Team:Heidelberg/Templates/Indigoidine week16

From 2013.igem.org


Contents

Preparation for T-Domain exchanges

We assemble pRB14, which is a version of pRB3, in which the T-Domain is exchanged by the whole insert of the pDONR vector. Since pRB13 grew on DH5alpha and TOP10 we hope that pRB14 won't. This vector will be used to exchange T-Domains and will be modified to get a version without the PPTase sfp and the cutting sites EcoRI and SpeI in indC. To check whether a successfully exchanged T-Domain can be activated by a defined PPTase, we build pSB2K3-derived plasmids with a single PPTase (sfp, svp, entD or delC) under control of the lac-Promotor and the RBS BBa_B0029.


Fragment Amplification

PCR of Fragments for PPTase plasmids

Table 12.x PCR for pRB15-18: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pSB2K3 RB21/63 in BioRAD old old
template: 3 ul pSB2K3 250 ng/ ul (distribution)
cyclestemperature (°C)time (s)
19810
10 981
62 ? 0.55
7240
25981
575
7240
172300
112-

pSB2K3 contains the 3'-part of the reversed Primer in an internal lac-Operator-structure, so the PCR product is just half of the backbone (2500 bp instead of 4800 bp). We will use the same primers but pSB3K3 of the 2013 spring distribution plate 5 well 5E; which is around 3000 bp.

Table 12.x PCR for pRB15-18: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pSB3K3 RB21/63 in BioRAD old old
template: 2 ul pSB3K3 250 ng/ ul (distribution)
cyclestemperature (°C)time (s)
19810
3 981
585
7250
32981
655
7250
172180
112-

We get unspecific product -> repetition with more stringent conditions

pSB3K3 RB21/63 in BioRAD T100
template: 0.5/1.5 ul pSB3K3 250 ng/ ul (distribution)
cyclestemperature (°C)time (s)
19810
10 981
65 -0.55
7250
25981
695
7250
172180
112-


PCR of Fragments for T-Domain exchange

Table 12.x PCR for pRB13: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pRB3?T KH3/4 in T100
template: 1 ul pRB3 1 ng/ ul
cyclestemperature (°C)time (s)
19810
10 981
62 ? 0.55
72120
25981
575
72120
172300
112-
KH9/10 in T100
template: 2 ul pDONR 1 ng/ ul
cyclestemperature (°C)time (s)
19810
8 981
70 ? 0.55
7215
28981
655
7215
17290
112-
T-Domains in BioRAD old
tycC6-T RB57/58 1 ul B. para overnight culture
tycA1-T RB55/56 1 ul B. para overnight culture
entF-T RB53/54 colony MG1655
delH4-T RB59/60 colony D. aci SPH-1
delH5-T RB61/62 colony D. aci SPH-1
cyclestemperature (°C)time (s)
198120
40981
655
725
17230
112-


Gel Extraction

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB-T

FragmentConcentration [ng/ ul]~ Fragment size [bp]Molarity [nM]
sfp329.6700713.42
svp44.975090.7
entD289.2650674.13
delC54.5700117.97
pSB3K3 26.8 3000 13.54
pRB3dT124.8680027.81
ccdB121.2700262.33
indC-T192.42001457.58
bpsA-T198.92001506.18
entF-T184.82001400.00
tycA1-T184.22001395.45
tycC6-T198.62001504.55
delH4-T175.02001325.76
delH5-T162.42001230.30

CPEC Assembly and Transformation

pRB14-18
PlasmidFragment 1Molarity [nM]Volume in MMFragment 2Molarity [nM]Volume in MM
pRB14pRB3dT27.813.8ccdB262.331.2
pRB15pSB3K313.544.5sfp713.420.5
pRB16pSB3K313.543.5svp90.71.5
pRB17pSB3K313.544.5entD674.130.5
pRB18pSB3K313.543.8delC117.971.2
pRB21indC(RB27/46)25.403.5pSB1C3(RB21/22)133.081.5

Table 12.x

CPEC Assembly pRB14-18
BioRAD T100
CyclesTemperature [°C]Time [s]
19810
5981
535
72120
172300
112-

Transforation according to standard protocol for chemical transformation

  • TOP10 with pRB15-18 (-> LB+Kan)
  • TOP10 with pRB15-18 and pMM64, respectively (-> LB+Kan+Amp)
  • TOP10 with pMM64 (-> LB+Amp)
  • OneShot with pRB14 (-> LB+Cm)
  • OneShot+pRB14: small colonies after 20 hours
  • TOP10+pRB15-18: colonies on all plates
  • TOP10+pMM64+pRB15-18: (almost) no colonies after 20 hours; hopefully due to indigoidine growth retardation
  • TOP10+pMM64 TraFo was efficient

We will perform PCR screening in triplicates of TOP10+pRB15-19 and OneShot+pRB14 to get a first impression on whether the assembly was successful.


Plasmid Validation

PCR Screening

We use forward primers of the insert and standard reversed primer VR to screen pRB14-18 in triplicates. We use iTaq DNA-polymerase in 20 ul PCR mix:

  • 10 ul iTaq 2x Master Mix
  • 2 ul Primer 10 uM each
  • 6 ul water
  • colony pick from plate


PlasmidPrimer fwPrimer rvExpected Fragment Size [bp]
pRB14VF2KH101000
pRB15VF2RB361050
pRB16VF2RB301100
pRB17VF2RB341000
pRB18VF2RB671050

Table 12.x

PCR screening pRB14-18
BioRAD T100
CyclesTemperature [°C]Time [s]
195120
359530
5330
7260
172300
112-

Annealing temperature according to NEB Tm calculator for Taq DNA polymerase and VF2

Table 12.x concentrations of pRB15-18 (NanoDrop ND-1000)
PlasmidConcentration [ng/ ul]~ size [bp]Molarity [nM]
pRB1598.7 3500
pRB16106.6 3500
pRB17188.3 3500
pRB18129.9 3500
Test Transformation

OneShot and TOP10-sells have been transformed with pRB14. We expect OneShot cells to grow as usual and no colonies on TOP10. As a control group we transformed OneShot as well as TOP10 with pRB3.

pRB19

pRB19 is a pRB14-derived plasmid without the PPTase sfp. The genotype is pSB1C3-lacPromotor-BBa_B0034-indCdT(ccdB)

Table 12.x

PCR Amplification of pRB14 for pRB19
BioRAD T100
RB21/46 from pRB14 miniprep 0.6 ng
CyclesTemperature [°C]Time [s]
19810
8981
TD 615
72100
30981
655
72100
172300
112-

Smear -> Annealing temperatures in the touchdown-part were too high, so RB21 3' end couldn't bind properly. Second run with milder conditions.

Table 12.x

PCR Amplification of pRB14 for pRB19 #2
BioRAD T100
RB21/46 from pRB14 miniprep 1 ng
CyclesTemperature [°C]Time [s]
19810
12981
TD 605
72110
25981
655
72110
172300
112-

Smear -> we will run another strategy, i.e. amplification of indC from pRB14 with RB27/46 to further assemble it with pSB1C3(RB21/22)

Table 12.x

PCR Amplification of indC(RB27/46) for pRB19 #3
BioRAD T100
RB27/46 from pRB14 miniprep 1 ng
CyclesTemperature [°C]Time [s]
19810
10981
TD 575
7260
25981
655
7260
172180
112-

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB19

FragmentConcentration [ng/ ul]~ Fragment size [bp]Molarity [nM]
indC-pRB14(RB27/46)133.0380053.03
pSB1C3(RB21/22)210.82400133.08
PlasmidFragment 1Molarity [nM]Volume in MM
!Fragment 2Molarity [nM]Volume in MM
pRB19indC-pRB14(RB27/46)pRB14(RB21/46)53.033.3
pSB1C3(RB21/22)133.081.7

Table 12.x

CPEC Assembly pRB19
BioRAD T100
CyclesTemperature [°C]Time [s]
19810
5981
535
72100
172300
112-

Transformation into OneShot Survival cells. Doing colony PCR for screening with primer KH9/VR which would give a fragment size of around 1.8 kbp: (Note: Throw picked colony 4 away since unintendently pooled 2 colonies in PCR tube). Used iTaq.

Table 12.x

PCR screening pRB19 and pRB21
Cycler 2
KH9/VR from picked colonies.
CyclesTemperature [°C]Time [s]
195120
359530
5330
72120
172300
112-

Colony PCR for pRB19 gave a lot of unspecific bands, probably because of less stringent parameters. Oculutated 6 ml of TB+Cm with colony 2 for MP preparation.

Biobricks

The PPTases sfp, svp, entD and delC and the indigoidine synthetase indC will be prepared for submission to the registry. indC contains two RFC10-cutting site, that have been removed during the assembly of pRB22.

pRB21 series

pRB21 is a pSB1C3-plasmid with lacPromotor, BBa_B0034 and indC. After the first assembly, EcoRI and SpeI cutting sites in indC will be removed to yield pRB22. After cutting site removal, the native T-Domain will be exchanged with ccdB to yield pRB22. pRB23 has the same genotype as pRB20 but with two PCR steps less.

Fragment Amplification

Table 12.x

PCR Amplification of indC for pRB21 #1
BioRAD T100
RB27/46 from P. luminescens pellet
CyclesTemperature [°C]Time [s]
19810
8981
TD 615
72120
25981
655
72120
172300
112-


Amplification was not successful. Next try from PCR product with short primers as a rePCR.

Table 12.x

PCR Amplification of indC for pRB21 #2
BioRAD T100
RB27/46 from indC short gel extraction
CyclesTemperature [°C]Time [s]
19810
8981
TD 565
7260
25981
655
7260
172180
112-
Gel Extraction

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB21

FragmentConcentration [ng/ ul]~ Fragment size [bp]Molarity [nM]
indC(RB27/46)63.7380025.40
pSB1C3(RB21/22)210.82400133.08
CPEC Assembly and Transformation
PlasmidFragment 1Molarity [nM]Volume in MMFragment 2Molarity [nM]Volume in MM
pRB21indC(RB27/46)25.403.5pSB1C3(RB21/22)133.081.5

Table 12.x

CPEC Assembly pRB21
BioRAD T100
CyclesTemperature [°C]Time [s]
19810
5981
535
7260
172180
112-
Validation

Colony PCR for pRB21 was unsuccessful (see gel picture), try again with less strigent parameters and positive control: KH5/VR on MP pRB3, which should give an amplicon size of around 2 kbp.

Table 12.x

PCR screening pRB21
T100 (right)
KH5/VR from picked colonies and 5 ng pRB3 MP
CyclesTemperature [°C]Time [s]
195120
359530
5030
7290
172150
112-

Colony PCR for pRB21 worked now as well as the positive control. Oculutated 6 ml of TB+Cm with colony 8 for MP preparation.


PPTase BioBricks

have to look up used primer/template combinations again Konrad (talk)

Table 12.x

PCR Amplification of PPTases for BioBrick submission
BioRAD T100
sfp: KH17/18 from pRB15
svp: KH19/20 from pRB16
entD: KH13/14 from pRB17
delC: KH15/16 from pRB18
CyclesTemperature [°C]Time [s]
19810
12981
TD 665
7215
25981
655
7215
17260
112-
Gel Extraction

Assembly and Transformation

Digest
Ligation
Transformation