Team:Heidelberg/Tyrocidine week19 biobrickmod

From 2013.igem.org

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==RFC10 Standardization of Tyc Modules==
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===Ligation of digested Fragments each with linearized pSB1C3===
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====DNA Concentration of Digestions====
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{| class="wikitable"
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|-
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! Fragment !! Source!! Protocol !! Concentration
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|-
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| Tyc AdCom|| AT_01/AT_02 || Reamplification 2013-08-31[[xxx]] || 5.0 ng/µl
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|-
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| Tyc B1|| AT_09/AT_10 || Reamplification 2013-08-31[[xxx]]|| 22.5 ng/µl
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|-
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| Tyc C5|| AT_11/AT_06 || Reamplification 2013-08-31[[xxx]]|| 19.2 ng/µl
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|-
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| Tyc C6|| AT_07/AT_08 || Reamplification 2013-08-31[[xxx]]|| 35.7 ng/µl
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|-
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| pSB1C3|| mediprep of ONC || - || 14.4 ng/µl
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|}
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====Reaction====
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'''1:1 Ratio of pSB1C3:insert'''
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{| class="wikitable"
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|-
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! Fragment !! Size [kb]!! Concentration [ng/µL]!! Amount for Reaction [µL]!! 10x T4 DNA Lig Buffer [µL]!! T4 DNA Ligase [µL] !! +ddH<sub>2</sub>O
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|-
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|colspan="3" style="text-align: center;" | '''add Backbone''' pSB1C3; 4.2 kb; 14.4 ng/µl || '''3.5''' ||
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|-
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| Tyc AdCom|| 3.2 || 5.0 ng/µl|| 7.6|| rowspan="4" style="text-align: center;"| 2 || rowspan="4" style="text-align: center;"| 1||5.9
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|-
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| Tyc B1|| 3.2 || 22.5 ng/µl||1.7||11.8
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|-
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| Tyc C5|| 3.2 || 19.2 ng/µl||2||11.5
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|-
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| Tyc C6|| 3.9 || 35.7 ng/µl||1.5||12
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|}
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===Transformation (Standard Protocol) into TOP10===
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2 plates per Ligation one with less one with more transformed cells.
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===Picking & Screening I===
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'''picked red clones'''
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Picked 3 clones per plate= 6 clones per construct
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====Colony PCR with Amplification Primers====
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Phusion Flash Colony PCR (see amplification protocols above)
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'''Result''' NO Product?!
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====Screening Colony PCR with VR_rv and VF2_fw====
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iTaq Colony PCR
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'''Result''' NO Product?!
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===Picking & Screening II===
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'''NEGATIVE RESULTS BEFORE BECAUSE: mrfp was replaced by insert thus the positive colonies are WHITE!!!'''
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Picked white colonies
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====Screening PCR====
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Screened all picked colonies of one fragment: positive
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Screened one clone per fragment: positive
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== Tyrocidine-Indigoidine-Fusion - Standardization of Constructs ==
== Tyrocidine-Indigoidine-Fusion - Standardization of Constructs ==
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===Results===
===Results===
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[[File:PCR_1'_2'_3'.png|thumb|right|100px]]
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[[File:Heidelberg_PCR_1'_2'_3'.png|thumb|right|100px]]
===CPEC===
===CPEC===

Latest revision as of 16:59, 4 October 2013

Contents

RFC10 Standardization of Tyc Modules

Ligation of digested Fragments each with linearized pSB1C3

DNA Concentration of Digestions

Fragment Source Protocol Concentration
Tyc AdCom AT_01/AT_02 Reamplification 2013-08-31xxx 5.0 ng/µl
Tyc B1 AT_09/AT_10 Reamplification 2013-08-31xxx 22.5 ng/µl
Tyc C5 AT_11/AT_06 Reamplification 2013-08-31xxx 19.2 ng/µl
Tyc C6 AT_07/AT_08 Reamplification 2013-08-31xxx 35.7 ng/µl
pSB1C3 mediprep of ONC - 14.4 ng/µl

Reaction

1:1 Ratio of pSB1C3:insert

Fragment Size [kb] Concentration [ng/µL] Amount for Reaction [µL] 10x T4 DNA Lig Buffer [µL] T4 DNA Ligase [µL] +ddH2O
add Backbone pSB1C3; 4.2 kb; 14.4 ng/µl 3.5
Tyc AdCom 3.2 5.0 ng/µl 7.6 2 15.9
Tyc B1 3.2 22.5 ng/µl1.711.8
Tyc C5 3.2 19.2 ng/µl211.5
Tyc C6 3.9 35.7 ng/µl1.512

Transformation (Standard Protocol) into TOP10

2 plates per Ligation one with less one with more transformed cells.

Picking & Screening I

picked red clones Picked 3 clones per plate= 6 clones per construct

Colony PCR with Amplification Primers

Phusion Flash Colony PCR (see amplification protocols above)

Result NO Product?!

Screening Colony PCR with VR_rv and VF2_fw

iTaq Colony PCR Result NO Product?!

Picking & Screening II

NEGATIVE RESULTS BEFORE BECAUSE: mrfp was replaced by insert thus the positive colonies are WHITE!!! Picked white colonies

Screening PCR

Screened all picked colonies of one fragment: positive Screened one clone per fragment: positive



Tyrocidine-Indigoidine-Fusion - Standardization of Constructs

So far, the Tyrocidine-Indigoidine-fusion constructs had two illegal cutting sites of RFC-10 restriction enzymes. Hence, we tried a CPEC-approach for which we reamplified a fragment from the vector with primers that introduced a mutation at the desired position.

Amplification

what µl
pPW05 (dil.) 1
RB68 2
RB69 2
Phusion Flash 2x Master Mix 10
ddH20 5
Cycles temperature [°C] Time [min:s]
1 98 0:05
35 98 0:05
59 0:10
72 3:00
1 72 10:00
1 10 inf

Results

Heidelberg PCR 1' 2' 3'.png

CPEC

After gel-extraction of the large fragment, the following procedure was followed:

  • 22µl H2O, 20µl of the large fragment, 2µl of the short fragment, 1µl DpnI and 5µl 10x CutSmart buffer were incubated for 2 hours at 37°C
  • Mixture was purified with isoprop, washed with ethanol and eluted in 10µl H2O
  • 10µl Phuson Flash Master Mix was added and the followin protocol was run:
Cycles temperature [°C] Time [min:s]
1 98 0:10
5 98 0:01
53 0:05
72 3:00
1 72 10:00
1 10 inf

Unfortunately the efficiency of this was not high enough, as no colonies were visible on the plates after heat-shock transformation in BAP-I cells. The Amplification and CPEC will be repeated next week.

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