Team:Heidelberg/Templates/Indigoidine week18

From 2013.igem.org

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File:Heidelberg_20130826_blllll.png|PCR #1 for indC for pRB19
 
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File:Heidelberg_20130826_blllll.png|PCR #2 for indC for pRB19
 
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Gel Extraction with QIAquick gel extraction kit.
Gel Extraction with QIAquick gel extraction kit.
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Digest ran for 1 hour at 37 °C, was put on a agarose gel, gel extracted and eluted in 15 ul water.
Digest ran for 1 hour at 37 °C, was put on a agarose gel, gel extracted and eluted in 15 ul water.
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File:Heidelberg_20130826_blllll.png|PCR synT
 
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File:Heidelberg_20130826_blllll.png|PCR synT
 
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Concentration was measured using NanoVue: 30 ng/ ul. 10 ul were put together with 10 ul of Phusion  
Concentration was measured using NanoVue: 30 ng/ ul. 10 ul were put together with 10 ul of Phusion  
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OneShot and TOP10 cellc were transformed using different amounts of CPEC product.
OneShot and TOP10 cellc were transformed using different amounts of CPEC product.
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File:Heidelberg_20130827_platte pRB19
 
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File:Heidelberg_20130827_platte pRB19
 
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Ten colonies were screened using iTaq with KH9/VR and RB35/VR, respectively, to see whether the assembly worked and sfp was kicked out.
Ten colonies were screened using iTaq with KH9/VR and RB35/VR, respectively, to see whether the assembly worked and sfp was kicked out.
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File:Heidelberg_20130827_platte pRB19
 
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File:Heidelberg_20130827_platte pRB19
 
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The screening was positive, so probe 10 was kept for 10 ml liquid culture (TB+Cm).
The screening was positive, so probe 10 was kept for 10 ml liquid culture (TB+Cm).
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File:Heidelberg_20130829_Gel TestPCR
 
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File:Heidelberg_20130826_blllll.png|PCR synT
 
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Gel extraction and measurement using NanvoVue. The final table of T-domain concentration is as  
Gel extraction and measurement using NanvoVue. The final table of T-domain concentration is as  
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File:Heidelberg_20130829_blllll.png|pSB3k3
 
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PCR was run with with a fragment of the Tyrocidine group with slightly suboptimal conditions. It will be repeated using otimal conditions.
PCR was run with with a fragment of the Tyrocidine group with slightly suboptimal conditions. It will be repeated using otimal conditions.
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File:Heidelberg_20130830_blllll.png|pSB3K3 #2
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File:Heidelberg_20130830_blllll.png|pSB3K3 #2
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Gel extraction yielded 144.2 ng/ ul (Nanodrop)
Gel extraction yielded 144.2 ng/ ul (Nanodrop)
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Cells were plated on LB+Kanamycin and incubated at 37 °C for 24 hours.
Cells were plated on LB+Kanamycin and incubated at 37 °C for 24 hours.
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File:Heidelberg_20130830_platten pRB15-18
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Five colonies of each plate were screened using iTaq polymerase and two primer combinations for each colony. This is VF2/PPTase_rv primer and PPTase_fw/VR primer.
Five colonies of each plate were screened using iTaq polymerase and two primer combinations for each colony. This is VF2/PPTase_rv primer and PPTase_fw/VR primer.
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[[Plasmids#pRB16|pRB16]] is obviously wrong. We checked the PCR product we used for the assembly and recognized that it's wrong, so we have to amplify it again from S. verticillus. DelC screenings show another small band, we have too check whether the primers bind elsewhere on the plasmid and run a negative control with those primers.
[[Plasmids#pRB16|pRB16]] is obviously wrong. We checked the PCR product we used for the assembly and recognized that it's wrong, so we have to amplify it again from S. verticillus. DelC screenings show another small band, we have too check whether the primers bind elsewhere on the plasmid and run a negative control with those primers.

Latest revision as of 15:45, 21 October 2013


Contents

Preparation for T-Domain exchange

Assembly of pRB19

PCR indC(ccdB) RB27/28 and pSB1C3 RB21/22 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 14.x PCR indC(ccdB) for pRB19
RB27/46 in BioRAD T100
cyclestemperature [°C]time [s]
19810
5981
555
7260
30981
7260
172180
112-


Table 14.x PCR indC(ccdB) for pRB19
RB27/46 in BioRAD T100
cyclestemperature [°C]time [s]
19810
10981
td 555
7270
25981
605
7270
172180
112-


Gel Extraction with QIAquick gel extraction kit.

Table 13.x CPEC Assembly Master Mix pRB19 second try
PlasmidFragment 1Molarity [nM]Volume in ulFragment 2Molarity [nM]Volume in MMDpnI

Master Mix total [ul]

pRB19indC(ccdB) from pRB1414.614pSB1C3 RB21/22105.3320

Digest ran for 1 hour at 37 °C, was put on a agarose gel, gel extracted and eluted in 15 ul water.


Concentration was measured using NanoVue: 30 ng/ ul. 10 ul were put together with 10 ul of Phusion

Flash HF Master Mix for standard CPEC assembly. OneShot and TOP10 cellc were transformed using different amounts of CPEC product.


Ten colonies were screened using iTaq with KH9/VR and RB35/VR, respectively, to see whether the assembly worked and sfp was kicked out.


The screening was positive, so probe 10 was kept for 10 ml liquid culture (TB+Cm). Miniprep of 4 ml yielded 69.4 ng/ ul in 50 ul. Miniprep was used for PCR with primers KH3/4 to get the fragment pRB19 without ccdB for insertion of T-Domains via CPEC. First we tried two different polymerases at two different conditions each. The first is Phusion Flash

HF 2x Master Mix (ThermoScientific) and the other is Phusion HF 2x Master Mix (NEB).

Table 14.x Test PCR KH3/4 from pRB19
KH3/4 in BioRAD T100 with Phusion HF (NEB)
CyclesTemperature [°C]Time [s]
19830
309815
57-6015
723.30
17210.00
112-
KH3/4 in BioRAD T100 with Phusion Flash HF (ThermoScientific)
CyclesTemperature [°C]Time [s]
19810
30981
57-605
722.00
1726.00
112-


Table 14.x synT-PCR
KH5/6 in BioRAD T100
cyclestemperature [°C]time [s]
19830
45985
6015
7215
17260
112-


Gel extraction and measurement using NanvoVue. The final table of T-domain concentration is as

follows:

Table 13.x Concentrations of T- and TTE-domains to be shuffled
FragmentConcentration [ng/ ul]~ Fragment size [bp]Molarity [nM]
ccdB121.2700262.33
indC-T192.42001457.58
bpsA-T198.92001506.18
entF-T64.5200488.6
tycA1-T184.22001395.45
tycC6-T198.62001504.55
delH4-T175.02001325.76
delH5-T162.42001230.30
plu2642-T79.0200598.5
plu2670-T31.5200238.6
synT143200325.8
synT237.5200284.1
synT340.5200306.8
synT436200272.7
synT537.5200284.1
synT637200280.3
synT740.5200306.8
bpsA-TTE92.91000140.8
entF-TTE-1000-
tycC6-TTE21100031.8
delH5-T22.9100034.7

Assembly of pRB15-18

pRB15-18 were unable to be sequenced with various primers. We now use pSB3K3 backbone from plate 3 of the 2013 spring distribution and assemble those PPTase-plasmids de novo.

Table 14.x PCR pSB3K3 with RB21/63 #1
RB21/63 in BioRAD T100
CyclesTemperature [°C]Time [s]
19810
14981
td 665
7260
16981
685
7260
172180
112-

PCR was run with with a fragment of the Tyrocidine group with slightly suboptimal conditions. It will be repeated using otimal conditions.

Table 14.x PCR pSB3K3 with RB21/63 #2
RB21/63 in BioRAD T100
CyclesTemperature [°C]Time [s]
19810
12981
td 615
7250
20981
655
7250
172180
112-


Gel extraction yielded 144.2 ng/ ul (Nanodrop)

CPEC was performed using pSB3K3 gel extraction and gel extractions made for assembly of pRB3-10. TOP10 cells were transformed using standard protocol.

Table 14.x CPEC Assembly pRB15-18
PlasmidFragment 1Molarity [nM]Volume [ul]Fragment 2Molarity [nM]Volume [ul]CPEC Master Mix total [ul]
pRB15pSB3K3(RB21/63)87.42 sfp713.416
pRB16pSB3K3(RB21/63)87.4 2 svp90.7412
pRB17pSB3K3(RB21/63)87.4 2entD674.116
pRB18pSB3K3(RB21/63)87.4 1 delC118.126

Cells were plated on LB+Kanamycin and incubated at 37 °C for 24 hours.

Five colonies of each plate were screened using iTaq polymerase and two primer combinations for each colony. This is VF2/PPTase_rv primer and PPTase_fw/VR primer.

pRB16 is obviously wrong. We checked the PCR product we used for the assembly and recognized that it's wrong, so we have to amplify it again from S. verticillus. DelC screenings show another small band, we have too check whether the primers bind elsewhere on the plasmid and run a negative control with those primers.

Assembly of pKH4

pKH4 is a pRB3 derived version of an indigoidine synthetase expression plasmid w/o sfp as activating PPtases. So the two plasmid strategy can be conducted (at first) with this plasmid instead to rely on pRB21. Basically the idea is to use restriction sites before and after sfp to discard it and to religate with another sequence to yield a functional backbone.

pRB3 has a BamHI before and a NheI cutting site after sfp. pMM64 and pMM65 both can be cut with a combination of BglII and XbaI to yield for example a 164 bp long fragment with compatible ends to the linearized pRB3. This fragment unfortunately contains a T7lac hybrid promoter which could interfere with our used primers, but still is worth a shot.

  • digest mix volume: 30 µl (3 µl NEBuffer 3.1, 6.5 µl pMM65 (~481 ng), 0.3 µl (BglII, XbaI) each, 19.9 µl H2O)

Resulting gel of digestion of pMM65 with BglII and XbaI in order to gain sequence with cohesive ends to linerized pRB3 w/o sfp.

  • digestion gave expected bands at 2 kbp and 2.4 kbp but band at ~160 bp is missing. Reaction was carried out with not enough DNA.
  • prepared ON for pMM64/65 MP as well as pRB3 MP
  • the next day ON were extended to 2x 4 ml LB cultures which were then

prepped after several hours of incubation at 37 °C

  • MP with cultures: DNA conc. measurement with NanoVue gave: 280

ng/µl (pMM64), 225 ng/µl (pMM65), 250 ng/µl (pRB3); analysis gel shows different concentration for pMM65 (~15 ng/µl ?)

Analytical gel for verification of MP pMM64/65 and pRB3: concentrations measured with NanoVue seem to be ok for pMM64 and pRB3, but much less for pMM65. 2log ladder lane contain 420 ng each; 100 V, TAE

  • prepare digestion (20 µl), 37 °C, 1.5 h:
    • pMM64: 2 µl NEBuffer 3.1, 17 µl pMM64, 0.5 µl (BglII, XbaI)
    • pRB3: 2 µl NEBuffer CutSmart, 10 µl pRB3, 0.5 µl (BamHI, NheI), 7

µl H2O

Digestion of pMM64 (BglII, XbaI) and pRB3 (BamHI, NheI) were desired bands for insert of ~170 bp and backbone-indC of ~6300 bp were cut out. 100 V, TAE

  • after gelextraction (elution in 20 µl) ligation was conducted with 2

µl 10x T4 ligase buffer, 1 µl T4 ligase, 2 µl pRB3 Dig_GE, 8 µl pMM64 Dig_GE and 7 µl H2O for 40 min at RT

Analytical gel of 1 µl of ligation mix, compared to 0.5 µl of pRB3 digestion with (BamHI, NheI). Additionally 1 µl of MP pMM65 was put on gel to verify previous concentration measurement. 1.5 µl of log2 ladder, 100 V, TAE

  • transformation in TOP10 with 1 µl and 5 µl of ligation mix
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