Team:Brown-Stanford/REGObricks/Transforming

From 2011.igem.org

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== '''Transformation''' ==
== '''Transformation''' ==
 +
=== '''Due Respect''' ===
 +
 +
Those familiar with the iGEM competition might remember a previous predecessor who also focused on the microbial carbonate precipitation - Bacilla Filla, Team Newcastle 2010’s project investigated the potential of Bacillus subtilis to remediate fractures in concrete architecture. However, due to the finicky nature of urease, they decided to focus on upregulating urease production inside the cell for their project. 
 +
''
 +
“Previous experiments involving up-regulating ureA, ureB and ureC in B. subtilis have not lead to an increase in urease production. This could be due to yet unidentified genes that are involved in the process. Therefore, we looked for another strategy.” – Newcastle 2010''
 +
 +
Since the last Jamboree, the study of microbial precipitation has risen in prominence beyond just remediation. As we suggest in our project, it is instead being championed as a new way to aggregate building materials entirely. As the previous graph referenced, this expansion is related to the robust urease function present in the new organism of study: S. pasteurii.
 +
 +
=== '''Motivation''' ===
 +
 +
 +
Though B. subitilis has more documentation of successful transformations, we decided to walk the path less traveled by. Characterization from recent research has shown numerous attractive features intrinsic to the S. pasteurii bacterium, including
 +
a. Robust function of its urease activity, even in 100% salinity [########]
 +
b. Its ability to utilize waste products and proliferate from cheap sources of nutrition (ie. Corn liquor, vegemite, etc).
 +
c. The success of mutagenic experiments to increase urease function: [########]
 +
Correspondingly, the motivation to investigate the transformation of S. pasteurii comes from several key benefits that would be accomplished if we were successful:
 +
i. The ability to confer antibiotic resistance to the cell culture, opening the door to
 +
i. More rigorous genetic controls cell culture fidelity**
 +
ii. Allowing continued selection in directed evolution experiments
 +
ii. The ability to introduce traits that will strengthen the bacterium profile for extraterrestrial space travel
 +
With a successful path to genetic recombination, we could add this organism to our list of powerful chassis’s, for use both terrestrial and cosmic use.
 +
 +
 +
 +
**During the course of our cell culture of S. pasteurii, we frequently encountered contamination of our media-rich culture, despite our best attempts at sterilization techniques under the UV hood.
 +
 +
[picture of UV hood]
 +
 +
The risk involved in this project selection was the existence of good reasons for the lack of published articles on S. pasteurii transformation. But in the words of Microsoft’s blue monster, we decided that we were going to either go big or go home.
 +
 +
[big blue monster]
 +
 +
 +
=== '''Experiment Design ''' ===
 +
 +
 +
Through all of the research we scoured, we found only one mention of the genetic transformation of S. pasteurii, from a symposium done by a then-undergraduate student in Arizona State University. In it she documented a seemingly successful transformation of S. pasteurii using protoplast transformation, giving us hope of its feasibility. Through fortuitous email correspondence, we were able establish contact with Alicia, who had had went on to become a practicing attorney. She kindly faxed us her old lab notebook sheets. We are still in awe of her organization skills. 
 +
 +
Several methods of transformation were available to choose from:
 +
 +
* Soil Transformation- relying on the natural competency of the bacteria to transfer genetic information
 +
*Protoplast Transformation- Digestion of the cell wall with lysozymes, followed by induction of DNA uptake by PEG 8000.
 +
*Electroporation- research suggests limited success with gram positive cells
 +
*Protoplast and Electroporation- a combination of two techniques, use of lysozymes to digest cell wall and electroporation to create entrances for DNA
 +
Because of S. pasteurii’s thick cell wall, the protoplast transformation seemed the most logical to succeed.
 +
 +
 +
=== '''Transformation''' ===
 +
 +
 +
S. pasteurii cells were obtained via ATCC 11859 stock.
 +
The transforming DNA was obtained from Dr. Yun-Peng Chao, Professor of Chemical Engineering at
 +
Feng Chia University in Taiwan. Plasmid pUB110 (not to be confused with pBU11 _______), was a shuttle vector between E. coli and B. subtilis, containing an Ampicillin cassette for selection of the former, and a Kanamycin cassette for the latter.
 +
 +
[picture of pUB110]
 +
 +
pUB110 was selected following the protocols suggested by Alicia’s notes, reasoning along the lines that the homology between the two bacteria (S. pasteurii was previously known as Bacillus pasteurii before a recategorization) would be sufficient to allow expression of the Kanamycin resistance cassette inside S. pasteurii. 
 +
 +
Newcastle iGEM’s 2009 Sporulation Part (BB-_______________) was also received via request to the Registry of Standardized Parts.
 +
 +
Protocols for the transformation can be found here. _______________
 +
 +
Lysozyme from chicken egg white (L3790 - 10 mg/mL) was obtained from Sigma-Aldrich.
 +
 +
=== '''Troubleshooting''' ===
 +
 +
Many attempts at protoplast transformation were carried out, but the results were unsuccessful: there was either no growth on the Kanamycin resistance plates, or there was excessive growth, with distinctly different colony features and tested negative on urea-phenol red test plates. This suggested that there was contamination due to 1) improperly prepared kanamycin plates or 2) kanamycin resistant strains colonizing the extremely rich recovery plates.
 +
 +
Troubleshooting suggested that the capacity of the protoplasts to regenerate their cell wall was a potential limiting factor. Viewing of the lysozyme digestion showed nearly complete digestion of the cell membranes (before and after). Procedures for collecting the protoplasts involved several centrifugation step at an unspecified “maximum speed” according to the protocol, though there was difficulty in collecting cell pellets at speeds below 8000 g, especially after the addition of viscous PEG 8000.
 +
 +
It is speculated that repeated centrifuge at high speeds was too traumatic for the digested protoplast cells, and the survival rate following the transformation was too low to produce colony forming units. 
 +
Additional difficulties also arose in the recovery phase of the protoplast transformation, as the lack of antibiotic selection sometimes compromised portions of our rich recovery plates. (The recovery plates contained over ______ g of sucrose/mL).
 +
 +
[picture of contaminated cells]
 +
 +
=== '''Conclusions''' ===
 +
 +
In retrospect, we were a little too ambitious in choosing the scale of our projects. The elucidation of successful transformation protocols required more time than we had available, and at the time of this wikifreeze, we are still trying different concentrations of PEG, and lysozyme digestion on S. pasteurii.
 +
 +
Our plan to characterize the function of 2009 Newcastle’s sporulation part is also currently unrealized, due to the difficulties in transforming S. past. Nevertheless, we believe that though it has not been realized for the cut-off date of this jamboree, sophisticated investigation of S. pasteurii as a genetic chassis is still a worthwhile endeavor. Particularly as the industrial commercialization of biocementation reaches fruition, the understanding and ability to regulate the genetic activity of urease in S. pasteurii will be an essential breakthrough optimizing and fine-tuning the biostructures we create.
{{:Team:Brown-Stanford/Templates/Foot}}
{{:Team:Brown-Stanford/Templates/Foot}}

Revision as of 06:13, 27 September 2011

Brown-Stanford
iGEM

Transformation

Due Respect

Those familiar with the iGEM competition might remember a previous predecessor who also focused on the microbial carbonate precipitation - Bacilla Filla, Team Newcastle 2010’s project investigated the potential of Bacillus subtilis to remediate fractures in concrete architecture. However, due to the finicky nature of urease, they decided to focus on upregulating urease production inside the cell for their project. “Previous experiments involving up-regulating ureA, ureB and ureC in B. subtilis have not lead to an increase in urease production. This could be due to yet unidentified genes that are involved in the process. Therefore, we looked for another strategy.” – Newcastle 2010

Since the last Jamboree, the study of microbial precipitation has risen in prominence beyond just remediation. As we suggest in our project, it is instead being championed as a new way to aggregate building materials entirely. As the previous graph referenced, this expansion is related to the robust urease function present in the new organism of study: S. pasteurii.

Motivation

Though B. subitilis has more documentation of successful transformations, we decided to walk the path less traveled by. Characterization from recent research has shown numerous attractive features intrinsic to the S. pasteurii bacterium, including a. Robust function of its urease activity, even in 100% salinity [########] b. Its ability to utilize waste products and proliferate from cheap sources of nutrition (ie. Corn liquor, vegemite, etc). c. The success of mutagenic experiments to increase urease function: [########] Correspondingly, the motivation to investigate the transformation of S. pasteurii comes from several key benefits that would be accomplished if we were successful: i. The ability to confer antibiotic resistance to the cell culture, opening the door to i. More rigorous genetic controls cell culture fidelity** ii. Allowing continued selection in directed evolution experiments ii. The ability to introduce traits that will strengthen the bacterium profile for extraterrestrial space travel With a successful path to genetic recombination, we could add this organism to our list of powerful chassis’s, for use both terrestrial and cosmic use.


    • During the course of our cell culture of S. pasteurii, we frequently encountered contamination of our media-rich culture, despite our best attempts at sterilization techniques under the UV hood.

[picture of UV hood]

The risk involved in this project selection was the existence of good reasons for the lack of published articles on S. pasteurii transformation. But in the words of Microsoft’s blue monster, we decided that we were going to either go big or go home.

[big blue monster]


Experiment Design

Through all of the research we scoured, we found only one mention of the genetic transformation of S. pasteurii, from a symposium done by a then-undergraduate student in Arizona State University. In it she documented a seemingly successful transformation of S. pasteurii using protoplast transformation, giving us hope of its feasibility. Through fortuitous email correspondence, we were able establish contact with Alicia, who had had went on to become a practicing attorney. She kindly faxed us her old lab notebook sheets. We are still in awe of her organization skills.

Several methods of transformation were available to choose from:

  • Soil Transformation- relying on the natural competency of the bacteria to transfer genetic information
  • Protoplast Transformation- Digestion of the cell wall with lysozymes, followed by induction of DNA uptake by PEG 8000.
  • Electroporation- research suggests limited success with gram positive cells
  • Protoplast and Electroporation- a combination of two techniques, use of lysozymes to digest cell wall and electroporation to create entrances for DNA

Because of S. pasteurii’s thick cell wall, the protoplast transformation seemed the most logical to succeed.


Transformation

S. pasteurii cells were obtained via ATCC 11859 stock. The transforming DNA was obtained from Dr. Yun-Peng Chao, Professor of Chemical Engineering at Feng Chia University in Taiwan. Plasmid pUB110 (not to be confused with pBU11 _______), was a shuttle vector between E. coli and B. subtilis, containing an Ampicillin cassette for selection of the former, and a Kanamycin cassette for the latter.

[picture of pUB110]

pUB110 was selected following the protocols suggested by Alicia’s notes, reasoning along the lines that the homology between the two bacteria (S. pasteurii was previously known as Bacillus pasteurii before a recategorization) would be sufficient to allow expression of the Kanamycin resistance cassette inside S. pasteurii.

Newcastle iGEM’s 2009 Sporulation Part (BB-_______________) was also received via request to the Registry of Standardized Parts.

Protocols for the transformation can be found here. _______________

Lysozyme from chicken egg white (L3790 - 10 mg/mL) was obtained from Sigma-Aldrich.

Troubleshooting

Many attempts at protoplast transformation were carried out, but the results were unsuccessful: there was either no growth on the Kanamycin resistance plates, or there was excessive growth, with distinctly different colony features and tested negative on urea-phenol red test plates. This suggested that there was contamination due to 1) improperly prepared kanamycin plates or 2) kanamycin resistant strains colonizing the extremely rich recovery plates.

Troubleshooting suggested that the capacity of the protoplasts to regenerate their cell wall was a potential limiting factor. Viewing of the lysozyme digestion showed nearly complete digestion of the cell membranes (before and after). Procedures for collecting the protoplasts involved several centrifugation step at an unspecified “maximum speed” according to the protocol, though there was difficulty in collecting cell pellets at speeds below 8000 g, especially after the addition of viscous PEG 8000.

It is speculated that repeated centrifuge at high speeds was too traumatic for the digested protoplast cells, and the survival rate following the transformation was too low to produce colony forming units. Additional difficulties also arose in the recovery phase of the protoplast transformation, as the lack of antibiotic selection sometimes compromised portions of our rich recovery plates. (The recovery plates contained over ______ g of sucrose/mL).

[picture of contaminated cells]

Conclusions

In retrospect, we were a little too ambitious in choosing the scale of our projects. The elucidation of successful transformation protocols required more time than we had available, and at the time of this wikifreeze, we are still trying different concentrations of PEG, and lysozyme digestion on S. pasteurii.

Our plan to characterize the function of 2009 Newcastle’s sporulation part is also currently unrealized, due to the difficulties in transforming S. past. Nevertheless, we believe that though it has not been realized for the cut-off date of this jamboree, sophisticated investigation of S. pasteurii as a genetic chassis is still a worthwhile endeavor. Particularly as the industrial commercialization of biocementation reaches fruition, the understanding and ability to regulate the genetic activity of urease in S. pasteurii will be an essential breakthrough optimizing and fine-tuning the biostructures we create.

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