Difference between revisions of "Team:Austin UTexas/Results"

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In order to design a construct increasing expression of PQQ-ADH and ALDH in <i>Ga. hansenii</i>, it was necessary to find the genome of the ATCC strain and identify the coding sequences for these genes. The whole genome shotgun sequence for our organism, ATCC 53582, is published on NCBI by J. Abbot (2015) with annotations regarding the functions of specific sequences. Coding sequences are annotated with proposed gene products. Though there are several aldehyde dehydrogenase genes annotated in the genome, there is only one which is described as membrane-bound, matching the description from Mamlouk and Gullo (2013). There are additionally multiple alcohol dehydrogenases. A known amino acid sequence for a homologous PQQ-ADH in ,i.Comamonas testosteroni</i> was compared against sequences in the <i>Ga. hansenii</i> genome using BLAST (<b>Table 1</b>). One ADH enzyme found in the <i>Ga. hansenii</i> genome sequence matches the <i>C. testosteroni</i> sequence with a query cover value of 94% and an E value of 0 (third line of <b>Table 1</b>).
 
In order to design a construct increasing expression of PQQ-ADH and ALDH in <i>Ga. hansenii</i>, it was necessary to find the genome of the ATCC strain and identify the coding sequences for these genes. The whole genome shotgun sequence for our organism, ATCC 53582, is published on NCBI by J. Abbot (2015) with annotations regarding the functions of specific sequences. Coding sequences are annotated with proposed gene products. Though there are several aldehyde dehydrogenase genes annotated in the genome, there is only one which is described as membrane-bound, matching the description from Mamlouk and Gullo (2013). There are additionally multiple alcohol dehydrogenases. A known amino acid sequence for a homologous PQQ-ADH in ,i.Comamonas testosteroni</i> was compared against sequences in the <i>Ga. hansenii</i> genome using BLAST (<b>Table 1</b>). One ADH enzyme found in the <i>Ga. hansenii</i> genome sequence matches the <i>C. testosteroni</i> sequence with a query cover value of 94% and an E value of 0 (third line of <b>Table 1</b>).
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<u>Creation of Golden Gate parts</u>
 
 
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[[File:T--Austin UTexas--ALDHlinmap.png|thumb|left|'''Figure 1''': Linear map of the coding sequence for membrane-bound ALDH with a Golden Gate type 3 prefix and suffix. BsmBI and BsaI sites are indicated. The restriction sites at either end are included in the prefix and suffix, but the internal BsaI site must be removed to create a functioning Golden Gate part.|600px]]
 
[[File:T--Austin UTexas--ALDHlinmap.png|thumb|left|'''Figure 1''': Linear map of the coding sequence for membrane-bound ALDH with a Golden Gate type 3 prefix and suffix. BsmBI and BsaI sites are indicated. The restriction sites at either end are included in the prefix and suffix, but the internal BsaI site must be removed to create a functioning Golden Gate part.|600px]]
 
[[File:T--Austin UTexas--ADHlinmap.png|thumb|left|'''Figure 2''': Linear map of the coding sequence for PQQ-ADH with a Golden Gate type 3 prefix and suffix. EcoRI, BsmBI, and BsaI sites are indicated. The centermost BsmBI restriction site is in the coding sequence and must be removed to create a functional Golden Gate part. EcoRI is not used in Golden Gate assembly, so those sites do not necessarily need to be removed.|600px]]
 
[[File:T--Austin UTexas--ADHlinmap.png|thumb|left|'''Figure 2''': Linear map of the coding sequence for PQQ-ADH with a Golden Gate type 3 prefix and suffix. EcoRI, BsmBI, and BsaI sites are indicated. The centermost BsmBI restriction site is in the coding sequence and must be removed to create a functional Golden Gate part. EcoRI is not used in Golden Gate assembly, so those sites do not necessarily need to be removed.|600px]]
 
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<u>Creation of Golden Gate parts</u>
 
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In order to assemble the construct, the coding sequences for the genes of interest must be amplified from the <i>Ga. hansenii</i> genome and edited such that they have the correct Golden Gate overhangs and no internal BsaI or BsmBI restriction sites. The sequences were uploaded to Benchling for analysis and planning. The coding sequence for the membrane-bound ALDH contains a BsaI restriction site near the middle of the gene (<b>Figure 1</b>), and the PQQ-ADH coding sequence contains a BsmBI restriction site near the end of the gene (<b>Figure 2</b>). To eliminate the BsaI site in ALDH, primers were designed that would introduce a point mutation at the restriction site. One set of primers, igem2016_KOM_EtOH_01 and igem2016_KOM_EtOH_02, amplifies the sequence upstream of the restriction site, adding a type 3 Golden Gate prefix and removing the restriction site. Another set, igem2016_KOM_EtOH_03 and igem2016_KOM_EtOH_04, amplifies the region downstream of the restriction site, introducing a mutation to the site and adding a type 3 Golden Gate suffix to the end of the gene. These two products will be used in an overlap PCR reaction to create a final product with no BsaI restriction sites and the correct prefix and suffix for assembly. To remove the BsmBI site from the PQQ-ADH coding sequence, a set of primers (igem2016_KOM_EtOH_05 and igem2016_KOM_EtOH_06) was designed to amplify the region upstream of the restriction site and add a Golden Gate type 3 prefix to the beginning of the sequence. The reverse primer additionally adds a mutation to existing BsmBI restriction site and creates a new BsmBI restriction site that will be used to join the piece to a double-stranded DNA, igem2016_KOM_EtOH_07, containing the rest of the gene’s coding sequence appended with a Golden Gate type 3 suffix. The assembly of the PQQ-ADH part will therefore take place in two reactions: one reaction in which the upstream piece of DNA is created, and one reaction in which it is ligated to the gBlock. <b>Table 2</b> contains more information about each of these oligonucleotides. All were ordered from IDT.
 
In order to assemble the construct, the coding sequences for the genes of interest must be amplified from the <i>Ga. hansenii</i> genome and edited such that they have the correct Golden Gate overhangs and no internal BsaI or BsmBI restriction sites. The sequences were uploaded to Benchling for analysis and planning. The coding sequence for the membrane-bound ALDH contains a BsaI restriction site near the middle of the gene (<b>Figure 1</b>), and the PQQ-ADH coding sequence contains a BsmBI restriction site near the end of the gene (<b>Figure 2</b>). To eliminate the BsaI site in ALDH, primers were designed that would introduce a point mutation at the restriction site. One set of primers, igem2016_KOM_EtOH_01 and igem2016_KOM_EtOH_02, amplifies the sequence upstream of the restriction site, adding a type 3 Golden Gate prefix and removing the restriction site. Another set, igem2016_KOM_EtOH_03 and igem2016_KOM_EtOH_04, amplifies the region downstream of the restriction site, introducing a mutation to the site and adding a type 3 Golden Gate suffix to the end of the gene. These two products will be used in an overlap PCR reaction to create a final product with no BsaI restriction sites and the correct prefix and suffix for assembly. To remove the BsmBI site from the PQQ-ADH coding sequence, a set of primers (igem2016_KOM_EtOH_05 and igem2016_KOM_EtOH_06) was designed to amplify the region upstream of the restriction site and add a Golden Gate type 3 prefix to the beginning of the sequence. The reverse primer additionally adds a mutation to existing BsmBI restriction site and creates a new BsmBI restriction site that will be used to join the piece to a double-stranded DNA, igem2016_KOM_EtOH_07, containing the rest of the gene’s coding sequence appended with a Golden Gate type 3 suffix. The assembly of the PQQ-ADH part will therefore take place in two reactions: one reaction in which the upstream piece of DNA is created, and one reaction in which it is ligated to the gBlock. <b>Table 2</b> contains more information about each of these oligonucleotides. All were ordered from IDT.

Revision as of 20:42, 18 October 2016

Austin_UTexas

Results


Click on one of the images below to learn more about our results!




Kombucha Strains

Conjugation

Recapitulation

Ethanol

pH Sensors