Difference between revisions of "Part:BBa K1231000:Experience"

Latest revision as of 18:50, 30 September 2024

Application NAWI_Graz 2017

Twin part BBa_K2348001 was created because information in this part was missing. Part itself works and asr promoter is activated at pH 5.0 and 4.5.

User Reviews

Oneonta iGEM 2024: phish and CHIPs

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.

Figure 1: Growth and expression of pASR-RFP expressing cells at different pHs.

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iGEM Dundee 2016

The function of this part was characterised in the hope of designing a pH-responsive system. We hooked up this asr promomoter to an excellent reporter system (BBa_E0840) and characterised its activity under this new composite biobrick (BBa_K1962014). The system showed some pH-dependent activity.

We also hooked up the asr promoter to biobricks encoding immunity proteins towards colicins. This should give some degree of regulation of transcription of the Im-Ia immunity protein (standard biobrick BBa_K1962001 and composite part BBa_K1962015) and the Im-E3 immunity protein (standard biobrick BBa_K1962004 and composite part BBa_K1962016).

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 __NOTOC__
-===Applications of BBa_K1231000===
 == Application NAWI_Graz 2017 ==
 Twin part [https://parts.igem.org/Part:BBa_K2348001 BBa_K2348001] was created because information in this part was missing. Part itself works and asr promoter is activated at pH 5.0 and 4.5.
 ===User Reviews===
+===Oneonta iGEM 2024: phish and CHIPs===
+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.
+:::::::::https://static.igem.wiki/teams/5097/parts/team-oneonta-2024-pasr-rfpgrowthstudy00.jpg
+::::::::::::::Figure 1: Growth and expression of pASR-RFP expressing cells at different pHs.
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