Team:Heidelberg/Tyrocidine week14 ms

From 2013.igem.org

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

--> 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

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.

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

Amplification of Fragment 12

Fusion/Q5

Fusion Touch Down

Analysis of DNA concentrations

• 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

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

• 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

• 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

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