Team:Heidelberg/Templates/Del week12 overview

From 2013.igem.org

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[[File:Heidelberg_Strategie3.png|300px|right|thumb| Vector map of [[:File:Heidelberg_Psb4k5+BBa J04450 DelRest.gb| pFSN plasmid]] including Primers FS_01 to FS_241 used for assembly of the desired genes from the ''D. acidovorans'' genome and the partsregistry backbone pSB4K5 .]]
[[File:Heidelberg_Strategie3.png|300px|right|thumb| Vector map of [[:File:Heidelberg_Psb4k5+BBa J04450 DelRest.gb| pFSN plasmid]] including Primers FS_01 to FS_241 used for assembly of the desired genes from the ''D. acidovorans'' genome and the partsregistry backbone pSB4K5 .]]
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As we discovered '''<span style="color:red"> Prediction Konrad </span>''' that there is a predicted promoter located right in front of DelO, we decided to include this promotor into our construct. This should ensure that the genes DelO, DelP and DelL are still expressed in the case that there is a terminator by the end of DelG. For this we designed primer FS_22 which binds right at the predicted promoter. However, this also meant, that we had to change the reverse primer for the DelG fragment in order to enable Gibson assembly of these fragments next to each oterh. Furthermore we created new primers for amplifying DelE, DelF and DelG as we were not successful in amplifying these fragments so far. These primers now offer more potential fragment combinations which can be tested. We hope to get the missing fragments with this  new strategy.
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As we discovered that there is a predicted promoter located right in front of DelO, we decided to include this promotor into our construct. This should ensure that the genes DelO, DelP and DelL are still expressed in the case that there is a terminator by the end of DelG. For this we designed primer FS_22 which binds right at the predicted promoter. However, this also meant, that we had to change the reverse primer for the DelG fragment in order to enable Gibson assembly of these fragments next to each oterh. Furthermore we created new primers for amplifying DelE, DelF and DelG as we were not successful in amplifying these fragments so far. These primers now offer more potential fragment combinations which can be tested. We hope to get the missing fragments with this  new strategy.
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Revision as of 18:12, 4 October 2013

Contents

Strategy

Vector map of pFSN plasmid including Primers FS_01 to FS_241 used for assembly of the desired genes from the D. acidovorans genome and the partsregistry backbone pSB4K5 .

As we discovered that there is a predicted promoter located right in front of DelO, we decided to include this promotor into our construct. This should ensure that the genes DelO, DelP and DelL are still expressed in the case that there is a terminator by the end of DelG. For this we designed primer FS_22 which binds right at the predicted promoter. However, this also meant, that we had to change the reverse primer for the DelG fragment in order to enable Gibson assembly of these fragments next to each oterh. Furthermore we created new primers for amplifying DelE, DelF and DelG as we were not successful in amplifying these fragments so far. These primers now offer more potential fragment combinations which can be tested. We hope to get the missing fragments with this new strategy.

Primer pairs, corresponding sequences and usage

Identifier Order date Note Sequence
FS_21: DelF_fw2013-07-13 Amplification of DelF from Delftia acidovorans Gibson Primer GACGCCATCTACCACCTGTG
FS_22: DelOP_short_fw2013-08-02 Amplification of DelOP from Delftia acidovorans inlcuding the recently predicted endogenous Promotor for DelOP Gibson Primer GATGACGCAGGGCGGCGGAATTTGTTCATC
FS_23: DelG_long_rv2013-07-13 Amplification of DelG from Delftia acidovorans Gibson primer with overhang to DelOP element including the recently predicted endogenous Promotor GATGAACAAATTCCGCCGCCCTGCGTCATCTCAGATATCTCCCAGAGTTTCGAGAAAG
FS_24: DelAE_rv2013-07-13 Amplification of DelAE from Delftia acidovorans Gibson Primer CAGAAGAATTCCCAGAAGGAGATGTCGAAG
FS_26: DelFG_rv2013-07-13 Amplification of DelFG from Delftia acidovorans Gibson Primer GAATTCATCCACGATGATCTGCATG

Amplification of Del Genes from D. acidovorans DSM-39 genome

Goals

This week we will try to amplify the sequence region of DelF-G with several combinations of primers including both, the intial primers and the recently ordered ones beeing FS_20, FS_21, FS_22, FS_23, FS_24 and FS_26. Furthermore we will continue opimtizing our protocol for the amplification of DelO-P. To evaluate whether the very weak bands on the corresponding agarose gels represent the intended amplicons, we decided to carry out re PCR and test restriction digests.

Results

PCRs from D.acidovorans DSM-39
Gene(s) Fragment Primer combination Successful?
DelA
DelB
DelC
DelD
DelE
DelF
DelG
DelA-E Primer FS_02 and FS_03
Heidelberg Tilde.png
DelE-G Primer FS_04 and FS_07
Heidelberg Yes check.svg.png
DelF-G Primer FS_20 and FS_07
Heidelberg Tilde.png
Primer FS_20 and FS_09
Heidelberg Red x.svg.png
Primer FS_21 and FS_09
Heidelberg Tilde.png
DelG Primer FS_08 and FS_09
Heidelberg Red x.svg.png
Primer FS_08 and FS_11_short
Heidelberg Red x.svg.png
DelO
DelP

DelL
DelO-P Primer FS_22 and FS_13
Heidelberg Red x.svg.png
Primer FS_22 and FS_13_short
Heidelberg Tilde.png
DelL-P Primer FS_13_short and FS_15_short
Heidelberg Red x.svg.png


Re-PCRs
Gene(s) Fragment Primer combination Successful?
DelE
DelF
DelG
DelE-G Primer FS_04 and FS_07
Heidelberg Red x.svg.png
DelF-G Primer FS_06 and FS_09
Heidelberg Red x.svg.png
Primer FS_21 and FS_09
Heidelberg Red x.svg.png
DelG Primer FS_08 and FS_09
Heidelberg Red x.svg.png
Primer FS_08 and FS_11_short
Heidelberg Red x.svg.png
DelO
DelP

DelL
DelO-P Primer FS_22 and FS_13
Heidelberg Red x.svg.png