Team:Heidelberg/Tyrocidine week15 ms

From 2013.igem.org


Contents

DNA concentrations of fragments

Analysis of DNA concentration

Heidelberg Tyr quantification 3 11 12 13 15.png
  • Concentrations obtained through quantification gel. There are 20µl of all fragments.
fragment concentration [ng/µl]
3 2
11 3
13 7
15 --
3 35
11 10
12 10
15 20

Complete List of DNA concentrations of all fragments

fragment concentration [ng/µl]
1 10 (25.7.), 30 (28.7.)
2 16 (25.7.), 12 (31.7.)
3 35 (04.8.)
4 32 (24.7.), 30 (28.7.)
5 12 (26.7.), 14 (31.7.)
6 8 (26.7.) (empty), 6.5 (03.8.)
7 12 (27.7.), 18 (31.7.)
8 8 (26.7.), 5 (03.8.)
9 30 (28.7.)
10 40 (28.7.)
11 5 (31.7.), 10 (04.8.)
12 10 (04.8.)
13 20 (31.7.), 7 (03.8.)
14 40 (28.7.)
15 11 (31.7.), 20 (04.8.)

Gibson Assembly

fragments (10µL) + 10 µL Gibson Mix


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

fragment concentration [ng/µl] volume for gibson assembly [µL]
1 30 3.14
2 16 4.71
3 35 2.15

Tripeptide I

fragment concentration [ng/µl] volume for gibson assembly [µL]
4 32 1.02
5 12 3.27
6 8 5.71

Tripeptide II

fragment concentration [ng/µl] volume for gibson assembly [µL]
4 32 1.25
7 18 1.77
8 8 6.98


Tetrapeptide II

fragment concentration [ng/µl] volume for gibson assembly [µL]
9 30 1.32
10 40 0.79
11 10 3.95
14 10 0.79
15 20 3.16


Pictures of plates

picture of colonies taken on 06.08.2013

missing: Tetrapeptide I

fragment concentration [ng/µl] volume for gibson assembly [µL]
9 30 0.77
10 40 0.46
11 10 2.31
12 10 2.77
13 20 3.69


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
what µl
DNA 4 (~200-800ng)
Cutsmart Mix 3
Enzyme 1
ddH20 ad 30 µl

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:

what µl
DNA 1200ng
Cutsmart Mix 2
Enzyme 1
ddH20 ad 20 µl

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

fragment concentration [ng/µl] volume for gibson assembly [µL]
9 30 0.77
10 40 0.46
11 10 2.31
12 10 2.77
13 20 3.69

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

restriction with Pst1 and EcoR1
restriction with MfeI
quantification gel of minipreps of different short-peptide-NRPSs
Probe name Available volume
Tet I 1a 1,3 ml
Tet I 1b 1,3 ml
Tet I 1c 1,3 ml
Tet I 2a 1,3 ml
Tet I 2b 1,3 ml
Tet I 2c 1,3 ml
Tet I 3a 0,845 ml
Tet I 3b 0,975 ml
Tet I 4a 1,3 ml
Tet I 4b 0,75 ml
Tet I 4c 0,845 ml
Tet II 2 11 0,750 ml
Tet II 2 14 0,975 ml
Tet II 3 15 0,750 ml
Tet II 2 12 0,975 ml
Di 1 0,975 ml
Tri II 1 0,975 ml
Di 2 1,075 ml
Tri II 2 0,975 ml
Di 3A 0,975 ml
Tri II 3 0,975 ml
Di 3b 0,975 ml

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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