Part:BBa_J23101

constitutive promoter family member
For video explanation on this promoter family and its use, visit [http://www.synbiotrails.org/#!/trail?id=org.clothocad.trails.youtube.TranscriptionModels here].

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

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_K2656007 which is this part adapted to the Golden Gate technology.

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]

USTC_2009's MEASUREMENT

BBa_K176012

Characterization by GreatBay_China 2018

Team GreatBay_China 2018 characterized J23119Part:BBa_J23119, Part:BBa_J23105, and Part:BBa_J23101 by assembling them with Part:BBa_B0034 and a sfGFP Part:BBa_I746916 on three vectors: pUC20 (copy number about 500/cell), pR6K (copy number about 15/cell), pSC101 (copy number about 2/cell). Then we measured the fluorescence by Flow Cytometry as a reference for the TALE stabilized promoter library.

The result indicate that the strength of J23119, J23105, and J23101 are about the same as described by team iGEM2006_Berkeley, and the fluorescence increases as the copy number of the vector increases

Baltimore Biocrew 2019 Characterization

Goal

We, the Baltimore Biocrew, decided to characterize some of the Anderson promoters. These promoters are highly used by iGEM but the relative expression of these promoters have been routinely determined by measuring the fluorescence of a reporter protein. However, the function of a promoter is to start transcription of a gene so it may be more informative to measure the amount of RNA (instead of protein) produced by a reporter gene. Therefore, we decided to further characterize a selection of the Anderson promoters (J23100, J23101, J23103, J23105, J23118) by measuring RNA using Quantitative Polymerase Chain Reaction (qPCR).

Results

We did data analysis using the Livak Method (a standard, comparative method) to determine the relative strength of the promoters from the qPCR data using rrSD as our reference gene, RFP as our target gene, and J23100 as our calibrator sample.

Example:

ΔCT(J23101) = CT(RFP, J23101) – CT(rrSD, J23101)

ΔΔCT(J23101) = ΔCT(J23101) – ΔCT(J23100)

2^(–ΔΔCT) = relative expression ratio

In our first trial of qPCR (8/03/19), we were able to measure the relative strengths for J23100, J23101, J23103, and J23105 which were 1.00, 0.00, 0.81, and 1.93, respectively. Since these strengths did not match the relative expression levels reported by iGEM2006_Berkeley, we repeated the qPCR (8/10/19) with the same cDNA. The strengths from this second trial were 1.00, 0.00, 0.37, and 0.20. We repeated it again and the relative strengths that we got on 10/12/19 for J23100, J23101, J23103, and J23103 were 1, 0, 2.91, and .32. Next, we made new cDNA by growing new liquid cultures, extracting RNA again, and repeating reverse transcription. From the new cDNA, we repeated the qPCR procedure two more times. The relative strengths for that we got on 9/28/19 for J23100, J23101, J23103, J23105, and J23118 were 1, 24.63, .36, 1.76, and .25. The relative strengths that we got on 10/12/19 for J23100, J23101, J23103, and J23105 were 1, 45.97, 3.20, and 1.26. In addition we measured promoter J23118 twice and got the strengths 1.13 and 1.32.

Here is the relative promoter strengths that we got from the qPCR. Baltimore BioCrew in blue compared to the 2006 Berkeley iGEM in orange.

To support our RNA measurements we also measured fluorescence of the liquid cultures we used to extract RNA. The cultures were grown overnight so we expected the bacteria to be at the stationary phase, but we measured OD to normalize any differences in growth.

Promoter	OD	fluorescence	fluorescence divided by OD	corrected relative expression	reported relative expression
BBa_J23100	0.876	250	285.38	1	1
BBa_J23101	0.674	255	378.33	1.33	0.7
BBa_J23103	1.1	230	209.09	0.73	0.01
BBa_J23105	1.08	215.74	209.09	0.76	0.24
BBa_J23118	1.04	238	228.84	0.80	0.56

Conclusion
After redoing our protocol many times and trying to compare our results to 2006 Berkeley iGEM team we concluded that our data doesn’t quite match theirs. The different results in strengths could be caused by many different factors while doing our protocols. However, we have succeeded in characterizing the different strength Andersons promoters (J23100, J23101, J23103, J23105, J23118) by measuring RNA using Quantitative Polymerase Chain Reaction (qPCR). In conclusion, we successfully reached our goal in bringing new data to the characterizations of 5 different Andersons Promoters. In the future, it would be good for other iGEM teams to try to measure RNA as well so there will be a standard qPCR protocol for iGEM.

{|width='100%' style='border:1px solid gray' |- |width='10%'| BBa_J23100 AddReview 5 University of Texas at Austin iGEM 2019 |width='60%' valign='top'| ====UT Austin iGEM 2019: Characterization of metabolic burden of the Anderson Series==== ===Description=== The 2019 UT Austin iGEM team transformed the Anderson Series promoters into our 'burden monitor' DH10B strain of E. coli which contains a constitutive GFP cassette in the genome of the cell. This GFP expression rate fluctuates depending on the number of ribosomes available for the host cell to sustain itself. Using this strain, the relative burden (percent reduction in growth rate) of each Anderson Series part was determined. Our results showed a range of growth rate reductions for each of these parts due to ribosomal reallocation from the genome of the host cell, towards the expression of RFP. Anderson Series parts with strong promoters are depicted with darker red colors and Anderson Series parts with weak promoters are depicted with lighter pink colors to show relative RFP expression. We saw a positive correlation between relative promoter strength and metabolic burden; parts with stronger promoters expressed less GFP and had a lower growth rate than parts with weaker promoters. The regression line for the graph below was constructed by measuring the burden of 5 parts that were created by the 2019 UT Austin iGEM team that each contained an Anderson Series promoter (J23104 or J23110), an RBS of varying strength, and a BFP reporter. For more information on characterization of these parts through the burden monitor, visit our team’s wiki page: [https://https://2019.igem.org/Team:Austin_UTexas]

Fig.1:Growth vs GFP Expression graph showing the relative burden positions of the Anderson Series promoters. The parts with strong promoters are depicted in dark red and are clustered near the bottom of the graph because they have lower growth rates and express lower levels of GFP as a result of high cellular burden. The parts with weaker promoter are depicted in light pink ad are clustered near the top of the graph because they have higher growth rates and express higher levels of GFP as a result of low cellular burden.

Table.1: Burden measurements for the Anderson Series promoters measured as percent reduction in growth rate ± 95% confidence interval.

Although often we cannot avoid using a specific burdensome part, knowing in advance that it is burdensome, and that it has a high chance of mutating into a non-functional genetic device, can help with troubleshooting and coming up with alternatives. In the specific case of fluorescent protein-expressing devices, Fluorescence-activated cell sorting (FACS) can be used to filter out individual cells that meet a certain fluorescence threshold. This way, the cells expressing lower levels of the fluorescent protein are weeded out of the population.