Difference between revisions of "Part:BBa K5097008:Experience"
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===Applications of BBa_K5097008===
 
===Applications of BBa_K5097008===
  
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The goal of pHish and CHIPS is to create a pH sensing and adjusting system for use in the remediation of wastewater produced during microprocessor fabrication.  We selected pARS as a pH sensitive genetic element that could be used to drive the expression of base producing genes to create a device that could sense acid and produce base.
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The goal of pHish and CHIPS is to create a pH sensing and adjusting system for use in the remediation of wastewater produced during microprocessor fabrication.  We selected pASR as a pH sensitive genetic element that could be used to drive the expression of base producing genes to create a device that could sense acid and produce base.
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Before creating such a device, we built this reporter circuit, to characterize the ability of pARS to regulate gene expression in response to acid.  We added pARS and a strong RBA (BBa_B0034) to RFP (BBa_E1010), and cloned this into pBS1C3. We then transformed E. coli DH5alpha cells with this construct.
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Before creating such a device, we built this reporter circuit, to characterize the ability of pASR to regulate gene expression in response to acid.  We added pASR and a strong RBA (BBa_B0034) to RFP (BBa_E1010), and cloned this into pBS1C3. We then transformed E. coli DH5alpha cells with this construct.
 
After confirming the sequence of the circuit by sequencing, we tested its functionality.  To do this, we prepared LB media buffered to a known pH with buffering agents.  MES (2-(N-morpholino)ethanesulfonic acid (pka 6.15)  was used to prepare pH 6.5 and pH 7.0, Stock HEPES (2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (pka 7.48)  buffer was used to prepare pH 7.5 and 8.0, and Sock Bicine (pka 8.26) buffer was used to prepare pH 8.5 and 9.0.  Media was supplemented with 1x Chloramphenicol before use.  
 
After confirming the sequence of the circuit by sequencing, we tested its functionality.  To do this, we prepared LB media buffered to a known pH with buffering agents.  MES (2-(N-morpholino)ethanesulfonic acid (pka 6.15)  was used to prepare pH 6.5 and pH 7.0, Stock HEPES (2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (pka 7.48)  buffer was used to prepare pH 7.5 and 8.0, and Sock Bicine (pka 8.26) buffer was used to prepare pH 8.5 and 9.0.  Media was supplemented with 1x Chloramphenicol before use.  
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Overnight cultures of pARS-RFP transfected cells were diluted to an OD600nm of 0.2 and added to the buffered media, and grown at 37°C with shaking to the indicated times. A 100 µL sample of the cultures, or a blank were loaded onto a 96 well plate. The OD600nm was measured, and then the fluorescence was measured on a microplate fluorescence spectrophotometer Ex 555nm, Em 596 nm.   
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Overnight cultures of pASR-RFP transfected cells were diluted to an OD600nm of 0.2 and added to the buffered media, and grown at 37°C with shaking to the indicated times. A 100 µL sample of the cultures, or a blank were loaded onto a 96 well plate. The OD600nm was measured, and then the fluorescence was measured on a microplate fluorescence spectrophotometer Ex 555nm, Em 596 nm.   
 
Data were processed by correcting the values to an LB blank, and then dividing the fluorescence value by the OD, and then determining the change from time 0.
 
Data were processed by correcting the values to an LB blank, and then dividing the fluorescence value by the OD, and then determining the change from time 0.
  
 
:::::::::https://static.igem.wiki/teams/5097/parts/team-oneonta-2024-pasr-rfpgrowthstudy00.jpg
 
:::::::::https://static.igem.wiki/teams/5097/parts/team-oneonta-2024-pasr-rfpgrowthstudy00.jpg
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::::::::::::Figure 1: Growth and expression of pARS-RFP expressing cells at different pHs.
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::::::::::::::Figure 1: Growth and expression of pASR-RFP expressing cells at different pHs.
  
  

Revision as of 17:56, 30 September 2024


Applications of BBa_K5097008

The goal of pHish and CHIPS is to create a pH sensing and adjusting system for use in the remediation of wastewater produced during microprocessor fabrication. We selected pASR as a pH sensitive genetic element that could be used to drive the expression of base producing genes to create a device that could sense acid and produce base. Before creating such a device, we built this reporter circuit, to characterize the ability of pASR to regulate gene expression in response to acid. We added pASR and a strong RBA (BBa_B0034) to RFP (BBa_E1010), and cloned this into pBS1C3. We then transformed E. coli DH5alpha cells with this construct. After confirming the sequence of the circuit by sequencing, we tested its functionality. To do this, we prepared LB media buffered to a known pH with buffering agents. MES (2-(N-morpholino)ethanesulfonic acid (pka 6.15) was used to prepare pH 6.5 and pH 7.0, Stock HEPES (2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (pka 7.48) buffer was used to prepare pH 7.5 and 8.0, and Sock Bicine (pka 8.26) buffer was used to prepare pH 8.5 and 9.0. Media was supplemented with 1x Chloramphenicol before use. Overnight cultures of pASR-RFP transfected cells were diluted to an OD600nm of 0.2 and added to the buffered media, and grown at 37°C with shaking to the indicated times. A 100 µL sample of the cultures, or a blank were loaded onto a 96 well plate. The OD600nm was measured, and then the fluorescence was measured on a microplate fluorescence spectrophotometer Ex 555nm, Em 596 nm. Data were processed by correcting the values to an LB blank, and then dividing the fluorescence value by the OD, and then determining the change from time 0.

team-oneonta-2024-pasr-rfpgrowthstudy00.jpg
Figure 1: Growth and expression of pASR-RFP expressing cells at different pHs.


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