Difference between revisions of "Team:Stanford-Brown/SB16 BioMembrane p-Aramid"
| Line 203: | Line 203: | ||
Since pABA is an intermediate in the folic acid pathway, pABA will be consumed in the pathway’s downstream chemical reactions. Dihydropteroate (DHP) synthase catalyzes the reaction that converts pABA to 7,8-dihydropteroate, a precursor to 7,8-dihydrofolate [3]. By knocking out the gene that expresses dihydropteroate synthase, folate production is halted, thus preventing the conversion of pABA to subsequent folate precursors. To carry this out, we planned to use CRISPR-Cas9 for excision of the gene and to transform into E. coli. To verify that the cells were successfully transfected, cells were grown in medium with selection markers, such as chloramphenicol and folate. Afterwards, the growing cells would be replated on medium containing only the selection marker. Colonies that did not survive on the replated medium are successful, because they are folate-dependent since their folate pathway was disrupted by the DHP synthase knockout. | Since pABA is an intermediate in the folic acid pathway, pABA will be consumed in the pathway’s downstream chemical reactions. Dihydropteroate (DHP) synthase catalyzes the reaction that converts pABA to 7,8-dihydropteroate, a precursor to 7,8-dihydrofolate [3]. By knocking out the gene that expresses dihydropteroate synthase, folate production is halted, thus preventing the conversion of pABA to subsequent folate precursors. To carry this out, we planned to use CRISPR-Cas9 for excision of the gene and to transform into E. coli. To verify that the cells were successfully transfected, cells were grown in medium with selection markers, such as chloramphenicol and folate. Afterwards, the growing cells would be replated on medium containing only the selection marker. Colonies that did not survive on the replated medium are successful, because they are folate-dependent since their folate pathway was disrupted by the DHP synthase knockout. | ||
| + | <br><br> | ||
| + | References:<br> | ||
| + | http://www.dupont.co.uk/content/dam/dupont/products-and-services/fabrics-fibers-and-nonwovens/fibers/documents<br>/Kevlar%C2%AE%20Technical%20Guide.pdf<br> | ||
| + | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC160886/pdf/070907.pdf<br> | ||
| + | http://biology.kenyon.edu/BMB/jsmol2012/Lyra_Rina/index.htm<br> | ||
</div> <!--END col-sm-12--> | </div> <!--END col-sm-12--> | ||
Revision as of 23:31, 15 October 2016
p-Aramid team member Anna introduces the p-aramid project
Why aramids?
For our exploration purposes, it is important to have a membrane that can withstand harsh environments without bursting. While our collagen-elastin fiber and latex polymer display promising properties of elasticity and strength, their ability to withstand high physical stress is limited. Due to its rigidity (70,500 MPa [1]), toughness (2,920 MPa [1]), and low density (1.44 g/cm³ [1]), Kevlar® is highly capable of meeting the unique needs of inflatable modules and habitats. It can act as both a lightweight balloon reinforcement and shield against atmospheric debris. In addition, it is also highly resistant to chemical, thermal, and physical wear--making it ideal for durability in a harsh environment.
Today, a Kevlar® composite is used as a protective outer covering for the Bigelow Space Module on the International Space Station for exactly these reasons. We wanted to make an aramid fiber similar in structure to Kevlar so that it can exhibit those same desirable properties that Kevlar possesses.
Current industrial production of Kevlar® is based on materials which are derived from petrochemicals. The long term use of fossil fuels is hazardous to the environment and will become unsustainable as our fuel sources are exhausted. By replacing the monomers 1,4-phenylene-diamine and terephthaloyl chloride with a single biologically produced monomer, para-aminobenzoic acid (pABA), our team can manufacture poly-pABA, a cost-efficient and ecologically sound p-aramid Kevlar® analogue.
Our primary goal is to optimize the production of pABA by identifying and isolating genes relevant for pABA synthesis and transforming them in Escherichia coli (E. coli).
Experimental Design
Our p-aramid production is focused on the manipulation of E. coli’s native Shikimate metabolic pathway to maximize the synthesis of our monomer p-aminobenzoic acid. In this pathway, pABA is an intermediate for folate synthesis [2].
First, we wanted to enhance the activity of the first enzyme of the Shikimate pathway. This enzyme is 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase. By introducing more copies of the gene and overexpressing this enzyme in cells, an increased amount of carbon will be funneled into the pathway. The pathway then proceeds and produces (after several intermediate products) chorismate, which is partially funneled to produce pABA. Blitzen blue plasmid was used to transform DAHP synthase into E. coli.
First, we wanted to enhance the activity of the first enzyme of the Shikimate pathway. This enzyme is 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase. By introducing more copies of the gene and overexpressing this enzyme in cells, an increased amount of carbon will be funneled into the pathway. The pathway then proceeds and produces (after several intermediate products) chorismate, which is partially funneled to produce pABA. Blitzen blue plasmid was used to transform DAHP synthase into E. coli.
Second, our introduction of genes PabA, PabB and PabC facilitates the production of enzymes that are responsible for the conversion of chorismate to pABA. Chorismate is transformed into para-aminobenzoic acid through chemical reactions involving the enzymes 4-amino-4-deoxychorismate (ADC) synthase and 4-amino-4-deoxychorismate (ADC) lyase, with the former encoded by PabA, and the latter encoded by PabB and PabC. Our approach was to introduce extra copies of genes PabA, PabB, and PabC into our cells. PabA and PabB together produce ADC synthase, which is responsible for the conversion of chorismate to 4-amino-4-deoxychorismate. Expression of PabC results in ADC lyase, the enzyme that would convert 4-amino-4deoxychorismate to pyruvate and our desired product, pABA.
Since pABA is an intermediate in the folic acid pathway, pABA will be consumed in the pathway’s downstream chemical reactions. Dihydropteroate (DHP) synthase catalyzes the reaction that converts pABA to 7,8-dihydropteroate, a precursor to 7,8-dihydrofolate [3]. By knocking out the gene that expresses dihydropteroate synthase, folate production is halted, thus preventing the conversion of pABA to subsequent folate precursors. To carry this out, we planned to use CRISPR-Cas9 for excision of the gene and to transform into E. coli. To verify that the cells were successfully transfected, cells were grown in medium with selection markers, such as chloramphenicol and folate. Afterwards, the growing cells would be replated on medium containing only the selection marker. Colonies that did not survive on the replated medium are successful, because they are folate-dependent since their folate pathway was disrupted by the DHP synthase knockout.
References:
http://www.dupont.co.uk/content/dam/dupont/products-and-services/fabrics-fibers-and-nonwovens/fibers/documents
/Kevlar%C2%AE%20Technical%20Guide.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC160886/pdf/070907.pdf
http://biology.kenyon.edu/BMB/jsmol2012/Lyra_Rina/index.htm
Since pABA is an intermediate in the folic acid pathway, pABA will be consumed in the pathway’s downstream chemical reactions. Dihydropteroate (DHP) synthase catalyzes the reaction that converts pABA to 7,8-dihydropteroate, a precursor to 7,8-dihydrofolate [3]. By knocking out the gene that expresses dihydropteroate synthase, folate production is halted, thus preventing the conversion of pABA to subsequent folate precursors. To carry this out, we planned to use CRISPR-Cas9 for excision of the gene and to transform into E. coli. To verify that the cells were successfully transfected, cells were grown in medium with selection markers, such as chloramphenicol and folate. Afterwards, the growing cells would be replated on medium containing only the selection marker. Colonies that did not survive on the replated medium are successful, because they are folate-dependent since their folate pathway was disrupted by the DHP synthase knockout.
References:
http://www.dupont.co.uk/content/dam/dupont/products-and-services/fabrics-fibers-and-nonwovens/fibers/documents
/Kevlar%C2%AE%20Technical%20Guide.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC160886/pdf/070907.pdf
http://biology.kenyon.edu/BMB/jsmol2012/Lyra_Rina/index.htm
