Difference between revisions of "Part:BBa K124002:Experience"

Revision as of 11:56, 3 October 2012

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_K124002

User Reviews

[http://2012.igem.org/Team:Berkeley/Project/Localization#Promoter%20CharacterizationUCBerkeley 2012 Team:] To assay their potential use in our project, we characterized the strength of several registry yeast promoters. We wanted to compare their consistency of expression and relative fluorescence, then quantify them against a standard promoter fluorescence. The promoters we used in our assay: pSTE5 (weak), pCYC1 (medium),pADH1 (strong), and pTDH3 (strongest).

Experimental Design: We designed our promoters to express a fluorescent protein so that we could quickly measure bulk fluorescence via TECAN. To test if downstream sequence affected expression, we cloned the promoter in front of two different fluorescent proteins, yellow fluorescent Venus and red fluorescent mKate in different strains. If expression was sequence-independent, we expected similar fluorescence intensity from both channels. The device was cloned on a backbone with Leu2 marker and Cen6 origin of replication and transformed into S228C S. cerevisiae. Ideally, we wanted the calibration curve to span several orders of magnitude and exhibit a linear relationship, which was true for all the promoters we characterized except for pCYC1.

Calibration curve of fluorescence intensity. Error bars are +/- one standard deviation.

Promoter	RFP/OD	YFP/OD
background	55±13	36±2
pSTE5	126±14	216±16
pCYC1	189±27	928±31
pADH1	1170±89	4360±100
pTDH3	22164±671	62531±1714

UNIQf4b3375a620f3828-partinfo-00000000-QINU UNIQf4b3375a620f3828-partinfo-00000001-QINU

@@ Line 1: / Line 1: @@
 __NOTOC__
 This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
@@ Line 7: / Line 6: @@
 ===User Reviews===
+[http://2012.igem.org/Team:Berkeley/Project/Localization#Promoter%20Characterization'''UCBerkeley 2012 Team:'''] To assay their potential use in our project, we characterized the strength of several registry yeast promoters. We wanted to compare their consistency of expression and relative fluorescence, then quantify them against a standard promoter fluorescence. The promoters we used in our assay: pSTE5 (weak), pCYC1 (medium),pADH1 (strong), and  pTDH3 (strongest). <br>
+'''Experimental Design:''' We designed our promoters to express a fluorescent protein so that we could quickly measure bulk fluorescence via TECAN. To test if downstream sequence affected expression, we cloned the promoter in front of two different fluorescent proteins, yellow fluorescent Venus and red fluorescent mKate in different strains. If expression was sequence-independent, we expected similar fluorescence intensity from both channels. The device was cloned on a backbone with Leu2 marker and Cen6 origin of replication and transformed into S228C <i>S. cerevisiae</i>. Ideally, we wanted the calibration curve to span several orders of magnitude and exhibit a linear relationship, which was true for all the promoters we characterized except for pCYC1.
+[[Image:UCBerkeley12_promoter_characterization.png |thumb|none|610px|Calibration curve of fluorescence intensity. Error bars are +/- one standard deviation.]]
+{| {{table}}
+| align="center" style="background:#f0f0f0;"|'''Promoter'''
+| align="center" style="background:#f0f0f0;"|'''RFP/OD'''
+| align="center" style="background:#f0f0f0;"|'''YFP/OD'''
+|-
+| background||55±13||36±2
+|-
+| pSTE5||126±14||216±16
+|-
+| pCYC1||189±27||928±31
+|-
+| pADH1||1170±89||4360±100
+|-
+| pTDH3||22164±671||62531±1714
+|}
 <!-- DON'T DELETE --><partinfo>BBa_K124002 StartReviews</partinfo>
 <!-- Template for a user review