Difference between revisions of "Part:BBa K3498030:Experience"

 
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{|width='80%' style='border:1px solid gray'
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<partinfo>BBa_K3498030 AddReview 5</partinfo>
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<I>IGEM FCB-UANL</I>
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PvanCC promotor + Rsn-3 RBS + Rsn-3 + Rsn-4 RBS + Rsn-4 + Rsn-5 RBS + Rsn-5 + Terminator.
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This composite part is regulated by vanillic acid sensitive promoter PvanCC. This part contains an histidine tag used for purification. All assembled in pSB3K3 plasmid, a low number of copies plasmid
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Introduction
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Ranaspumin-3-5.
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Ranaspumin-3-5, also known as Rsn-3-5 is recombinantly produced protein in E. coli Top 10. Its foam stability capacity is being tested for fire fighting foam production purposes. It is believed that the stability effect improves with the three ranaspumin working together
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Results.
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Protein expression in E. coli Top 10.
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A characterization of the expression in E. coli was made. For this, different concentrations of inducer (vanillic acid) and different final growing times were tested. Concentrations were determined by imageJ pixel density analyzer program
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Firstly 100 and 1000 mM concentrations were tested, but with higher concentrations the expressed protein was lower, so lower concentrations were tested out
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75 mM seemed to be the best candidate for the expression of ranaspumin 3-5, so it was tested out for 24 hours
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[[File:T-FCB-UANL--RSN3-5-I.png|200px|thumb|left|Figure 1: Induction conditions for Rsn-3-5]]
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The results indicate that the best induction condition for Rsn-3-5 are 75 mM and 12 hours of growing at 37°C with a production of 0.2014 mg/mL
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Foamability
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The foamability of the expressed Rsn-3-5 was determined by testing the sonicated cultures, manually agitating a 15 mL corning tube containing 5 mL of the sonicated protein and observing the volume of the foam produced
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Measurements were taken at 0, 1, 3, 5, 10, 15 and 30 minutes, after that, the measurements were taken every 30 minutes until 12 hours after the agitation
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[[File:T--FCB-UANL--PE.png|200px|thumb|left|Figure 2: Water (negative control), Rsn-2, Rsn-3, Rsn 3-5, SDS (positive control), all agitated 30 second]]
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The foamability is obtained by FV/SV where FV is foam volume and SV is solution volume. Ranaspumin-3 made a Foam volume of 4.5 mL with a 5 mL solution, hence its foamability is of 0.9 (relative number)
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[[File:T--FCB-UANL--E-GC.png|200px|thumb|left|Figure 3: Foamability of Rsn-3-5]]
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Passing the 12 hours the behaviour of the foam was analyzed in order to see how durable it is
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[[File:T--FCB-UANL-E-GB.png|200px|thumb|left|Figure 4: Foam volume over 12 hours]]
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It took 9 hours for the foam to go from 4.5 mL to  1.5 mL (lower than the half life), and at the final measurement it still had 1 mL of foam
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Latest revision as of 21:28, 20 October 2021


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K3498030

User Reviews

UNIQ7094855dc4031ef0-partinfo-00000000-QINU UNIQ7094855dc4031ef0-partinfo-00000001-QINU

•••••

IGEM FCB-UANL

PvanCC promotor + Rsn-3 RBS + Rsn-3 + Rsn-4 RBS + Rsn-4 + Rsn-5 RBS + Rsn-5 + Terminator.

This composite part is regulated by vanillic acid sensitive promoter PvanCC. This part contains an histidine tag used for purification. All assembled in pSB3K3 plasmid, a low number of copies plasmid

Introduction

Ranaspumin-3-5.

Ranaspumin-3-5, also known as Rsn-3-5 is recombinantly produced protein in E. coli Top 10. Its foam stability capacity is being tested for fire fighting foam production purposes. It is believed that the stability effect improves with the three ranaspumin working together

Results.

Protein expression in E. coli Top 10.

A characterization of the expression in E. coli was made. For this, different concentrations of inducer (vanillic acid) and different final growing times were tested. Concentrations were determined by imageJ pixel density analyzer program

Firstly 100 and 1000 mM concentrations were tested, but with higher concentrations the expressed protein was lower, so lower concentrations were tested out

75 mM seemed to be the best candidate for the expression of ranaspumin 3-5, so it was tested out for 24 hours

Figure 1: Induction conditions for Rsn-3-5











The results indicate that the best induction condition for Rsn-3-5 are 75 mM and 12 hours of growing at 37°C with a production of 0.2014 mg/mL

Foamability

The foamability of the expressed Rsn-3-5 was determined by testing the sonicated cultures, manually agitating a 15 mL corning tube containing 5 mL of the sonicated protein and observing the volume of the foam produced Measurements were taken at 0, 1, 3, 5, 10, 15 and 30 minutes, after that, the measurements were taken every 30 minutes until 12 hours after the agitation


Figure 2: Water (negative control), Rsn-2, Rsn-3, Rsn 3-5, SDS (positive control), all agitated 30 second















The foamability is obtained by FV/SV where FV is foam volume and SV is solution volume. Ranaspumin-3 made a Foam volume of 4.5 mL with a 5 mL solution, hence its foamability is of 0.9 (relative number)

Figure 3: Foamability of Rsn-3-5










Passing the 12 hours the behaviour of the foam was analyzed in order to see how durable it is

Figure 4: Foam volume over 12 hours










It took 9 hours for the foam to go from 4.5 mL to 1.5 mL (lower than the half life), and at the final measurement it still had 1 mL of foam