Team:Colombia Uniandes/Parts

From 2013.igem.org

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==Glucocorticoid sensor==
==Glucocorticoid sensor==
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{| border="1" align="center"
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Here are the parts sent to the registry:
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|+'''Individual Parts Checklist*'''
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|-
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[[File:partsGC.jpg|700px|center|]]
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| style="text-align: center;" |'''Part'''
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| style="text-align: center;"|'''Nickname'''
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| style="text-align: center;" |'''Status''' 
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| style="text-align: center;" |tVP16
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| style="text-align: center;" |'''A'''
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| style="background:#D2D809; text-align: center;"|''Extracted''
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|-
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| style="text-align: center;" |GCR HBD
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| style="text-align: center;" |'''B'''
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| style="background:#D2D809; text-align: center;"|''Extracted''
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|-
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| style="text-align: center;" |pBAP2
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| style="text-align: center;" |'''C'''
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| style="background:#D2D809; text-align: center;"|''Extracted''
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|-
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| style="text-align: center;" |GAL4
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| style="text-align: center;" |'''D'''
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| style="background:#D2D809; text-align: center;"|''Extracted''
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|-
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| style="text-align: center;" |pGAL1 with 1 UAS box
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| style="text-align: center;" |'''E''' or '''E1'''
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| style="background:#D2D809; text-align: center;"|''Extracted''
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|-
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| style="text-align: center;" |pGAL1 with 3 UAS boxes
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| style="text-align: center;" |'''E2'''
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| style="background:#36A9E1; text-align: center;"|''Synthesised''
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|-
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| style="text-align: center;" |pGAL1 with 6 UAS boxes
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| style="text-align: center;" |'''E3'''
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| style="background:#36A9E1; text-align: center;"|''Synthesised''
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|-
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| style="text-align: center;" |pGAL1 with 9 UAS boxes
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| style="text-align: center;" |'''E4'''
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| style="background:#36A9E1; text-align: center;"|''Synthesised''
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|-
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| style="text-align: center;" |Yeast Terminator
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| style="text-align: center;" |'''F'''
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| style="background:#36A9E1; text-align: center;"|''Synthesised''
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|-
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| style="text-align: center;" |mCherry
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| style="text-align: center;" |'''G'''
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| style="background:#D2D809; text-align: center;"|''Extracted''
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|}
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{| border="1" align="center"
{| border="1" align="center"
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|+'''Fusion Checklist*'''
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|+'''Name table'''
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| style="text-align: center;" |pGAL1 with 1 UAS box + mCherry + Terminator
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| style="text-align: center;" |Part Name
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| style="background:#D2D809; text-align: center;"|''Fused''
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| style="text-align: center;" |Registry Number
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|-
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| style="text-align: center;" |E1
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| style="text-align: center;" |BBa_K1144001
|-  
|-  
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| style="text-align: center;" |pGAL1 with 3 UAS boxes + mCherry + Terminator
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| style="text-align: center;" |E2
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| style="background:#D2D809; text-align: center;"|''Fused''
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| style="text-align: center;" |BBa_K1144002
|-  
|-  
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| style="text-align: center;" |pGAL1 with 6 UAS boxes + mCherry + Terminator
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| style="text-align: center;" |E3
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| style="background:#D2D809; text-align: center;"|''Fused''
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| style="text-align: center;" |BBa_K1144003
|-  
|-  
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| style="text-align: center;" |pGAL1 with 9 UAS boxes + mCherry + Terminator
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| style="text-align: center;" |E4
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| style="background:#D2D809; text-align: center;"|''Fused''
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| style="text-align: center;" |BBa_K1144004
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|-
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| style="text-align: center;" |E1GF
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| style="text-align: center;" |BBa_K1144005
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|-
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| style="text-align: center;" |E2GF
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| style="text-align: center;" |BBa_K1144006
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|-
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| style="text-align: center;" |E3GF
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| style="text-align: center;" |BBa_K1144007
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|-
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| style="text-align: center;" |E4GF
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| style="text-align: center;" |BBa_K1144008
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|-
|}
|}
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You can see the confirmation gels of the parts below:
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 +
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[[File:Gel1GC.jpg|410px|thumb|left|''' PCR confirmation: E1GF-E1GF(2)-E2GF-E2GF(2)-WM-E3GF-E3GF(2)-E4GF-E4GF(2)''']]  [[File:Gel2GC.jpg|400px|thumb|center|'''Co-transformation: WM-E1GF2-E2GF-E3GF1-E3GF2-E4GF-WM- E3GF3-E4GF2-E2GF2
 +
''']]
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[[File:GEL3.jpg|400px|thumb|center|'''PCR confirmation of the parts cloned in the pSB1C3 backbone using the specifically designed primers 15 and 31 (see primers)''']]
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==='''Characterization of reporters'''===
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 +
To assess the strength of our promoters when induced with Dexamethasone, we performed a fluorometric assay using mCherry as our reporter.Since we don't have our transactivating protein ready yet, we co-transformed our parts into the ''E. coli'' DH10B strain with the  pAT7002 vector (Aoyama and Chua, 1997), which contains a well characterized Glucocorticoid Responsive Element that also uses the GAL4 DNA binding domain.
 +
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[[File:Response1hr.jpg|700px|thumb|center|''' Unsaturated curve where we see how the mCherry reporter appears after the addition of 10 uM dexamethasone, a glucocorticoid. Emmision intensity was measured at 607nm after excitation at 586nm''']]
 +
 +
 +
[[File:Response3hr.jpg|700px|thumb|center|''' Saturated curve for fluorescence. After three hours of the addition of 10 uM dexamethasone the reporter (mCherry) reaches its highest signal. We can see the different strengths depending on the number of UAS boxes. Emmision intensity was measured at 607nm after excitation at 586nm''']]
 +
 +
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We also decided to visually inspect our induced transformants. Here two of the images taken using a epifluorescent microscopy with a TRITC filter.
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[[File:E2GF.jpg|370px|thumb|left|''' Cells with the E2GF part (BBa_K1144006) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']]
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[[File:E4GF.jpg|370px|thumb|right|''' Cells with the E4GF part (BBa_K1144008) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC''']]
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 +
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====References====
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Aoyama, T. & Chua N. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. ''The Plant Journal, 11''(3): 605-612.
<html>
<html>
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==Nickel Removal System==
==Nickel Removal System==
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{| border="1" align="center"
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Here are the parts sent to the registry:
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|+'''Individual Parts Checklist*'''
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| style="text-align: center;" |PrcnR
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| style="background:#36A9E1; text-align: center;"|''In progress''
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|-
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| style="text-align: center;" |rcnR
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| style="background:#36A9E1; text-align: center;"|''In progress''
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|-
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| style="text-align: center;" |PrcnA
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| style="background:#D2D809; text-align: center;"|''Extracted''
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|-
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| style="text-align: center;" |hoxN
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| style="background:#D2D809; text-align: center;"|''Extracted''
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|-
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| style="text-align: center;" |mRFP
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| style="background:#D2D809; text-align: center;"|''Extracted''
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|-
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|}
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[[File:partsNi.jpg|700px|center|]]
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[[File:Prcna_hoxN.jpg|400px|thumb|center|Prcna-HoxN and HoxN-RFP]]
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{| border="1" align="center"
 
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|+'''Fusion Checklist*'''
 
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| style="text-align: center;" |RBS+hoxN+mRFP+STOP
 
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| style="background:#D2D809; text-align: center;"|''Fused''
 
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|-
 
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| style="text-align: center;" |prcnA+RBS+hoxN
 
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| style="background:#D2D809; text-align: center;"|''Fused''
 
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|-
 
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| style="text-align: center;" |prcnA+RBS+hoxN+mRFP+STOP
 
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| style="background:#D2D809; text-align: center;"|''Fused''
 
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|-
 
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| style="text-align: center;" |prcnR+rcnR+prcnA+RBS+hoxN+mRFP+STOP
 
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| style="background:#E30D18; text-align: center;" |''Stand by''
 
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|}
 
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</html>
</html>
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<nowiki>* Updated September 24</nowiki>
 

Latest revision as of 03:40, 28 September 2013

Parts

Glucocorticoid sensor

Contents

Glucocorticoid sensor

Here are the parts sent to the registry:

PartsGC.jpg


Name table
Part Name Registry Number
E1 BBa_K1144001
E2 BBa_K1144002
E3 BBa_K1144003
E4 BBa_K1144004
E1GF BBa_K1144005
E2GF BBa_K1144006
E3GF BBa_K1144007
E4GF BBa_K1144008

You can see the confirmation gels of the parts below:


PCR confirmation: E1GF-E1GF(2)-E2GF-E2GF(2)-WM-E3GF-E3GF(2)-E4GF-E4GF(2)
Co-transformation: WM-E1GF2-E2GF-E3GF1-E3GF2-E4GF-WM- E3GF3-E4GF2-E2GF2
PCR confirmation of the parts cloned in the pSB1C3 backbone using the specifically designed primers 15 and 31 (see primers)


Characterization of reporters

To assess the strength of our promoters when induced with Dexamethasone, we performed a fluorometric assay using mCherry as our reporter.Since we don't have our transactivating protein ready yet, we co-transformed our parts into the E. coli DH10B strain with the pAT7002 vector (Aoyama and Chua, 1997), which contains a well characterized Glucocorticoid Responsive Element that also uses the GAL4 DNA binding domain.

Unsaturated curve where we see how the mCherry reporter appears after the addition of 10 uM dexamethasone, a glucocorticoid. Emmision intensity was measured at 607nm after excitation at 586nm


Saturated curve for fluorescence. After three hours of the addition of 10 uM dexamethasone the reporter (mCherry) reaches its highest signal. We can see the different strengths depending on the number of UAS boxes. Emmision intensity was measured at 607nm after excitation at 586nm


We also decided to visually inspect our induced transformants. Here two of the images taken using a epifluorescent microscopy with a TRITC filter.


Cells with the E2GF part (BBa_K1144006) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC
Cells with the E4GF part (BBa_K1144008) showing their fluorescent reporter, mCherry, after induction with 10 uM dexamethasone! The filter used was TRITC


References

Aoyama, T. & Chua N. (1997). A glucocorticoid-mediated transcriptional induction system in transgenic plants. The Plant Journal, 11(3): 605-612.

Nickel Removal System

Nickel Removal System

Here are the parts sent to the registry:

PartsNi.jpg
Prcna-HoxN and HoxN-RFP


Retrieved from "http://2013.igem.org/Team:Colombia_Uniandes/Parts"