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

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Applications of BBa_K3498030

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UNIQf4c2cdea7c0bdf10-partinfo-00000000-QINU UNIQf4c2cdea7c0bdf10-partinfo-00000001-QINU

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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)

[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

@@ Line 46: / Line 46: @@
 mM seemed to be the best candidate for the expression of ranaspumin 3-5, so it was tested out for 24 hours
+[[File:T-FCB-UANL--RSN3-5-I.png|200px|thumb|left|Figure 1: Induction conditions for Rsn-3-5]]
-https://parts.igem.org/File:T-FCB-UANL--RSN3-5-I.png
-Figure 1: Induction conditions for Rsn-3-5
@@ Line 60: / Line 58: @@
-https://parts.igem.org/File:T--FCB-UANL--PE.png
+[[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]]
-Figure 2: Water (negative control), Rsn-2, Rsn-3, Rsn 3-5, SDS (positive control), all agitated 30 second
@@ Line 68: / Line 64: @@
-https://parts.igem.org/File:T--FCB-UANL--E-GC.png
-Figure 3: Foamability of Rsn-3-5
+[[https://parts.igem.org/File:T--FCB-UANL--E-GC.png|200px|thumb|left|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
-https://parts.igem.org/File:T--FCB-UANL-E-GB.png
+[[File:T--FCB-UANL-E-GB.png|200px|thumb|left|Figure 4: Foam volume over 12 hours]]
-Figure 4: Foam volume over 12 hours