Difference between revisions of "Part:BBa J23105"

Revision as of 17:33, 20 October 2019

constitutive promoter family member

 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

Manchester 2017 used this part to create part LowPromoter_PduD(1-20)_mCherry (BBa_K2213006). This promoter was combined with PduD(1-20) to create a tag with lower expression levels. The mCherry tagged PduD(1-20) localisation tag displayed lower fluorescence levels under the low promoter as compared to under medium (BBa_K2213007) and high strength (BBa_K2213008) promoters, demonstrating correct function.

More information can be found here: https://parts.igem.org/Part:BBa_K2213006

GreatBay_China 2018:
Team GreatBay_China 2018 characterized J23119, Part:BBa_J23105, and Part:BBa_J23101 by assembling them with Part:BBa_B0034 and a sfGFPPart: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

Thessaly 2019 Characterization

Thessaly 2019 sought to characterize the coding sequence of TEM-optimized β-lactamase (Part:BBa_I757010) under the regulation of the constituve Anderson Family promoters Part:BBa_J23100, Part:BBa_J23105, Part:BBa_J23106, Part:BBa_J23119. β-lactamase is an enzyme that hydrolyses β-lactams (e.g. ampicillin) and is naturally found in prokaryotic cells. A colorimetric assay has been developed using nitrocefin as a substrate which after hydrolysis from β-lactamase changes the reaction color, from yellow (380nm) to red (490nm).

To achieve that, the coding sequence was assembled with each promoter, a universal RBS (Part:BBa_B0034) and a double terminator(Part:BBa_B0015). The parts were cloned in pSB1C3 and pSB1K3 and transformed into E. coli DH5a competent cells.

In the photo below you can see the results of the primer addition using overhang PCR:

Figure 1. The results obtained after the PCR with the overhang primers for the different promoters of the Anderson family. We tested different annealing temperatures (45, 47 & 53℃) aiming for clear results. The expected band is at 1119bp and the ladder used was the 100bp DNA ladder by NEB.

For protein expression, the plasmids were transformed into E. coli BL21 (DE3) competent cells.

For the beta-lactamase assay, we set up the following experimental design:

1. Grow BL21 (DE3) pre-culture overnight in 5ml LB (~16h) at a shaking incubator, 37 degrees C / 210rpm

2. The following morning, measure the OD600 of overnight cultures

3. Dilute all cultures to OD600¬ = 0.05 in M9 minimal medium

4. Grow cells 37 degrees C /210 RPM until OD600=0.4-0.6 (~2h)

5. Dilute all cells to the same OD600 (e.g. 0.4)

6. Load 160 of culture in a 96-well plate (do triplicates). Add 40 ul 0.5 uM nitrocefin for a final concentration of 100nM

7. Measure the absorbance at 490nm (for nitrocefin hydrolysis) and 600nm (for cell growth) every 30 seconds for 2 hours in a microplate reader. Shake between readings. Because plateau was reached within the first 30 minutes of the reaction, only those are depicted in the graph.

To ensure that the absorbance shown corresponds only to enzymatic activity by β-lactamase, we included 3 controls in the experiment. The first control has M9 medium only (no cells) and nitrocefin, the second has empty BL21 (DE3) cells (no plasmid) and nitrocefin, while the third has BL21 (DE3) cells containing the plasmid but not the part (empty plasmid). To obtain comparable results, we normalized all values by dividing OD490 by OD600.

The results are shown in the graph below

Figure 2. The hydrolysis of nitrocefin enabled by the expression of the β-lactamase gene, under the control of different promoters (J23100, J23105, J23106 & J23119) of the Anderson family. The substrate (nitrocefin) hydrolysis (490nm) is divided by cell growth (600nm), in order to normalize all values.

The maximum expression of β-lactamase was observed under control of the J23119 (brown line) which is the wild type promoter of the Anderson family. The expression is reduced with the J23100 and J23106 (yellow and purple line respectively), while the lowest expression levels were observed with the J23105 promoter (blue line). These results are in accordance with those from previous teams that measured fluorescence and the same pattern is observed. The controls conditions (pSB1C3 and BL21, or light purple and light blue respectively) confirm that the absorbance measured derives from β-lactamase activity only, both quantitatively and visually.

Below you can see the 96-well plate of the assay:

Figure 3.The observed color change due to the hydrolyzation of nitrocefin due to the production of β-lactamase, after a 2-hour enzymatic assay.

Note that the picture was taken after the plate-reader assay was completed and all conditions had reached a plateau, except the controls.

The maximum expression of beta-lactamase was observed under control of the J23119 (brown line) which is the wild type promoter of the Anderson family. The expression is reduced with the J23100 and J23106 (yellow and purple line respectively), while the lowest expression levels were observed with the J23105 promoter (blue line). These results are in accordance with those from previous teams that measured fluorescence and the same pattern is observed. The controls conditions (pSB1C3 and BL21, or light purple and light blue respectively) confirm that the absorbance measured derives from beta-lactamase activity only, both quantitatively and visually.

Below you can see the 96-well plate of the assay:

Figure 3.The observed color change due to the hydrolyzation of nitrocefin due to the production of b-lactamase, after a 2-hour enzymatic assay.

Note that the picture was taken after the plate-reader assay was completed and all conditions had reached a plateau, except the controls.

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.