Team:Austin UTexas/Results

Conjugation

We have attempted to conjugate GFP into both G. oxydans and G. hansenii with a Diaminopimelic Acid (DAP) auxotrophic strain of E. coli . The plasmid contains the vector pMMB67EH, the promoter PA-1, GFP and a spectinomycin resistance gene.

The first conjugation was done with KOM strains 4 (G. oxydans) 5 ( G. oxydans and 15 ( L. fermentati ). We attempted these conjugations before sequencing the recipient strains, so that is why we tried to conjugate into L. fermentati . First, a mixture between a KOM strain and the DAP auxotroph strain were plated on a LB+DAP solid medium to allow for conjugation to occur. After 24 hours of incubation, I scraped up the growth and plated each conjugation mixture onto a LB+Spec plate.

LB+SPEC plates that contain conjugation mixtures of KOM 4, 5 and 15 ( L. fermentati )

Next, we viewed the potential transconjugants on a fluorescence microscope.

Using a fluorescent microscope, this was a picture taken of the G. oxydans strain, KOM 4, without the plasmid that contains GFP.

Using a fluorescent microscope, this was a picture taken of the potential transconjugant, KOM 4, with the plasmid GFP. 16s sequencing was still needed to confirm successful conjugation.

We then picked these glowing colonies and then after streaking them out onto more LB+Spec plates, we attempted to use 16s sequencing to confirm successful conjugation. After troubleshooting our 16s procedure, we were finally able to obtain a viable sequencing result. However, all of the glowing colonies were identified as E. coli. For the next round of conjugation, we used a strain of both G. oxydans and Gluconacetobacter hansenii from the American Type Culture Collection (ATCC).

This is a LB+DAP plate on a dark reader that has four different conjugations occurring at one time. The two left quadrants have the same ATCC strain of G. oxydans,, while the two quadrants on the right have Ga. hansenii.

These growths were then scraped up and plated onto a LB+Spec plate.

These are my potential transconjugants on a LB+DAP plates. The dark reader was used when taking this picture. The top two are G. oxydans while the bottom two are G. hansenii.

We then picked isolated colonies and streaked them out onto LB+DAP plates. After using 16s sequencing on the potential transconjugants, we encountered an anomaly. Instead of amplifying the 16s gene, we recieved the sequence of the L,D Transpeptidase gene of E. coli . We plan on repeating the 16s procedure.

Ethanol Reduction

Identifying genes of interest

In order to design a construct increasing expression of PQQ-ADH and ALDH in Ga. hansenii, 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 Comamonas testosteroni was compared against sequences in the Ga. hansenii genome using BLAST (Table 1,). One ADH enzyme found in the Ga. hansenii genome sequence matches the C. testosteroni sequence with a query cover value of 94% and an E value of 0 (third line of table 1).

*Need to insert table 1 here*

Creation of Golden Gate parts

In order to assemble the construct, the coding sequences for the genes of interest must be amplified from the Ga. hansenii 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 (Figure 1), and the PQQ-ADH coding sequence contains a BsmBI restriction site near the end of the gene (Figure 2). 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. Table 2 contains more information about each of these oligonucleotides. All were ordered from IDT.

*Will need to insert additional tables and figures. Numbering of tables and figures may need to be adjusted. If anyone is willing to help insert figures and tables in this section, contact me (Stratton) and I'll send everything to be inserted.