Difference between revisions of "Part:BBa K2282006"

Revision as of 11:58, 1 November 2017

AmilCP with DSbox (BBa_K2282001) supplemented with BBa_J23100, BBa_B0034 and a double terminator.

Usage and Biology

The DSBox (Downstream Box) increases the translation of the mRNA at low temperature, probably by binding to the 30s subunit via ribosomal protein S1 (Studer et al 2006, Duval et al, 2013, Qu et al 2012). The DSBox is unusual among 5' regulatory sequence motifs in that it appears after the start codon and is translated into the protein product at the N-terminus. The double terminator provides robust termination. AmilCP is a coral chromoprotein which displays a strong blue color. With this part we can evaluate the impact of the DSBox on the coding sequence of the protein and see if amilCP expression is modified as well as its properties (colors, time of folding).

Source of this part

This part is the same as BBa_K2282005 with the slight difference that we prepended the DSBox sequence (Mitta et al, 1997) from the E.coli genome to the amilCP coding sequence. The DSBox size as defined in Mitta et al, 1997 is 14bp. In order to avoid introducing a frame shift error we added an adenine nucleotide at the 3’ end to increase the size from 14bp to 15bp by completing the 3’ codon. The sequences of the DSBox and other cold-induced translational regulatory elements are very similar to the parts of the iGEM10_Mexico-UNAM-CINVESTAV that can be found here : http://partsregistry.org/Part:BBa_K328003

Design consideration

Adding 15 nucleotides to a protein coding sequence raised some concern about its effect on protein stability and activity. We addressed this problem with molecular modelling to optimize the placement of the DS Box (see our wiki) and confirmed the results in the lab.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Characterization IONIS_PARIS 2017

Group & Author: IONIS_PARIS /La Paillasse/2017

Objective: Verify that the DS box inclusion inside the amilCP sequence does not impact the protein expression.

After having constructed our recombinant plasmid composed of the part BBa_K2282006 ligated into the pSB1C3 backbone, we compared the visual aspect and the expression rate of amilCP in comparison with the same plasmid without the DS box.

You can find the complete detailed characterization protocols on our dedicated section on our wiki page (IONIS_PARIS 2017)

Results :

DH5-α E.Coli bacteria were transformed with plasmids containing either part BBa_K2282005 containing native amilCP or part BBa_K2282005 containing amilCP added with the DS box.

Impact of the DS box on amilCP visual aspect

Agar plates Liquid cultures Culots

Impact of the DS box on amilCP absorbance properties

After an overnight bacterial culture at 37°C, the protein content was extracted and the absorbance spectrum of the obtained solution was measured. We corrected the values obtained with a blank consisting of wild-type culture extracted proteins. The results are presented in the following graphs:

The absorption spectrum of the native AmilCP from part BBa_K2282005:

The absorption spectrum of the DS box-added AmilCP from part BBa_K2282006:

Interpretation:

The native amilCP as well as the amilCP added with the DS box both give the same absorbance peak at 588nm. This is consistent with the existing data (https://parts.igem.org/Part:BBa_K592009), and shows that the DS box does not alter the chromoprotein absorption profile, and therefore its color properties. This is in accordance with the DS box location outside the chromophore domain. We therefore made the hypothesis that the slight color difference observed between pellets might be due to a lower protein expression. This was verified through a kinetics measure of OD588nm, representative of the amilCP expression, at different temperatures.

Impact of the DS box on amilCP expression rate

Ionis-paris-2017-seq_2_%C3%A0_37%C2%BAC.png

Interpretation :

Both bacterial batches express functional amilCP as the signal at OD588nm shows. This confirm that the presence of the DS box does not impact the protein properties. The same protein expression pattern is observable at both temperatures, with an overall lower expression at 18°C. This predictable result confirms the absence of enhancing properties on cold protein expression from the DS box alone.

This proves that this lower expression rate possibly induced by the presence of the DS box is not problematic when combined with the CspA promoter and 5’UTR sequence, allowing its activation.

Finally, we attempted to sequence this part but did not obtain any satisfying result.

@@ Line 29: / Line 29: @@
 Objective: Verify that the DS box inclusion inside the amilCP sequence does not impact the protein expression.
-We modelled the amilCP protein structure in 3D in order to assess where in its sequence the DS box could be inserted without compromising the protein conformation and functions. This modeling work led to the assumption that the amilCP structure would not be changed and that the chromophore would still be expressed despite the presence of the DS box. We wanted to confirm this hypothesis by performing laboratory experiments.
-Method :
 After having constructed our recombinant plasmid composed of the part BBa_K2282006 ligated into the pSB1C3 backbone, we compared the visual aspect and the expression rate of amilCP in comparison with the same plasmid without the DS box.
@@ Line 60: / Line 58: @@
 https://static.igem.org/mediawiki/parts/thumb/8/8f/IMG_20171010_192933.jpg/800px-IMG_20171010_192933.jpg
-Impact of the DS box on amilCP expression rate
+Interpretation:
-Courbes Nicolas --> C'est pas sur la seq 7 ..? ou on met les deux ?
+The native amilCP as well as the amilCP added with the DS box both give the same absorbance peak at 588nm. This is consistent with the existing data (https://parts.igem.org/Part:BBa_K592009), and shows that the DS box does not alter the chromoprotein absorption profile, and therefore its color properties. This is in accordance with the DS box location outside the chromophore domain. We therefore made the hypothesis that the slight color difference observed between pellets might be due to a lower protein expression. This was verified through a kinetics measure of OD588nm, representative of the amilCP expression, at different temperatures.
+Impact of the DS box on amilCP expression rate
+https://static.igem.org/mediawiki/2017/6/66/Ionis-paris-2017-seq_2_%C3%A0_37%C2%BAC.png
+ https://static.igem.org/mediawiki/2017/7/7a/Ionisigem-laboratory-seq2-2.jpeg
 Interpretation :
-Through experiment, we characterized the sequence 2 and observed there were no difference on the AmilCP and its choromophore expression at 588nm. We could then conclude there is no influence of the DSBox on the protein properties.
-However, the pelets of the seq 2 were more pale and the pelets of the seq 1 after the cold characterization. It can mean that the DSBox can have an influence on the mRNA folding of AmilCP.
+Both bacterial batches express functional amilCP as the signal at OD588nm shows. This confirm that the presence of the DS box does not impact the protein properties.
+The same protein expression pattern is observable at both temperatures, with an overall lower expression at 18°C. This predictable result confirms the absence of enhancing properties on cold protein expression from the DS box alone.
-Finally, we can say that the DSBox does not influence the AmilCP properties but influences its expression.
-This result was the predicted one thanks to the modelling, so this confirms the results.
+This proves that this lower expression rate possibly induced by the presence of the DS box is not problematic when combined with the CspA promoter and 5’UTR sequence, allowing its activation.
-Also, we attempted to sequence this part, but we did not obtain any satisfying result.
+Finally, we attempted to sequence this part but did not obtain any satisfying result.