Team:Heidelberg/Del week 11 overview

From 2013.igem.org

Contents

Week 11

Strategy

Vector map of pFSN plasmid including Primers FS_01 to FS_16 used for assembly of the desired genes from the D. acidovorans genome as well as the partsregistry backbone pSB4K5 .

We had various problems with the amplification of the fragments located in theregion DelF-G and DelO-P. For these particular PCRs long primers containing the Gibson overlaps had to be used. As these long primers often form complex secondary structures decreasing annealing efficiency we designed short version of these primers, having the same sequence region complementary to the genomic target, but lacking any longer overhangs. Consequently these primesrs will serve only for amplification of the desired genes from the genome of D. Acidovorans to obtain specific templates. This specific template afterwards will be used to introduce the designated overlap using the long primers again. In total 3 short primer versions FS_11s, FS_13s and FS_15s were ordered.


The ordered primers, their functions and sequences are listed below:

Identifier Order date Note Sequence
FS_11: DelAG_5_short_rv2013-05-07 Amplification of DelAG from Delftia acidovorans. Gibson Primer TCAGATATCTCCCAGAGTTTCGAGAAAG
FS_13: DelOP_short_rv2013-05-07 Amplification of DelOP from Delftia acidovorans. Gibson Primer with overhang to DelLTCACACCGTGGTGTCTGCAGGCG
FS_15: DelL_mRFP_pSB4K5__short_rv2013-05-07 Amplification of DelL. Gibson Primer with overhang to BBa_J04450TCAGTCCTGCAGCGCCAGCTGTTCTGTG

Amplification of Del Genes from DSM-39 genome

Goals

The PCRs which worked in the previous week will be repeated to obtain higher product yields, as high concentrations of the long amplicons are needed for a successfull Gibson Assembly. Furthermore we will try to establish PCRs for the obtained short Primers and thereby provide specific templates for the second amplification step using the corresponding primers introducing the required Gibson overlaps. Amplifications of the sequence region DelF-G will be tried with different protocols to optimize PCR conditions, as we did not obtain any amplicons at all last week. Therefore it is likely that these primers are only able to bind in a very small frame of annealing temperature or even that the used primer combination do not posses a common annealing temperature and therefore amplification with the desired combinations is not possible.

As the amplification of DelO-P also turned out to be difficult, we decided to try a backup strategy which not only includes the desired genes DelO and DelP but also DelL and, since they are located between DelP and DelL, DelM and DelN. These two genes would increase our amplicon size by only 2.2 kb, but we hoped that on the other hand the corresponding primers would work better.

Results

In the following table the outcomes of this weeks PCRs are summarized.

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-G FS_02 and FS_07
Heidelberg Red x.svg.png
DelE-G FS_04 and FS_07
Heidelberg Yes check.svg.png
FS_04 and FS_11s
Heidelberg Red x.svg.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
FS_06 and FS_11s
Heidelberg Red x.svg.png
DelG FS_08 and FS_11s
Heidelberg Tilde.png
FS_10 and FS_11
Heidelberg Yes check.svg.png
FS_10 and FS_11s
Heidelberg Yes check.svg.png
DelO
DelP

DelL
DelO-P FS_12 and FS_13
Heidelberg Red x.svg.png
FS_12 and FS_13s
Heidelberg Red x.svg.png
DelL FS_14 and FS_15
Heidelberg Yes check.svg.png
DelL-P FS_13 and FS_15s
Heidelberg Red x.svg.png
pSB4K5 pSB4K5 FS_01 and FS_16
Heidelberg Yes check.svg.png


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

DelL
DelL-P Primer FS_13_short and FS_15_short
Heidelberg Red x.svg.png