Difference between revisions of "Team:MIT/Chutch"

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Cytophaga Hutchinsonii

Background on C. hutchinsonii

Cytophaga hutchinsonii is an aerobic and mesophilic soil bacterium that rapidly degrades crystalline cellulose. It is capable of using cellulose as its sole carbon and energy source, and it grows better on cellulose than on any other carbon source (Wilson 2009). However, the mechanisms of its cellulose degradation are still largely unknown (Zhou 2015). Researchers have observed that it requires direct contact with the cellulosic substrate, most of its cellulases are cell-associated, its ability to degrade cellulose is associated with its ability to glide, and that it contains no obvious homologs of many known crucial cellulolytic enzymes (Xie et al. 2007; Wilson 2009; Zhu 2010). C. hutchinsonii either uses a novel mechanism or novel processive enzymes different from both the free cellulase system and the cellulosome complex (Wilson 2009; McBride 2014; Zhu et al. 2015). Researchers have theorized a novel mechanism in which C. hutchinsonii uses proteins to bind cellulose and facilitate initial digestion by cell-surface cellulases, and then transports cellobiose and longer cellodextrins into the periplasm for further digestion (McBride et al. 2014; Wilson 2009; Xie et al. 2007). However, recently researchers have shown that the transport proteins thought to be involved in transporting the cellodextrins are not crucial for cellulose degradation (Zhu et al. 2015). Thus, C. hutchinsonii either uses unknown transport proteins to import cellodextrins or uses novel and poorly uncharacterized surface enzymes to digest cellulose extracellularly (Zhu et al. 2015). This made C. hutchinsonii very challenging to model.

C. hutchinsonii’s breakdown of cellulose

When C. hutchinsonii is grown on pure crystalline cellulose, soluble sugars (including glucose, cellobiose, cellotriose, and cellotetraose) accumulate extracellularly. Small amounts of these soluble sugars are detected in cultures in exponential growth phase, and larger amounts are detected in cultures incubated with resting cells. Most of these soluble sugars are cellotriose and cellobiose, and smaller amounts are glucose and cellotetrose (Zhu et al. 2010). When C. hutchinsonii is grown on filter paper, a yellow slime is produced that consists of both hexose and pentose saccharides (such as polysaccharides composed of glucose, mannose, arabinose, and xylose) (Liu 2012). Although filter paper is only around 16% pentose sugars, around twice as many pentoses accumulate in solution that hexoses (Liu 2012). This is consistent with reports that C. hutchinsonii is able to break down polymers of pentoses (such as xylooligosaccharides), but is unable to utilize the resulting soluble sugars (i.e. xylose) as a sole carbon source (Xie et al. 2007). It is hypothesized C. hutchinsonii tightly couples an efficient process of cellulose degradation with assimilation of the resulting cellooligosaccharides.

Handling C. hutchinsonii

We developed protocols to grow C. hutchinsonii, including plating techniques and media formulations.

It forms yellow-orange colonies due to flexirubin production (McBride 2014).

There is a long lag phase of 72 - 240 hours if C. hutchinsonii is switched from growing on glucose to filter paper or cellodextrins (Zhu et al. 2010). For cellulose-inoculated cells, lag phase on filter paper is about 3 days, on glucose about days, and on cellobiose about 7 days (Liu 2012). Under starvation conditions, the long, flexible rod cell of Cytophaga hutchinsonii would bend and turn into circular cell, which fails to produce carboxymethyl cellulase (Han et al 2009). (Problems with using flow cytometry)

We investigated hypothesized natural multiple-antibiotic resistance in C. hutchinsonii by culturing it in YPD along with Ampicillin, Kanamycin, Chloramphenicol, Spectinomycin and Tetracycline. We found that it does not display resistance to any of these antibiotics.

Transforming C. hutchinsonii

In order to be able to engineer C. hutchinsonii, we first had to demonstrate that we could perform a genetic transformation. Having shown that C. hutchinsonii did not show any native resistance to chloramphenicol or kanamycin (our MoClo selection markers), we decided to start by transforming C. hutchinsonii with those resistance genes. To do this, we inserted our C. hutchinsonii oriC part (see Part Design pages) into two standard MoClo cloning vectors - DVK and DVC (modified versions of pSB1K3 and pSB1C3 respectively). Using E. coli based cloning vectors allowed us to grow up large amounts of DNA in a better characterized cloning chassis and the oriC insert allowed the plasmid to be maintained and hopefully expressed in C. hutchinsonii once transformed. Both plasmids were transformed into C. hutchinsonii via electroporation (see Protocols page) and based on existing literature(Ji et al. 2014, Wang et al. 2014) the DVC and DVK transformants were plated on 10 ug/mL Cm and 30 ug/mL Kan respectively. The DVC transformation was successful fairly early on and we were able to demonstrate that DVC+ C. hutchinsonii were capable of growing on concentrations of chloramphenicol up to 10 ug/mL whereas the wild-type did not demonstrate any chloramphenicol resistance (see picture below). We believe that we were able to successfully transform DVK into C. hutchinsonii as well but did not have sufficient time for characterization. Based on this experiment, we believe that both chloramphenicol and kanamycin could be used as selection markers for maintaining plasmids in C. hutchinsonii. This means that it would be feasible to use the MoClo system to assemble plasmids using DVK/DVC along with the oriC part and transcriptional units containing heterologous genes for further characterization.

References

Ji et al, Applied and Environmental Microbiology, 80(15):4511-4518 (2014)

Wang et al, Applied and Environmental Microbiology, 80(19):6037-6045 (2014)

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 In order to be able to engineer <i>C. hutchinsonii</i>, we first had to demonstrate that we could perform a genetic transformation.  Having shown that <i>C. hutchinsonii</i> did not show any native resistance to chloramphenicol or kanamycin (our MoClo selection markers), we decided to start by transforming <i>C. hutchinsonii</i> with those resistance genes.  To do this, we inserted our <i>C. hutchinsonii</i> oriC part (see Part Design pages) into two standard MoClo cloning vectors - DVK and DVC (modified versions of pSB1K3 and pSB1C3 respectively).  Using <i>E. coli</i> based cloning vectors allowed us to grow up large amounts of DNA in a better characterized cloning chassis and the oriC insert allowed the plasmid to be maintained and hopefully expressed in <i>C. hutchinsonii</i> once transformed.  Both plasmids were transformed into <i>C. hutchinsonii</i> via electroporation (see Protocols page) and based on existing literature(Ji et al. 2014, Wang et al. 2014) the DVC and DVK transformants were plated on 10 ug/mL Cm and 30 ug/mL Kan respectively.  The DVC transformation was successful fairly early on and we were able to demonstrate that DVC+ <i>C. hutchinsonii</i> were capable of growing on concentrations of chloramphenicol up to 10 ug/mL whereas the wild-type did not demonstrate any chloramphenicol resistance (see picture below).  We believe that we were able to successfully transform DVK into <i>C. hutchinsonii</i> as well but did not have sufficient time for characterization.  Based on this experiment, we believe that both chloramphenicol and kanamycin could be used as selection markers for maintaining plasmids in <i>C. hutchinsonii</i>.  This means that it would be feasible to use the MoClo system to assemble plasmids using DVK/DVC along with the oriC part and transcriptional units containing heterologous genes for further characterization.
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