Team:Heidelberg/Templates/Del week10 overview

From 2013.igem.org

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==Strategy==
==Strategy==
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[[File:Heidelberg_Strategie1.png|300px|right|thumb|Vector map of [[:File:Heidelberg_Psb4k5+BBa J04450 DelRest.gb| pFSN plasmid]] including Primers FS_01 to FS_16 used for assembly of the desired genes from ''D. acidovorans'' genome and the partsregistry backbone pSB4K5 .]]In order to transfer the delftibactin NRPS cluster from ''D. acidovorans'' in ''E. coli'' for efficient production of delftibactin, we designed the plasmid pFSN. It comprises the above listed genes as inserts. Furthermore, it includes an IPTG-inducible promoter, an origin of replication, compatible to those of the plasmids pDelH and pIK8.6 (required for delftibactin expression in ''E. coli'') expression plasmids as well as a kanamycin resistance marker. The choice of the ori was critical, as three plasmids PFSN, pDelH and PIK8.6 would have to be cotransformed in order to obtain the final ''E. coli'' delftibactin production strain and all three plasmids needed to be stabely propagated.<br/>Additionally, mRFP was added as last cds behind the DelRest genes onto the pFSN construct in oder to have an easy control for expression of the DelRest genes located in front of mRFP.<br/><br/> In the Delftibactin cluster the genes DelA, DelB, DelC, DelD, DelE, DelF and DelG are encoded as a single polycistronic operon. However, it is most likely not possible to amplify the entire coding sequence of DelA to DelG in one single PCR amplicon, as its corresponding size of 22.8 kb in combination with the complex GC-rich sequence is most likely not suitable for commonly available polymerases and PCR protocols. Consequently, we will use various primer combinations to amplify the genes in different sub-fragments. These will be assemled again using Gibson cloning<br/><br/>DelO and DelP are encoded in reverse-complementary direction on the Delftibactin cluster. However, in our construct these two genes will be assembled in the same orientation as the other ones and thereby added to the DelA-G operon. As DelO and DelP are also located next to each other and they will therefor be amplified together as a single 2.7 kb amplicon<br/><br/> DelL is the somehow lonesome rider, not surrounded by any of our desired del genes and will thus also be amplified seperately (fragment size 1.4 kb). <br/>As complications are most likely to occure for the very long PCR amplicons, we will start with the huge DelA-G region and optimize PCR conditions for the corresponding amplicons.<br/><br/>We decided to use the DSM-39 substrain of ''Delftia acidovorans'' as our genomic template since our reference paper from Johnston ''et al.'' (2013) showed the gold precipitation expreiments using this substrain. The only full-length genomic sequence available for ''D. acidovorans'' is, however, the SPH-1 substrain sequence. Nevertheless, as the abovementioned paper also based their analysis on the SPH-1 strain sequence, we also based our primer designs on the available SPH-1 substrain genomic sequence, hoping the Del cluster sequence would be highly conserved among different ''D. acidovorans'' strains.<div style="clear:both"></div><br/><br/>====Plasmid backbone part from the partsregistry====<br/>
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[[File:Heidelberg_Strategie1.png|300px|right|thumb|Vector map of [[:File:Heidelberg_Psb4k5+BBa J04450 DelRest.gb| pFSN plasmid]] including Primers FS_01 to FS_16 used for assembly of the desired genes from ''D. acidovorans'' genome and the partsregistry backbone pSB4K5 .]]In order to transfer the delftibactin NRPS cluster from ''D. acidovorans'' in ''E. coli'' for efficient production of delftibactin, we designed the plasmid pFSN. It comprises the above listed genes as inserts. Furthermore, it includes an IPTG-inducible promoter, an origin of replication, compatible to those of the plasmids pDelH and pIK8.6 (required for delftibactin expression in ''E. coli'') expression plasmids as well as a kanamycin resistance marker. The choice of the ori was critical, as three plasmids PFSN, pDelH and PIK8.6 would have to be cotransformed in order to obtain the final ''E. coli'' delftibactin production strain and all three plasmids needed to be stabely propagated.<br/>Additionally, mRFP was added as last cds behind the DelRest genes onto the pFSN construct in oder to have an easy control for expression of the DelRest genes located in front of mRFP.<br/><br/> In the Delftibactin cluster the genes DelA, DelB, DelC, DelD, DelE, DelF and DelG are encoded as a single polycistronic operon. However, it is most likely not possible to amplify the entire coding sequence of DelA to DelG in one single PCR amplicon, as its corresponding size of 22.8 kb in combination with the complex GC-rich sequence is most likely not suitable for commonly available polymerases and PCR protocols. Consequently, we will use various primer combinations to amplify the genes in different sub-fragments. These will be assemled again using Gibson cloning<br/><br/>DelO and DelP are encoded in reverse-complementary direction on the Delftibactin cluster. However, in our construct these two genes will be assembled in the same orientation as the other ones and thereby added to the DelA-G operon. As DelO and DelP are also located next to each other and they will therefor be amplified together as a single 2.7 kb amplicon<br/><br/> DelL is the somehow lonesome rider, not surrounded by any of our desired del genes and will thus also be amplified seperately (fragment size 1.4 kb). <br/>As complications are most likely to occure for the very long PCR amplicons, we will start with the huge DelA-G region and optimize PCR conditions for the corresponding amplicons.<br/><br/>We decided to use the DSM-39 substrain of ''Delftia acidovorans'' as our genomic template since our reference paper from Johnston ''et al.'' (2013) showed the gold precipitation expreiments using this substrain. The only full-length genomic sequence available for ''D. acidovorans'' is, however, the SPH-1 substrain sequence. Nevertheless, as the abovementioned paper also based their analysis on the SPH-1 strain sequence, we also based our primer designs on the available SPH-1 substrain genomic sequence, hoping the Del cluster sequence would be highly conserved among different ''D. acidovorans'' strains.<div style="clear:both"></div><br/><br/>
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====Plasmid backbone part from the partsregistry====
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| pSB4K5 || J04450 || Spring 2012 || 1|| 5G || Backbone for DelA-G,OP,L || Kanamycin
| pSB4K5 || J04450 || Spring 2012 || 1|| 5G || Backbone for DelA-G,OP,L || Kanamycin
|}
|}
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<div style="clear:both"></div><br/>====Primer pairs, corresponding sequences and usage===<br/>
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<div style="clear:both"></div><br/>
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====Primer pairs, corresponding sequences and usage===
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{| class="wikitable"
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Revision as of 18:04, 4 October 2013

Contents

Strategy

Vector map of pFSN plasmid including Primers FS_01 to FS_16 used for assembly of the desired genes from D. acidovorans genome and the partsregistry backbone pSB4K5 .
In order to transfer the delftibactin NRPS cluster from D. acidovorans in E. coli for efficient production of delftibactin, we designed the plasmid pFSN. It comprises the above listed genes as inserts. Furthermore, it includes an IPTG-inducible promoter, an origin of replication, compatible to those of the plasmids pDelH and pIK8.6 (required for delftibactin expression in E. coli) expression plasmids as well as a kanamycin resistance marker. The choice of the ori was critical, as three plasmids PFSN, pDelH and PIK8.6 would have to be cotransformed in order to obtain the final E. coli delftibactin production strain and all three plasmids needed to be stabely propagated.
Additionally, mRFP was added as last cds behind the DelRest genes onto the pFSN construct in oder to have an easy control for expression of the DelRest genes located in front of mRFP.

In the Delftibactin cluster the genes DelA, DelB, DelC, DelD, DelE, DelF and DelG are encoded as a single polycistronic operon. However, it is most likely not possible to amplify the entire coding sequence of DelA to DelG in one single PCR amplicon, as its corresponding size of 22.8 kb in combination with the complex GC-rich sequence is most likely not suitable for commonly available polymerases and PCR protocols. Consequently, we will use various primer combinations to amplify the genes in different sub-fragments. These will be assemled again using Gibson cloning

DelO and DelP are encoded in reverse-complementary direction on the Delftibactin cluster. However, in our construct these two genes will be assembled in the same orientation as the other ones and thereby added to the DelA-G operon. As DelO and DelP are also located next to each other and they will therefor be amplified together as a single 2.7 kb amplicon

DelL is the somehow lonesome rider, not surrounded by any of our desired del genes and will thus also be amplified seperately (fragment size 1.4 kb).
As complications are most likely to occure for the very long PCR amplicons, we will start with the huge DelA-G region and optimize PCR conditions for the corresponding amplicons.

We decided to use the DSM-39 substrain of Delftia acidovorans as our genomic template since our reference paper from Johnston et al. (2013) showed the gold precipitation expreiments using this substrain. The only full-length genomic sequence available for D. acidovorans is, however, the SPH-1 substrain sequence. Nevertheless, as the abovementioned paper also based their analysis on the SPH-1 strain sequence, we also based our primer designs on the available SPH-1 substrain genomic sequence, hoping the Del cluster sequence would be highly conserved among different D. acidovorans strains.


Plasmid backbone part from the partsregistry

Backbone Part Distribution Plate Well Usage Resistance
pSB4K5 J04450 Spring 2012 1 5G Backbone for DelA-G,OP,L Kanamycin

=Primer pairs, corresponding sequences and usage

Identifier Order date Note Sequence
FS_01: pSB4K5_DelA_rv20-13-06-28Amplification of pSB4K5 from the iGEM Distribution. Gibson Primer with overhang to DelA introducing the RBS BBa_B0035 TCGCGGCGATCCGGTACTGCGCCTCTGTTGAACATCTGATATTCT
CCTCTTTAATCGACAGATTGTGTGAAATTGTTATCCGCTCAC
FS_02: DelAG_1_fw2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. TTCAACAGAGGCGCAGTACCGGATC
FS_03: DelAG_1_rv2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. GTCGGAGACGATGTGGTGCATCAC
FS_04: DelAG_2_fw2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. CTGCAGGCCAATGAGCACATCCTG
FS_05: DelAG_2_rv2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. CACAGGTGGTAGATGGCGTC
FS_06: DelAG_3_fw2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. ATTGCGAGGACTTGCTCGATG
FS_07: DelAG_3_rv2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. TTTGCTGCAGCGCCAGCACATCGAG
FS_08: DelAG_4_fw2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. GTACGGCCTATCACATCAGCG
FS_09: DelAG_4_rv2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. GAAGCTCAGCAGGTTGGGCGAGACG
FS_10: DelAG_5_fw2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. GAATTTTGTTCCACCACCTGCTG
FS_11: DelAG_5_rv2013-06-28 Amplification of DelA-G from Delftia acidovorans genome. Gibson Primer with overhang to DelOP CTTGAGCAGGCGCAGTACCTCGGAGGGCGGTCGGCTGGCGTTTTCCATGATTCAGG
TTTCCTGTGTGAAGCTCATCTCAGATATCTCCCAGAGTTTCGAGAAAG
FS_11: DelAG_5_short_rv2013-08-02 Amplification of DelA-G from Delftia acidovorans genome. TCAGATATCTCCCAGAGTTTCGAGAAAG
FS_12: DelOP_fw2013-06-28 Amplification of DelO-P from Delftia acidovorans genome. GAATCATGGAAAACGCCAGCCGAC
FS_13: DelOP_rv2013-06-28 Amplification of DelO-P from Delftia acidovorans genome. Gibson Primer with overhang to DelL CAATGTTGGAGGGGCCGAAGCCGATGCCGATCAGCGGGTGGGTTTGCATGGAAGGT
CCTTTCATTGGGTCGATGCGTCCAGTGTCACACCGTGGTGTCTGCAGGCG
FS_13: DelOP_short_rv2013-06-28 Amplification of DelO-P from Delftia acidovorans genome. Gibson Primer with overhang to DelLTCACACCGTGGTGTCTGCAGGCG
FS_14: DelL_fw2013-06-28 Amplification of DelL from Delftia acidovorans genome. CAAACCCACCCGCTGATCGGCATC
FS_15: DelL_mRFP_pSB4K5_rv2013-06-28 Amplification of DelL from Delftia acidovorans genome. Gibson Primer with overhang to BBa_J04450 GAAACGCATGAACTCTTTGATAACGTCTTCGGAGGAAGCCATCTAGTATTTCTCCTC
TTTCTCTAGTATCAGTCCTGCAGCGCCAGCTGTTCTGTG
FS_15: DelL_mRFP_pSB4K5__short_rv2013-06-28 Amplification of DelL from Delftia acidovorans genome. Gibson Primer with overhang to BBa_J04450TCAGTCCTGCAGCGCCAGCTGTTCTGTG
FS_16: mRFP_pSB4K5_fw (Del)2013-06-28 Amplification of pSB4K5 from iGEM Distribution. Gibson Primer GCTTCCTCCGAAGACGTTATC

Amplification of Del Genes from DSM-39 genome

Goals

This week primers FS_01 to FS_16 arrived. As the aim of this week is to amplify the backbone pSB4K5 with Gibson overlaps matching our initial and very last insert fragment, we will firstly use the pSB4K5 backbone including the mRFP cassette as template for this purpose. Furthermore amplification of the desired genes from the Del-cluster of D.acidovorans DSM-39 will be carried out in parallel.

Results

The following table summarizes the outcomes of this weeks PCRs.

PCRs from D.acidovorans DSM-39
Gene(s) Fragment Primer combination Successful?
DelA
DelB
DelC
DelD
DelE
DelF
DelG
DelA-E FS_02 and FS_03
Heidelberg Yes check.svg.png
DelA-F FS_02 and FS_05
Heidelberg Yes check.svg.png
DelA-G FS_02 and FS_07
Heidelberg Red x.svg.png
DelE-G FS_04 and FS_07
Heidelberg Tilde.png
DelF-G FS_06 and FS_07
Heidelberg Red x.svg.png
FS_06 and FS_09
Heidelberg Red x.svg.png
FS_06 and FS_11
Heidelberg Red x.svg.png
DelG FS_08 and FS_11
Heidelberg Red x.svg.png
DelO
DelP

DelL
DelO-P FS_12 and FS_13
Heidelberg Red x.svg.png
DelL FS_14 and FS_15
Heidelberg Yes check.svg.png
pSB4K5 pSB4K5 FS_01 and FS_16
Heidelberg Yes check.svg.png