracted by gel extraction; fragment 15 missing -> Optimization

Amplification of fragment 5

A

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5

B

PCR Results of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and extracted with Qiagen Gel Extraction Kit

Amplification of fragment 6

A

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5

B

PCR Results of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and extracted with Qiagen Gel Extraction Kit

Amplification of fragment 7

A

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5

B

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction

PCR redone over night as gel was clumpy and did not show the expected fragments.

Amplification of fragment 8

A

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5

B

PCR Results of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and extracted with Qiagen Gel Extraction Kit

Amplification of fragment 9

A

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction

PCR redone over night as gel was clumpy and did not show the expected fragments.

B

all constructs extracted by gel extraction; fragment 15 missing -> Optimization

Amplification of fragment 10

A

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction

PCR redone over night as gel was clumpy and did not show the expected fragments.

B

Amplification of fragment 11

A

amplification of fragments 11 & 12. Fragment 11 is clearly visible, fragment 12 is missing

Amplification of fragment 12

A

amplification of fragments 11 & 12. Fragment 11 is clearly visible, fragment 12 is missing

Amplification of fragment 13

A

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction

PCR redone over night as gel was clumpy and did not show the expected fragments.

B

all constructs extracted by gel extraction; fragment 15 missing -> Optimization

Amplification of fragment 14

A

all constructs extracted by gel extraction; fragment 15 missing -> Optimization

Amplification of fragment 15

A

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction

PCR redone over night as gel was clumpy and did not show the expected fragments.

B

Analysis of DNA concentrations of previously amplified fragments

Analytical Gel Electrophoresis 0.8% agarose Scheme: each 1µL DNA (eluation in 20µL water)+ 3 µL loading dye
2log ladder, 4 µL 24.7:4, 25.7:1,2,3,7, 26.7.:3,5,6,8,3', 28.7.1,2,4,9,10,13,14 2log ladder, 4 µL

quantification gel of the fragments extracted from gel. See table for precise amounts

Amplifications Round I

Analysis of DNA concentrations of fragments amplified so far

Analytical Gel Electrophoresis 0.8% agarose Scheme: each 1µL DNA (eluation in 20µL water)+ 3 µL loading dye
2log ladder, 4 µL 24.7:4, 25.7:1,2,3,7, 26.7.:3,5,6,8,3', 28.7.1,2,4,9,10,13,14 2log ladder, 4 µL

quantification gel of the fragments extracted from gel. See table for precise amounts

--> next steps:

• reamplify fragments 3, 13 & 15 (optimize)
• reamplify fragments 5, 6, 7 & 8 for higher yield
• Plan B: Re-PCR from low-yield fragments

Reamplification of low yield fragments: 2, 7, 6, 8, 5

• fragments 2 & 7 (Two-Block A)
• fragments 6 & 8 (T 100)
• fragment 5 (Two-Block B)

For all amplifications 2x 20µl PCRs were used. PCR was run with Q5

PCRs were conducted 2°C lower than Tm for all constructs. Good yields were obtained for fragments 2, 5 & 7, unspecific bands for fragments 6 & 8. All bands with right size cut out for gel-extraction.

The PCRs were run 2°C under Tm for the primers, as specific products were obtained in the previous amplifications, but yield after gel-extraction was low.

Amplification of 3, 5, 15

PCRs of Fragments 3, 5 and 15 with phusion flash. And same PCR touchdown program. Two-block A.

PCRs of fragments 3, 13 and 15. 2x20µl were used for increase of yield

fragment 3: IK15+13 fragment 5: IK12+PW11 fragments 15: Ik12+Pw13

Fragment 12 PCR

Amplification of Fragment 12

Fusion/Q5

Fusion Touch Down

Analysis of DNA concentrations

quantification gel with 4 µl 2log ladder. 1 µl sample was mixed with 3 µl loading dye

• Gel-extraction of fragments 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, 15
• DNA was eluted with 30 µl instead of 20 µl

• Reamplify fragments 6 & 8 and use gel-extraction kit for long fragments
• Optimize conditions for fragment 12

Amplification of fragments 6, 8, 12

Q5 6 IK12,18 8 IK18,20

Phusion Flash, Touchdown 12 PW09,10

Assembly Round I

Tripeptide-I-NRPS Assembly

Gibson Assembly to Tripeptide-I-NRPS

• • DO ALL WORK ON ICE!!
  • mix 1,08 µl of fragment 4, 3; 24 µl of fragment 5 & 5.68 µl of fragment 6
  • add 10 µl of Gibson master mix
  • let incubate for 60 minutes at 50°C
  • split up 20 µl into:
    • 15 µl for isopropanol / ethanol DNA precipitation
      • add 60 µl (4 volumes) of isopropanol and centrifuge for 20 mins at full speed
      • discard supernatant
      • wash pellet in ethanol, centrifuge for 5 mins and discard supernatant, let dry
      • let resuspend in 10 µl H20 and electroporate
    • to the remaining 5 µl, add 10µl ddH2O
      • electroporate with 1 µl
      • electroporate with 14 µl
  • use a negative control (10 µl H2O)

CPEC Assembly of Tripeptide-I-NRPS

• • DO ALL WORK ON ICE!!
  • mix fragment - DNA aequimolarly --> 10 µl
  • add 10 µl of Phusion flash master mix

• • split up 20 µl into:
    • 5 µl for chemical transformation
    • 10 µl for isopropanol / ethanol DNA-precipitation
      • see above
    • add 10 µl ddH2O to the remaining 5 µl
      • electroporate 1 µl
      • electroporate 14µl

Gibson Assembly Results

• evaluation of the plates obtained by electroporation our first assembly our first assembly
• picking of colonies from positive plates
  • 6 colonies were picked: 3 from plate 5 and 3 from plate 6
• plates:
  • 1: Cloning with CPEC - Electroporation with cleaned up DNA
  • 2: Cloning with CPEC - Electroporation with 1µl DNA + H2O
  • 3: Cloning with CPEC - Electroporation with 14µl DNA + H2O
  • 4: Cloning with Gibson - Electroporation with cleaned up DNA
  • 5: Cloning with Gibson - Electroporation with 1µl DNA + H2O
  • 6: Cloning with Gibson - Electroporation with 14µl DNA + H2O
  • 7: Negative Control - Electroporation with H2O
  • 8: Cloning with CPEC - Chemical Transformation

plate 1	plate 2	plate 3	plate 4
plate 5	plate 6	plate 7	plate 8

PCR of fragments 3, 12, 6, 8, 11, 13 & 15 - the latter three were conducted with 3 samples each in order to gain more yield

We noticed that we mixed up fragments 1 and 4 thus neither the Gibson nor the CPEC could have worked. We are going to repeat this step next week

Amplifications Round II

Fragment 3

3 IK12,15

Fragment 6

primer: IK18 & IK12

Fragment 8

primer: IK20 & IK12

Fragment 12

12 PW09,10

Amplifications Round III

• Reamplification of fragments 3, 6, 8, 11, 12, 13 & 15
  • Primers used:
  • fragment 3: IK15 & IK12
  • fragment 6: IK18 & IK12
  • fragment 8: IK20 & IK12
  • fragment 11: PW07 & PW08
  • fragment 12: PW09 & PW10
  • fragment 13: PW11 & IK12
  • fragment 15: PW13 & IK12

OPTIMIZATION FOR FRAGMENTS 3 & 15 NEEDED, all other fragments obtained

PCR of fragments 3 and 15 for optimization. Phusion Flash Touchdown PCR was used on 02.08.13. Gel was run and picture taken on 03.08.13

• PCRs of Fragments 3 and 15 with phusion flash. T100.

fragment 3: IK15+IK12 fragments 15: IK12+PW13

• gel-extraction of fragments 6, 8, 11, 12 & 13 using long fragment kit

DNA Concentration Measurement

• Quantification Gel of Gibson Assembly Colony Minipreps and fragments 6,8,11,12,13

fragments 6, 8, 11, 12 and 13 were extracted from gel with long fragment kit. The minipreps of the Gibson transformants contain plasmids that are way too low - the reason for this is that primers for fragments 1 and 4 were mixed up

• Gel extraction of fragments 3, 15 and the two unknown samples in the freezer
• Quantification gel of those two fragments
  • unfortunately the quantification gel did not show any usable concentrations
  • --> reamplification of fragments 3 & 15 - and, as yield was low of fragments 11 & 12

Fragment 3

fragment 3: IK15+IK12 fragments 15: IK12+PW13

Fragment 11

11 PW07,08

Fragment 12

12 PW09,10

Fragment 15

fragments 15: IK12+PW13

Results Amplification Round III

PCR of fragments 3, 11, 12, 13. All lanes were bright and have the right size.

• Samples of Amplification Round III (fragments 3, 11, 12 & 13) were run on gel
• gelextraction

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DNA concentrations of fragments

Analysis of DNA concentration

• Concentrations obtained through quantification gel. There are 20µl of all fragments.

Complete List of DNA concentrations of all fragments

Gibson Assembly

fragment date length

1. 29.7 2500
2. 29.7 2000
3. 5.8. 4000
4. 29.7 2500
5. 29.7 3000
6. 29.7 7000
7. 31.7 2000
8. 29.7 7000
9. 29.7 2500
10. 29.7 2000
11. 5.8. 5000
12. 5.8. 3000
13. 31.7 4000
14. 29.7 2000
15. 5.8. 4000

Dipeptide

Tripeptide I

Tripeptide II

Tetrapeptide II

Pictures of plates

picture of colonies taken on 06.08.2013

missing: Tetrapeptide I

overview over Gibson constructs

Excel-Document with constructs, names, DNA-concentrations and important indications

Validation of Gibson Assembly

Restriction digest of Tripetide I, II and Tetrapeptide II

Samples were digested with 0.5 µl NotI. Lanes 2, 8, 10, 11, 20, 25, 37, 40, 43 seem to be positive and are used for futher analysis by restriction with other enzymes

• Minipreps of colonies picked on 06.08.2013
• Restriction digest of DNA obtained by minipreps
  • with NotI
• reamplifications of fragments 6 and 8 according to Protocol-page
  • did not lead to any usable DNA-sample

Results

Gel-Picture taken on 07.08.2013. Different peptide constructs are annotated. For exact reference to picked colonies, see: our overview over our Gibson colonies

Restriction Digest of Tri- Di and Tetrapeptides

Samples were digested with 1 µl SphI (2 years over due date) (for tri- and tetrapeptides) and MfeI (for dipeptide. All lanes show unexpected lines, however, adding the fragment-sizes leads to expected plasmid size for tetrapeptide

Restriction with MfeI and NotI was performed with samples from miniprep on 07.08.2013 that appeared positive:

Gibson Assembly was performed with fragments needed for Tetrapeptide I according to the protocol described above.

Results

As the observed bands were not expected, other colonies (Tripeptide I and II) were picked for further analysis. Samples will be send to sequencing (which????xxxxxxxxx).

Proving the results of Gibson Assembly

restriction with NotI

restriction with SphI

• Colonies from Gibson-Assembly were picked and grown in 3 ml 2x YT medium
• Minipreps from Tripeptide-Colonies picked the day before that were grown in 2x YT medium
• Restriction digests:
  • NotI (~0.8µl per sample)
  • SphI (~0.5µl per sample) (enzyme 2 years over due-date)

Results

• ==> Tripeptide I shows a good band for the insert and the backbone, Tripeptide II doesn't show any positive bands
• ==> the SphI-digest was not successful - maybe because enzyme was not working
• for reference, see the overview Excel-document
• samples were sent to sequencing:
  • Dipeptide 1 (lane 2) --> from plate Dipeptide 1 (colony B) (picked on 05.08.)
  • Dipeptide 2 (lane 8) --> from plate Dipeptide 3 (colony B) (picked on 05.08.)
  • Tripeptide I 1 (lane 10) --> from plate Tripeptide I 1 (colony A) (picked on 05.08.)
  • Tripeptide I 2 (lane 11) --> from plate Tripeptide I 1 (colony B) (picked on 05.08.)
  • Tripeptide II 1 (lane 20) --> from plate Tripeptide II 1 (colony B) (picked on 05.08.)
  • Tripeptide II 2 (lane 25) --> from plate Tripeptide II 3 (colony A) (picked on 05.08.)
  • Tetrapeptide II 1 (lane 37) --> from plate Tetrapeptide II' 1 (colony A) (picked on 05.08.)
  • Tetrapeptide II 2 (lane 40) --> from plate Tetrapeptide II' 2 (colony A) (picked on 05.08.)
  • Tripeptide I new 1 (lane 5 on gel image 09.08.2013) --> from plate Tripeptide I 3 (colony C) (picked on 08.08.)
  • Tripeptide I new 2 (lane 6 on gel image 09.08.2013) --> from plate Tripeptide I 3 (colony D) (picked on 08.08.)

Glycerol Stock

Glycerol Stocks of all colonies that were sent to sequencing

Glycerol Stocks of newest Di-, Tri- and Tetrapeptide - colonies ->the Gibson construct overview table for reference

Restriction Digest of Tetrapeptide-I-NRPSs

• Minipreps of Tetrapeptide I colonies and new Di-, Tri- & Tetrapeptide colonies
• --> see the Gibson construct overview table for reference

restriction with Pst1 and EcoR1

restriction with MfeI

quantification gel of minipreps of different short-peptide-NRPSs

Results

Restriction digest I of Tetrapeptide I with PstI and EcoR1-HF was probably positive for probe 2b,so we drove another Restriction digest with MfeI for Validation. The result was not clear, perhaps because of the extremely high DNA concentration of our Miniprep or because of the old enzyme.

We decided to sent this probe to sequencing to be sure.

Second Restriction digest with MFE 1 for validation of 2B.

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Validation of constructs by sequencing

• The sequences of for our NRPSs arrived: The alignments for the Di-, TriI-, TriII- and TetII - constructs and the new TriI - constructs.
• We also have positive Alignments, you can find them here
• So far, we have positive alignments for:
  • Tripeptide I
  • Tetrapeptide II
  • one direction for Tripeptide II (the other direction - with IK13 - is not yet finished)

Restriction Digest of Dipeptide-NRPS from Minipreps

Enzymes used

• PstI
• EcoRI
• expected fragments: ~6000 & ~2000 bp

Protocol

restriction with EcoRI and PstI

Results

• the obtained band does not match the expected size
• => optimize Gibson Assembly for Dipeptide

Gibson Assembly of missing short-peptide NRPS

Protocols

• According to the problems detected by sequencing, the following changes were performed
• Dipeptide: lower backbone-to-insert-ratio (less backbone used), as most cells were transformed with religated backbone-constructs
• Tripeptide II: lower backbone-to-insert-ratio, as religations or false ligations occured
• Tetrapeptide I: fragment 12 was given in excess, as it was missing in the sequenced plasmid
• The protocols used were the following:

Dipeptide

Tripeptide II

Tetrapeptide I

Tetrapeptide II

Transformation

• DH10β cells were transformed with electroporation:
  • Dipeptide: purified DNA (15µl Gibson Mix purified with Isopropanol and EtOH, eluted in 10µl water)
  • Dipeptide: raw Gibson mix (5µl Gibson Mix diluted in 10µl water)
  • Tripeptide II: purified DNA (15µl Gibson Mix purified with Isopropanol and EtOH, eluted in 10µl water)
  • Tripeptide II: raw Gibson mix (5µl Gibson Mix diluted in 10µl water)
  • Tetrapeptide I: purified DNA (15µl Gibson Mix purified with Isopropanol and EtOH, eluted in 10µl water)
  • Tetrapeptide I: raw Gibson mix (5µl Gibson Mix diluted in 10µl water)
  • Tetrapeptide II: 1 µl of 5 µl raw Gibson Mix diluted in 10 µl water
  • Tetrapeptide II: 14 µl of 5 µl raw Gibson Mix diluted in 10 µl water
  • negative control: 10 µl water

Results

pictures of plates

• white colonies were picked and grown overnight in 2x YT-medium with Chloramphenicol

Verification of correct constructs

Procedure

• 2 ml of overnight culture were used for miniprep and obtained DNA was quantified on Nano-drop
• Restriction digests with Not I

• Sequencing of positive samples

Results

restriction digest with NotI. Lanes 1 to 8: Dipeptide-NRPS, lanes 9 to 15: Tripeptide II-NRPS, lanes 16 to 23: Tetrapeptide-I-NRPS.

Restriction digest with NotI. Lanes 1 to 3: Tripeptide-II-NRPS

Heidelberg pPW02 DH10β ACD.png Restriction digest with PstI & EcoRI. All lanes: Tetrapeptide-II-NRPS

Heidelberg pPW02 DH10β B.png Restriction digest with PstI & EcoRI. All lanes: Tetrapeptide-II-NRPS

• 2 samples for the Dipeptide-NRPS (lane 3 and lane 5) and 2 samples for the Tetrapeptide I-construct (lane 16 and lane 23) were sent to sequencing.
• Positive alignments -> linkout
• All colonies that were transformed with Tripeptide II were negative, hence three additional colonies were picked

Tetrapeptide II was abandoned

Transformation of BAP I cells

Protocol

• thaw chemically competent BAP I cells on ice
• add ~50-100 ng of DNA
• let incubate for 20-30 minutes
• heat-shock at 42°C for 40 seconds
• let cool down on ice for 2 minutes
• add 1 ml 2x YT-medium
• let incubate for 60 minutes at 37°C
• centrifuge at 6000 rpm for 2:30 minutes
• decant medium and resuspend cells in remaining medium
• plate cells on agar plates with Chloramphenicol

Tripeptide I

Results

As the first digest with NotI (figure XY) did not give any positive results - in fact the bands lead to the conclusion that the digest did not work, as one can see the bands that are characteristic for coiled-coil, knicked and linearized plasmid - further colonies were picked and grown as an overnight-culture. Miniprep and restriction digest with NotI on the next day lead to a positive result (figure XY). In this case, the validated plasmid that was used for transformation was used as a positive control. We use Glycerol stocks of both BAP I cultures that were transformed with the NRPS for Tripeptide I for conservation.

Dipeptide

Results

A first restriction digest with PstI & EcoRI only lead to faint positive bands (figure XY), hence further colonies were picked and grown as an overnight culture. After miniprep and restriction digest - again with PstI & EcoRI - a clearer validation was obtained (figure XY). We created Glycerol Stocks of the cells containing the validated plasmid for the Dipeptide-NRPS.

Tetrapeptide I

After a first, negative PstI-EcoRI-restriction digest from minipreps of BAPI-cells that were transformed with the validated plasmid for the Tetrapeptide-I-NRPS (figure XY), further colonies from the same plate were picked and tested, following the same procedure. This time the plasmid that was validated by sequencing and used for transformation was added as a positive contol (figure XY). However, the expected bands were not visible on the gel. Hence, the transformation was done once again, following the protocol described in the section above.

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RFC10 Standardization of Tyc Modules

Primer Design

Amplification of Tyc A

TycA&B1 after PCR

Result There was a band at the right length but we were not able to cut.

Amplification of Tyc B1

A

TycA&B1 after PCR

Result no band in gel, empty lane; optimization

Final validation of BAP-I-cells transformed with pIK03 and pIK04

Restriction Digests

In order to select the samples that we will test on SDS-PAGE and in Mass-Spec, we decided to perform restriction digests and to send the positive samples to sequencing. Restriction digest was performed in a 20µl volume, using 2µl 10x CutSmart buffer, 1µl enzyme(s) and ~500ng DNA per reaction. In order to see both, the purity of the sample, as well as the correct sizes of insert and backbone, we digested every sample twice, once with EcoRI and PstI (in order to see, if the insert has the appropriate size) and once more with EcoRI alone (to see, if there is only one single band). At this stage, all plasmids were already transformed into our expression strain BAP-I.

The restriction digests look very promising for the samples of the Dipeptide- and the Tripeptide-I-NRPS. We hence decided to use all of them on an SDS-PAGE. On the first two images, one can nicely see the expected cutting patterns. The third image was thought as comparison between the positive Di- and Tripeptide-NRPS samples and the Tetrapeptide-I-NRPS samples that we wanted to use.

SDS-PAGE

Recipe for Running Buffer:

• 100 mM Tris-HCl
• 4% SDS
• 0.2% bromophenol-blue
• 20% glycerol
• 200 mM DTT

Unfortunately there were no bands showing up on the gel, which is most probably due to a wrong pH of the loading buffer. Hence this was changed for further optimization.

Tetrapeptide I

Unfortunately, the restriction digest described above did not reveal the expected and desired fragment sizes:

At first, we believed that the chemically competent cells could not incorporate the 16kb plasmid in a heat-shock transformation, therefore we started a Co-transformation of pPW01 (Tetrapeptide I) and the PPTase sfp so that we could "stay" in DH10β-cells where we had electrocompetent cells available.

Even though screening by restriction digest is not that easy when two plasmids are present, it is clear that the approach did not work and that there has to be another problem.

Amplifications

In the course of this project, we'll have to amplify the following fragments: Fragments for Tyrocidine-Indigoidine-Fusion

For reasons of clarity, the gel-pictures obtained by PCR are shown here in this gallery:

Comment on the second File:Heidelberg_

The normal gel-image before the cut out was not properly saved on the PC that is connected to the camera. The bands that were visible (one can estimate them by the cut-out) were at about 2.4 to 2.5 kbp, which is the expected size of the backbone

Fragment A

A

File:Heidelberg ...

Results

The gel was overloaded, which might be due to a too low dilution of Medi-Prep and hence too much template. For optimization use less template and take it from a more diluted sample.

B

Results

Still the gel is overloaded, though less template was used. For the next time, reduce cycle repetitin from 35 to 30 and increase annealing temperature by 1°C

C

Results

Fragment B

A

File:Heidelberg ...

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

Results

Fragment C

A

File:Heidelberg ...

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

Results

Fragment D

A

For obtaining more product and optimizing the conditions simultaneously, a gradient PCR with touch-down was carried out. 4 wells were used at 62°C, 61.4°C, 60.6°C and 60°C for touchdown, with steady temperatures at 59°C, 58.4°C, 57.6°C and 57°C.

Results

Fragment E

A

File:Heidelberg ...

Results

The gel was overloaded, which might be due to a too low dilution of Medi-Prep and hence too much template. For optimization use less template and take it from a more diluted sample.

B

Result

Still the gel is overloaded, though less template was used. For the next time, reduce cycle repetitin from 35 to 30 and increase annealing temperature by 1°C

C

Results

Fragment F

A

File:Heidelberg ...

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

Results

Fragment G

A

File:Heidelberg ...

Results

The gel was overloaded, which might be due to a too low dilution of Medi-Prep and hence too much template. For optimization use less template and take it from a more diluted sample.

B

Results

Still the gel is overloaded, though less template was used. For the next time, reduce cycle repetitin from 35 to 30 and increase annealing temperature by 1°C

C

Results

Fragment H

A

File:Heidelberg ...

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

Results

Fragment I

A

File:Heidelberg ...

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

Results

Quantification-gels

For the further work, the amount of DNA available has to be quantified. We decided to do this by a quantification gel in order to check the right sizes of the constructs. The fragments A, E and G from the first PCR were not used due to the big smear and the double-bands, instead the bands obtained in the second PCR were taken for the quantification.

The bands did not have a definite size, hence they are not useful to work with due to unwanted byproducts that are obviously present. After the optimizations regarding annealing-temperature and cycle-repetition, the following bands were obtained during quantification

Here, the bands have a definded size and a sufficient quantity to work with. The quantification for fragment D was carried out later, as the amplification of those fragments happened later.

RFC10 Standardization of Tyc Modules

Tyc C5

Tyc C6

Tyc A 2013-08-20

Since neither Tyc B1, C5, C6 where succesfully amplified and Tyc A was rather a light band we decided to first find the optimal protocol for Tyc A. Also the Q5 Polymerase was contaminated we decided to use Phusion Flash which is faster and cheaper than the Q5.

Optimiziation A

Reaction

Condition I

Conditions II

Conditions III

Tyc B1 2013-08-21

Reaction

Condition

Tyc C5 2013-08-21

Condition

Tyc C6 2013-08-21

Condition

Tyc B1 2013-08-22

Reaction

Conditions II

Tyc C5 2013-08-22

Reaction

Conditions II

Tyc B1 2013-08-23

B 23-08-2013

Reaction

Conditions

Tyc C5 2013-08-23

B 23-08-2013

Reaction I

Reaction II

Conditions

Final validation of BAP-I-cells and Mass-Spectrometry

SDS-PAGEs of positive BAP-I-cells (Dipeptide & Tripeptide I)

As the SDS-PAGE carried out the week before was unfortunately not successful (no bands were showing up at all), we concluded that the use of another loading buffer, in which the samples are boiled would be useful.

Recipe for loading buffer

The new recipe is as follows:

• 800 µl ddH20
• 250 µl 0.5M Tris-HCl (pH = ~7.3) (i.e. dissolve 302.5 mg Tris in 5 ml and bring to appropriate pH with HCl)
• 400 µl Glycerol 50%
• 400 µl SDS 10%
• 100 µl β-Mercaptoethanol 99%
• bromophenol-blue

Procedure

• Take 1 ml culture in 1.5 ml Eppi
• centrifuge at 13,000 rpm for 10 minutes
• discard supernatant
• resuspend pellet in 100 µl loading buffer (eventually OD-normalize amount of loading buffer)
• boil with closed lids at 98°C for at least 10 minutes under the hood
• cool down (or freeze samples and thaw them again)
• centrifuge at 13,000 rpm for 10 minutes
• load max. 25 µl in SDS-Gel
• use 10 µl BSA-Standard
• let run for 1 hour at 180 V

Results

Two seperate SDS-PAGEs were conducted using the protocol described above.

blue arrows indicate the desired synthetases. For the synthetases of Dipeptide and the Tripeptide-I 212 kDa and 380 kDa bands were expected. The golden arrows indicate a possible lane for the NRPS. For the 380 kDa construct, it is not 100% clear, where one should find it on the gel.

Screening for positive Tetrapeptide-I-colonies

As we did not succeed in finding a positive colony in BAP-I-cells, we decided to continue screening for positive colonies. This however proved to be ineffective as one can see in the image below. All colonies that were grown in liquid culture, prepped and digested with EcoRI and PstI are negative for the insert.

So far, restriction digests were neagtive for Tetrapeptide-I (pPW01) transformed cells, i.e. only the backbone was detectable. For whatever reasons, we had somehow lost the positive plasmids (or couldn't transform them in chemically competent BAP-I cells due to their size. We therefore analyzed the glycerol-stocks we had prepared and performed restriction digests on the plasmids obtained after miniprep.

Both glycerol stocks, the one for DH10β (left image), as well as the one for BAP-I (right image) were clearly negative.

As we however have to functional plasmids, we will focus on the analysis of those and abandon our work on the Tetrapeptide-I-construct pPW01.

Tyrocidine-Indigoidine-Fusion - Cloning & Validation

Gibson Assembly

Gibson assemblies were prepared according to the following formulas:

pPW03 (Phe-Ind)

pPW04 (Asn-Ind)

pPW05 (Val-Ind)

Transformation

The constructs prepared by gibson assembly were chemically transformed (heat shock) into competent DH10ß.

Restriction Digest

Procedure

DH10ß - colonies were picked and grown overnight in LB with Chloramphenicol. After a miniprep, a restriction digest with EcoRI was performed, the expected bands were:

• pPW03 (Phe-Ind): 5858 and 3183
• pPW04 (Asn-Ind): 5858 and 3582
• pPW05 (Val-Ind): 5858 and 3105

Results

All samples showed the expected bands.

pPW03 and pPW04 digested with EcoRI, samples A, B, C & D for pPW03 and B, C & D for pPW04 show the expected bands.

pPW05 digested with EcoRI, all samples show the expected bands. The six lanes on the left are neglectable.

Discussion

Clones pPW03 A, B, C & D; pPW04 B, C & D; pPW05 A, B, C, D, E & F show the expected bands, hence two of each clone were sent to sequencing.

Sequencing & Transformation into BAP-I

Vectors were sequenced with the Primers used for the assembly. All sequences turned out to be positive, hence the minipreps that were sent to sequencing, were used for chemical transformation into BAP-I-cells accoding to the standard protocol. For each transformation 10µl and the rest were plated seperately. After less then two days, blue colonies occured on all plates, hence colony-PCR were performed on 5 colonies of each plate (3 blue ones and 2 white ones).

In parallel, BAP-I-cells were transformed with the remaining Gibson-Mix for pPW03, pPW04 and pPW05. Cells were picked and grown in LB with Chloramphenicol overnight. After miniprep, restricion digest with EcoRI was performed in 20 µl volume. Again, the expected bands were:

• pPW03 (Phe-Ind): 5858 and 3183
• pPW04 (Asn-Ind): 5858 and 3582
• pPW05 (Val-Ind): 5858 and 3105

As these results were not as clear as for the sequenced samples, we did not continue work on the direct BAP-transformants.

Screening PCR for positive BAP-I-colonies

Procedure

In order to screen for positive BAP-I-colonies, blue and white colonies were picked and used in colony-PCR.

Results

Screening for the Tyc-part in blue (B1, B2, B3) and white (W1, W2) colonies from different plates with cells that were transformed with pPW03, pPW04 or pPW05

Screening for the Tyc-part in blue (B1, B2, B3) and white (W1, W2) colonies from different plates with cells that were transformed with pPW03, pPW04 or pPW05 (left of black line) and screening for the Ind-part (right of black line)

Almost all colonies were positive for both modules, except the colonies that were transformed with pPW03 (Phe-Ind). For these constructs, the bands that show up do not match the expected bands:

• expected: xxxx
• obtained: yyyy

Induction, SDS-PAGE & TLC

Procedure

Cells were grown at 37°C and induced at OD600 = ~0.5 with 1mM IPTG. Cultures that were transformed with pPW05 turned blue after about 30 to 60 minutes. Cells were kept at 30°C overnight and the synthetized NRP was extracted and purified from samples. Herefore, 1ml culture was centrifuged at 14,000 rpm for 15 minutes, supernatant was kept for further analysis. The pellet was washed in 1ml Methanol and centrifuged at 14,000 rpm for 15 minutes. Supernatant was kept for further analysis and pellet was resuspended in 500µl DMSO. The crude supernatant, the methanol-wash and the purified NRP were used for TLC. As a control, purified indigoidine (treated with the same protocol) and its washes were used.

As solvent, a mixture of 95% Dichloromethane and 5% Methanol was used. However, all samples were running at the solvent front, hence a less polar solvent, i.e. 100% Dichloromethane was used, and qualitatively better results were obtained.

For the SDS-PAGE, remaining culture was treated as described previously and prepared for SDS-PAGE. Not only pPW05-cells, but also pPW03 and pPW04-positive cells were tested, in order to check for proper expression of the NRPSs.

Results

first TLC - one biological replicate for Val-Ind and one biological replicate for Indigoidine, including the crude supernatants and methanol-washes were used.	second TLC	third TLC	fourth TLC
fifth TLC - showing the running behavior for three biological replicates of Val-Ind and four technical replicates of Indigoidine. Samples were applied alternatingly in order to exclude the possibility of different running behavior due to placement on the TLC.	SDS-PAGE, stained with Coomassie. The bands for the expected Val-Ind-NRPS are visible as indicated by the blue arrows. The golden arrows mark highly probable bands for the other NRPSs that are however qualitatively not 100% clear due to e.g. smear.

Discussion

Especially the last TLC-picture nicely shows that the indigoidine-control has a significantly and reproducibly different sunning behavior on the TLC. Different biological replicates (different clones) were used for Valin-Indigoidine and in all cases, it is running lower than the control - as expected.

As far as the SDS-PAGE is concerned, the expected protein sizes are:

• Phe-Ind-NRPS: 247 kDa
• Asn-Ind-NRPS: 261 kDa
• Val-Ind-NRPS: 242 kDa
• Note: the indigoidine-synthetase alone: 145 kDa

As indicated by the blue arrows, the Val-Ind-NRPS is clearly present in all of the tested samples. For the other two NRPSs, there are bands at the expected height, as indicated by the golden arrows, these are however qualitatively not as good as the ones for the Val-Ind-NRPS due to smear or low expression.

RFC10 Standardization of Tyc Modules

new Primer for B1 and a new fw Primer for TycC5

Amplification Tyc B1 and Tyc C5

29-08-2013

Reaction I

Conditions II

Results of Gel extraction Tyc B1 and Tyc C5

Restriction Digest

Tyc ADeltaCom, B1, C5, C6 (AT_01 to AT02, AT_09 to AT_10, AT_11 to AT_06, AT_07 to AT_08; xx kb; xx kb ; xx kb; xx kb) with EcoRI-HF and PstI

Inkubate 37°C 2h

Restriction Digest of pSB1C3 (xx kb) with EcoRI-HF and PstI

Inkubate 37°C 2h

Nucleotide Removal and Concentration Measurement of digested pSB1C3, Tyc ADeltaCom, B1, C5, C6

Concentration measurements showed that DNA was lost with Nucleotide Removal.

Reamplification and RFC10_Modules#Restriction_Digestas above

Detection of short synthetic NRPs

03.09.2013 Pre-test

Pre-test: solubilize the synthetic dipeptide in Methanol; result: very bad solubility => not used for MS comparisons

03.09. - 04.09.2013 Preparation of samples 1-10 for mass spectrometry at neonate screening

Inocculation of BAP1 (Dipeptide I) and BAP1 (Tripeptide I) [Both tested on NRPS content with SDS-PAGE] in LB media. Washing and transfer of cells to M9 minimal media (evening 01.09.2013) and IPTG induction (noon 02.09.2013) of 50 ml (each) culture.

Taking first 15 ml samples after 2.5 hours. Centrifugation (3750 rpm, 4°C, 45 min) and separation of pellet and supernatant (2x ~7,5ml). Freezing with nitrogen and Lyophilization for 12 hours at ~-45°C and ~0,1 mbar.

Second sample (taken 12 hours after induction) and third one (21 hours after induction) have been treated as the first one.

Inocculation of native BAP1 cells in LB medium, washing in M9 and growing of cultures in M9 as negative reference for mass spec. Same treatment as positive samples.

Samples 1-6 contained different pellets, which have been resuspended in 500 µL PBS Buffer (1X) and treated with 3x20[s] ultra-sonification. Finally the samples have been centrifuged and supernatant decanted to 1.5 ml tubes for mass spectrometry at neonate screening.

Samples 7-10 contained lyophilized Medium and have been resuspended in 500 µL methanol for MS screening. As samples 1-6, supernatant was separated from solid contents and decanted to 1.5ml tubes. 100 µL were handed for MS at neonate screening. The rest has been frozen for hydrolysis.

05.09.-06.09.2013 Preparation of samples 11-21 for mass spectrometry at neonate screening

Samples treated as samples 1-10.

Samples with "H" are hydrolysates (6 M HCL at 105° over night). Those were hydrolysed over night, evaporated and neutralized for MS. Resolved supernatant for "media" samples has been analysed separately for the methanol supernatant and solid phase (marked with "sol").

09.09.-11.09.2013 further preparation for MS at chemistry

Preparation of cultures containing di- and tripeptide for MS (inocculation of LB, washing in M9, transfer to M9 and IPTG induction). IPTG induction at 12 p.m. 10.09., samples taken after 8.5 and 24 and 48 hours. Procedure as mentioned above (lysis, lophylisation etc.), but took pellet from 10 ml and lyophilized 7.5 ml supernatant again.

Results of hydrolysis (05.09.-06.09): A Maillard reaction occurred! Everything brown and many different products (as bonds between amino acids and sugars) arised during cooking in HCL (this protocol seems to be only used for pure peptide solutions). Therefore we have to search for other enzymatic reactions or other strategies to assess ornithine concentrations. Tried to rescue samples by an alkaline reaction with 5 M NaOH, but samples got only slightly lighter.

12.09.further preparation and delievery to MS at chemistry

Resuspension of lyophilized samples in 500 microliters methanol. Delievery of samples 25 and 26 to MS. Other samples to neonate screening.

Team:Heidelberg/Tyrocidine week19 interms

RFC10 Standardization of Tyc Modules

Ligation of digested Fragments each with linearized pSB1C3

DNA Concentration of Digestions

Reaction

1:1 Ratio of pSB1C3:insert

Transformation (Standard Protocol) into TOP10

2 plates per Ligation one with less one with more transformed cells.

Picking & Screening I

picked red clones Picked 3 clones per plate= 6 clones per construct

Colony PCR with Amplification Primers

Phusion Flash Colony PCR (see amplification protocols above)

Result NO Product?!

Screening Colony PCR with VR_rv and VF2_fw

iTaq Colony PCR Result NO Product?!

Picking & Screening II

NEGATIVE RESULTS BEFORE BECAUSE: mrfp was replaced by insert thus the positive colonies are WHITE!!! Picked white colonies

Screening PCR

Screened all picked colonies of one fragment: positive Screened one clone per fragment: positive

Tyrocidine-Indigoidine-Fusion - Standardization of Constructs

So far, the Tyrocidine-Indigoidine-fusion constructs had two illegal cutting sites of RFC-10 restriction enzymes. Hence, we tried a CPEC-approach for which we reamplified a fragment from the vector with primers that introduced a mutation at the desired position.

Amplification

Results

CPEC

After gel-extraction of the large fragment, the following procedure was followed:

• 22µl H2O, 20µl of the large fragment, 2µl of the short fragment, 1µl DpnI and 5µl 10x CutSmart buffer were incubated for 2 hours at 37°C
• Mixture was purified with isoprop, washed with ethanol and eluted in 10µl H2O
• 10µl Phuson Flash Master Mix was added and the followin protocol was run:

Unfortunately the efficiency of this was not high enough, as no colonies were visible on the plates after heat-shock transformation in BAP-I cells. The Amplification and CPEC will be repeated next week.

Short-Peptide-NRPSs: Mass-Spec

Since we did not receive all results from neonate screening yet, we chose two pellet samples(25 and 26) for delievery to Mass spectrometry.

Result

Too much background, therefore no distinctive peaks could be seen. This could be due to the solvent (DMSO) and other cell lysis products. But at least peaks were displayed, thus salts do not overlap the signal! We will wait for the results from neonate screening to deliever medium samples (solubilized in methanol).

Tyrocidine-Indigoidine fusion - extended

As Valin-Indigoidine and its synthetase could be detected by TLC and SDS-PAGE respectively, we decided to increase the number of Fusion-constructs in order to test Indigoidine as possible tag for NRPs and their synthesis. Herefore, we aim to build the following 7 NRPs:

Amplifications

Protocols

A

B

C

D