Difference between revisions of "Part:BBa J23102"
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<p>We wanted to add new, normalised RFU data for two promoters from the Anderson family of constitutive promoters BBa_J23102 (Strong constitutive promoter) and BBa_J23106 (weak constitutive promoter) respectively; and a T7 promoter BBa_K199118 all expressing mRFP1. Another interest that we had was to test how OD was affected by RFP production, therefore we performed our measurements twice, at OD600 and OD660 respectively. This was done because it has been shown that OD660 gives a more accurate representation of bacterial growth in RFP-producing bacteria (Hecht et al., 2016). </p>
 
<p>We wanted to add new, normalised RFU data for two promoters from the Anderson family of constitutive promoters BBa_J23102 (Strong constitutive promoter) and BBa_J23106 (weak constitutive promoter) respectively; and a T7 promoter BBa_K199118 all expressing mRFP1. Another interest that we had was to test how OD was affected by RFP production, therefore we performed our measurements twice, at OD600 and OD660 respectively. This was done because it has been shown that OD660 gives a more accurate representation of bacterial growth in RFP-producing bacteria (Hecht et al., 2016). </p>
 
<p>In order to obtain our results, we grew our cell cultures up to an OD600/660 of ~0.6. After the desired OD had been reached, cultures were induced with IPTG (for BBa_K199118) and anhydrotetracycline (for BBa_K092300). Then they were placed on a microplate reader (CLARIOstar®, BMG Labtech). The specific script conditions can be seen below. The machine was set to measure  OD600/660 and RFU overnight every 15 minutes. The data was then analysed and plotted. This experiment was performed both in transformed E. coli DH5a as well as BL21.</p>
 
<p>In order to obtain our results, we grew our cell cultures up to an OD600/660 of ~0.6. After the desired OD had been reached, cultures were induced with IPTG (for BBa_K199118) and anhydrotetracycline (for BBa_K092300). Then they were placed on a microplate reader (CLARIOstar®, BMG Labtech). The specific script conditions can be seen below. The machine was set to measure  OD600/660 and RFU overnight every 15 minutes. The data was then analysed and plotted. This experiment was performed both in transformed E. coli DH5a as well as BL21.</p>
−
<p style="font-size: 115%; font-weight: bold; ">OD600:</p>
+
<p style="font-size: 110%; font-weight: bold; ">OD600:</p>
 
<p style="margin: 0!important;">Discrete wavelengths, 1</p>
 
<p style="margin: 0!important;">Discrete wavelengths, 1</p>
 
<p style="margin: 0!important;">Wavelength: 600</p>
 
<p style="margin: 0!important;">Wavelength: 600</p>
 
<p style="margin: 0!important;">Well scan: spiral average, 5mm diameter</p>
 
<p style="margin: 0!important;">Well scan: spiral average, 5mm diameter</p>
−
<p style="font-size: 115%; font-weight: bold;">OD660:</p>
+
<p style="font-size: 110%; font-weight: bold;">OD660:</p>
 
<p style="margin: 0!important;">Discrete wavelengths, 1</p>
 
<p style="margin: 0!important;">Discrete wavelengths, 1</p>
 
<p style="margin: 0!important;">Wavelength: 660</p>
 
<p style="margin: 0!important;">Wavelength: 660</p>
 
<p style="margin: 0!important;">Well scan: spiral average, 5mm diameter</p>
 
<p style="margin: 0!important;">Well scan: spiral average, 5mm diameter</p>
−
<p style="font-size: 115%; font-weight: bold;">RFP Fluorescence:</p>
+
<p style="font-size: 110%; font-weight: bold;">RFP Fluorescence:</p>
 
<p style="margin: 0!important;">Focal Height: 7.5</p>
 
<p style="margin: 0!important;">Focal Height: 7.5</p>
 
<p style="margin: 0!important;">Gain: 1000</p>
 
<p style="margin: 0!important;">Gain: 1000</p>

Revision as of 19:17, 12 October 2019

constitutive promoter family member

BerkiGEM2006-PromotersEppendorfs.jpg
BerkiGEM2006-Promoters.jpg

 
 Variant RFP (au)
 J23112           1
 J23103           17
 J23113           21
 J23109           106
 J23117           162
 J23114           256
 J23115           387
 J23116           396
 J23105           623
 J23110           844
 J23107           908
 J23106           1185
 J23108           1303
 J23118           1429
 J23111           1487
 J23101           1791
 J23104           1831
 J23102           2179
 J23100           2547
PBca1020-r0040.jpg

Constitutive promoter family
Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. J23119 is the "consensus" promoter sequence and the strongest member of the family. All parts except J23119 are present in plasmid J61002. Part J23119 is present in pSB1A2. This places the RFP downstream of the promoter. Reported activities of the promoters are given as the relative fluorescence of these plasmids in strain TG1 grown in LB media to saturation. See part BBa_J61002 for details on their use.

These promoter parts can be used to tune the expression level of constitutively expressed parts. The NheI and AvrII restriction sites present within these promoter parts make them a scaffold for further modification. JCAraw

Contribution

Group: Valencia_UPV iGEM 2018
Author: Adrián Requena Gutiérrez, Carolina Ropero
Summary: We adapted the part to be able to assemble transcriptional units with the Golden Gate assembly method
Documentation: In order to create our complete [http://2018.igem.org/Team:Valencia_UPV/Part_Collection part collection] of parts compatible with the Golden Gate assembly method, we made the part BBa_K2656005 which is this part adapted to the Golden Gate technology.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Manchester 2019 Characterisation:

We wanted to add new, normalised RFU data for two promoters from the Anderson family of constitutive promoters BBa_J23102 (Strong constitutive promoter) and BBa_J23106 (weak constitutive promoter) respectively; and a T7 promoter BBa_K199118 all expressing mRFP1. Another interest that we had was to test how OD was affected by RFP production, therefore we performed our measurements twice, at OD600 and OD660 respectively. This was done because it has been shown that OD660 gives a more accurate representation of bacterial growth in RFP-producing bacteria (Hecht et al., 2016).

In order to obtain our results, we grew our cell cultures up to an OD600/660 of ~0.6. After the desired OD had been reached, cultures were induced with IPTG (for BBa_K199118) and anhydrotetracycline (for BBa_K092300). Then they were placed on a microplate reader (CLARIOstar®, BMG Labtech). The specific script conditions can be seen below. The machine was set to measure OD600/660 and RFU overnight every 15 minutes. The data was then analysed and plotted. This experiment was performed both in transformed E. coli DH5a as well as BL21.

OD600:

Discrete wavelengths, 1

Wavelength: 600

Well scan: spiral average, 5mm diameter

OD660:

Discrete wavelengths, 1

Wavelength: 660

Well scan: spiral average, 5mm diameter

RFP Fluorescence:

Focal Height: 7.5

Gain: 1000

Excitation: 574-15

Emission: 618-22

Well scan: Matrix scan 3x3 1mm diameter