Difference between revisions of "Part:BBa K2213007"

Latest revision as of 00:49, 2 November 2017

MediumPromoter_PduD(1-20)_mCherry

Figure1. Circuit diagram of BBa_K2213007.

The PduD(1:20) tag is one of many tags that has been proved to localise inside a Pdu microcompartment. This was proved by the iGEM Dundee 2011 team http://2011.igem.org/Team:Dundee. To develop on this, Manchester2017 Characterised the localisation of this tag within a EUT microcompartment consisting of BBa_K2213000, BBa_K2213001 and BBa_K2213002. (https://parts.igem.org/Part:BBa_K2213000 https://parts.igem.org/Part:BBa_K2213001 https://parts.igem.org/Part:BBa_K2213002)

Improvements

This part is an improved version of https://parts.igem.org/Part:BBa_K562001, originally submitted by team Dundee 2011. The original part contained an illegal XbaI site which has been removed to make it biobrick compatible. This part has been expressed under different strength promoters. A medium strength Anderson promoter here, low strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213006), and a high strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213008).

Characterisation

The PduD tag was combined with the medium strength Anderson promoter (https://parts.igem.org/Part:BBa_J23108) and mCherry.

Figure 2. Fluorescence microscopy images of Low, Medium and High strength Anderson promoter-PduD construct associated mCherry (OD600: 0.2) expressed in the absence of Eut.

A gradient of fluorescence is evident when compared to low and high promoters. When expressed the distribution of the mCherry signal was homogeneous and fluorescence level was slightly less than the high promoter. .

Figure 3. Optical Density (600nm) for Low, Medium and High strength Anderson promoter constructs with RFU values after 30 hours.

The expression levels compared to low and high are as shown. As the medium strength promoter had similar expression levels to the high promoter, it was not expressed alongside any Eut subunits. This result was unexpected and should be taken into consideration when choosing either the medium or high promoter.

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]

@@ Line 7: / Line 7: @@
 <b>Figure1.</b> Circuit diagram of BBa_K2213007.
 <br>
+The PduD(1:20) tag is one of many tags that has been proved to localise inside a Pdu microcompartment. This was proved by the iGEM Dundee 2011 team http://2011.igem.org/Team:Dundee. To develop on this, Manchester2017 Characterised the localisation of this tag within a EUT microcompartment consisting of BBa_K2213000, BBa_K2213001 and BBa_K2213002. (https://parts.igem.org/Part:BBa_K2213000 https://parts.igem.org/Part:BBa_K2213001 https://parts.igem.org/Part:BBa_K2213002)
 ===Improvements===
-<br>
-This part is an improved version of https://parts.igem.org/Part:BBa_K562001, originally submitted by team Dundee 2011. The original part contained an illegal XbaI site which has been removed to make it biobrick compatible.<br>
 <br>
 This part is an improved version of https://parts.igem.org/Part:BBa_K562001, originally submitted by team Dundee 2011. The original part contained an illegal XbaI site which has been removed to make it biobrick compatible. This part has been expressed under different strength promoters. A medium strength Anderson promoter here, low strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213006), and a high strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213008).<br>
+<br>
+===Characterisation===
 <br>
 The PduD tag was combined with the medium strength Anderson promoter (https://parts.igem.org/Part:BBa_J23108) and mCherry.<br>
 <br>
 https://static.igem.org/mediawiki/2017/e/e4/PromoterComparison800p.png<br>
+<br>
+<b>Figure 2.</b> Fluorescence microscopy images of Low, Medium and High strength Anderson promoter-PduD construct associated mCherry (OD600: 0.2) expressed in the absence of Eut.<br>
 <br>
 A gradient of fluorescence is evident when compared to low and high promoters. When expressed the distribution of the mCherry signal was homogeneous and fluorescence level was slightly less than the high promoter.
@@ Line 25: / Line 29: @@
 <br>
 https://static.igem.org/mediawiki/2017/3/31/TagExpression500p.jpg
+<br>
+<b>Figure 3.</b> Optical Density (600nm) for Low, Medium and High strength Anderson promoter constructs with RFU values after 30 hours.<br>
 <br>
 The expression levels compared to low and high are as shown. As the medium strength promoter had similar expression levels to the high promoter, it was not expressed alongside any Eut subunits. This result was unexpected and should be taken into consideration when choosing either the medium or high promoter.<br>