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From 2013.igem.org

Contents 1 Validation of constructs by sequencing 2 Restriction Digest of Dipeptide-NRPS from Minipreps 2.1 Enzymes used 2.2 Protocol 2.3 Results 3 Gibson Assembly of missing short-peptide NRPS 3.1 Protocols 3.1.1 Dipeptide 3.1.2 Tripeptide II 3.1.3 Tetrapeptide I 3.1.4 Tetrapeptide II 3.2 Transformation 3.3 Results 3.4 Verification of correct constructs 3.4.1 Procedure 3.4.2 Results 4 Transformation of BAP I cells 4.1 Protocol 4.2 Tripeptide I 4.2.1 Results 4.3 Dipeptide 4.3.1 Results 4.4 Tetrapeptide I

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

what	µl
DNA	6
Cutsmart Mix	2
EcoRI	0.75
PstI	0.75
ddH20	10.5

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

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
1	30	0.80
2	16	4.81
3	35	4.39

Tripeptide II

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
4	32	0.38
7	18	1.08
8	8	8.54

Tetrapeptide I

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
9	30	0.35
10	40	0.42
11	10	4.20
12	10	3.36
13	20	1.68

Tetrapeptide II

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
9	30	0.25
10	40	0.61
11	10	6.09
14	40	0.61
15	20	2.44

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

what	µl
DNA	600ng, alternatively 4 µl
Cutsmart Mix	2
Enzyme	0.5
ddH20	ad 20 µl

• 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.