Part:BBa_K1321306

Komagataeibacter rhaeticus iGEM

Gluconacetobacter xylinus is a obligate aerobe Gram negative cellulose-producing bacterial species. Imperial iGEM 2014 team isolated a strain of G.xylinus (named G. xylinus KI for Kombucha Isolate) from the Kombucha tea seed culture (Kombucha tea seed culture is a mixture of yeast, G.xylinus and other organisms, and is used to make the increasingly popular Kombucha tea). G. xylinus KI produces cellulose readily on different carbon feedstocks. Although G. xylinus KI does not produce as high amounts of cellulose as G. xylinus ATCC 53582 (see cellulose productivity on different feedstocks), it is more amenable to genetic engineering and testing of genetic constructs.

G.xylinus KI genome sequence

G.xylinus strain KI genome was sequenced as a part of Imperial 2014 iGEM project. The genome is about 3.5Mbp in size, with a GC content of about 60%.

Culturing G. xylinus KI

G.xylinus ATCC KI growth rate varies depending on the culturing conditions. Growth rate is highest when G.xylinus is cultured at 30degC, 180rpm shaking in a well-aerated setting. However, in shaking conditions, the prevalence of cellulose non-producing mutants (cel- mutants) increases, which causes a decrease in the total yield of produced cellulose. For this reason, when high cellulose production is required, G.xylinus is often grown in static conditions, at 30degC, in a well-aerated setting. In shaking cultures, KI strain reaches stationary phase in 48-72 hours, whereas in static cultures, depending on the volume of the medium, it can take up to 15 days. When grown on HS-agar plates at 30degC, cellulose-producing colonies become visible within 48-72 hours, cel- mutant colonies between 24 and 48 hours. Cellulose producing colonies can be distinguished from cel- mutants by colony morphology: cel+ have a rough colony morphology, whereas cel- mutants have a smooth colony morphology (see Figure 1). The most commonly used medium for G.xylinum culturing is Hestrin-Schramm (HS) medium. HS medium contains the following components (for a full protocol for G.xylinus culturing, see G.xylinus culturing on [http://2014.igem.org/Team:Imperial/Protocols Imperial 2014 wiki]: 2% (w/v) glucose 0.5% (w/v) yeast extract 0.5% (w/v) peptone 0.27% (w/v) Na2HPO4 0.15% (w/v) citric acid

Figure 1. Different colony morphologies of cel+ and cel- G.xylinus colonies. Cel+ colonies have a rough colony morphology (left), whereas cel- have a smooth colony morphology(right). Plates were inoculated using 50µl of seed cultures grown to stationary phase, and incubated inverted at 30C for 8 days.

Cellulose productivity of G.xylinus KI

G.xylinus KI cellulose productivity depends strongly on the carbon feedsource used. In HS-glucose media, grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity is approximately one fourth of that of ATCC 53582 strain (see Figure 1 for comparison of ATCC53582 and KI strains and experimental details). However, cellulose productivity is increased in HS-glycerol media and HS-sucrose media (see Feedstocks for G.xylinus KI).

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Figure 3. Cellulose production of G.xylinus KI and ATCC 53582 strains in HS-glucose medium. Note that HS-glycose is not the optimal media for cellulose production using G.xylinus KI. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.

Feedstocks for G.xylinus KI

G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. have found that KI cellulose productivity is highest when grown on glycerol, and higher than ATCC 53582 when grown on sucrose (see Figure 2.)

Natural antibiotics resistance of G.xylinus KI

See Figure 3.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]