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| | ===Applications of BBa_J10050=== | | ===Applications of BBa_J10050=== |
| | + | Lambert_GA 2019 used this part in experiments to characterize the effect of different strengths of promoters and ribosomal binding sites on expression. The data obtained can aid in the design of constructs by giving information about controlling expression. |
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| | ===User Reviews=== | | ===User Reviews=== |
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| − | <partinfo>BBa_R0071 AddReview 4</partinfo>
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| − | <I>ETH Zurich 2014</I>
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| − | = Characterization of two-order crosstalk on the promoter =
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| − | == Background information == | + | = Lambert_GA 2019 Characterization = |
| | + | ==Characterization of weak promoter (BBa_J23113) and weak RBS (BBa_B0031)== |
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| − | We used an ''E. coli'' TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell). The first plasmid contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter [https://parts.igem.org/Part:BBa_R0079 pLas (BBa_R0079)] followed by [https://parts.igem.org/Part:BBa_B0034 RBS (BBa_B0034)] and superfolder green fluorescent protein (sfGFP). In general, for spacer and terminator sequences the parts [https://parts.igem.org/Part:BBa_B0040 BBa_B0040] and [https://parts.igem.org/Part:BBa_B0015 BBa_B0015] were used, respectively. The second plasmid contained the pBR322 origin (pMB1), which yields a stable two-plasmid system together with p15A, an ampicillin resistance gene, and a strong promoter [https://parts.igem.org/Part:BBa_J23100 (BBa_J23100)] chosen from the [https://parts.igem.org/Promoters/Catalog/Anderson Anderson promoter collection] followed by one of the three different regulators ([https://parts.igem.org/Part:BBa_C0062 LuxR], [https://parts.igem.org/Part:BBa_C0179 LasR], and [https://parts.igem.org/Part:BBa_C0171 RhlR]) used in the experiments in order to quantify crosstalk with [https://parts.igem.org/Part:BBa_I14017 pRhl]. The detailed regulator construct design and full sequences (piG0040, piG0041, piG0042, piG0060) are [http://2014.igem.org/Team:ETH_Zurich/lab/sequences available here]. In the following, we describe the experimental set-up and all the different levels of crosstalk we have assessed.
| + | Differences in promoter strength had more effect on enzymatic expression compared to differences in RBS strengths as evidenced in the graphs below. Lambert_GA 2019 had originally thought that BBa_J10050 would result in the least amount of expression out of the tested combinations, but this part, along with BBa_J10051 and BBa_J10052, all shared the lowest expression out of the combinations. This is due to the translation rate not being an important factor in the determination of expression of ONP; however, the promoter strength is a significant factor. In other words, expression values are significantly different with different promoters but not with different RBS strengths. |
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| − | = Experimental Set-Up =
| + | <br> |
| | + | <center>[[File:T--Lambert GA--tuning1a.png|800px]]</center> |
| | + | <br> |
| | + | <center><i>Figure 1: This sequence is to be used as a proof of concept, and can be used to develop biosensor control devices. The highlighted points in yellow represent BBa_J10050 (weak promoter/ weak RBS) compared to BBa_J10051 (weak promoter/ strong RBS) and BBa_J10052 (weak promoter/ medium RBS), both in light blue. The weak, medium, and strong RBS have similar expression levels when in combination with the weak promoter.</i></center> |
| | + | <br> |
| | + | <br> |
| | + | <center>[[File:T--Lambert GA--tuning1b.png|800px]]</center> |
| | + | <br> |
| | + | <center><i>Figure 2: The highlighted points represent the BBa_J10050 composite part (weak promoter/ weak RBS). As the promoter strength increases, the expression increases as well.</i> |
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| | + | <br> |
| | + | The graphs above show that RBS strength has a negligible effect on expression, whereas increased promoter strength increases expression. </center> |
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| − | The above described ''E. coli'' TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (50 μg/mL) and ampicillin (200 μg/mL) to an OD<sub>600</sub> of about 1.5 (37 °C, 220 rpm). As a reference, a preculture of the same strain lacking the sfGFP gene was included for each assay. The cultures were then diluted 1:40 in fresh LB containing the appropriate antibiotics and measured in triplicates in microtiter plate format on 96-well plates (200 μL culture volume) for 10 h at 37 °C with a Tecan infinite M200 PRO plate reader (optical density measured at 600 nm; fluorescence with an excitation wavelength of 488 nm and an emission wavelength of 530 nm). After 200 min we added the following concentrations of inducers ([[3OC6HSL|3OC6-HSL]], [[AHL|3OC12-HSL]], and [[AHL|C4-HSL]]): 10<sup>-4</sup> nM and 10<sup>4</sup> nM (from 100 mM stocks in DMSO). Attention: All the dilutions of [[AHL|3OC12-HSL]] should be made in DMSO in order to avoid precipitation. In addition, in one triplicate only H<sub>2</sub>O was added as a control. From the the obtained kinetic data, we calculated mean values and plotted the dose-response-curves for 200 min past induction.
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| − | == First-order crosstalk ==
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| − | In the first order crosstalk section we describe crosstalk of [https://parts.igem.org/Part:BBa_I14017 pRhl] due to [https://parts.igem.org/Part:BBa_C0171 RhlR] binding to inducers different from [[AHL|C4-HSL]] or [https://parts.igem.org/Part:BBa_I14017 pRhl] itself binding a regulator-inducer pair different from [https://parts.igem.org/Part:BBa_C0171 RhlR]-[[AHL|C4-HSL]].
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| − | === First Level crosstalk: RhlR binds to different HSL and activates the promoter ===
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| − | In the conventional system [[AHL|C4-HSL]] binds to its corresponding regulator, [https://parts.igem.org/Part:BBa_C0171 RhlR], and activates the pRhl promoter (figure 2, green). However, RhlR can potentially also bind other AHLs and then activate pRhl (Figure 2, [[AHL|3OC12-HSL]] in red and [[3OC6HSL|3OC6-HSL]] in light blue). This leads then to unwanted gene expression (crosstalk).
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| − | [[File:ETH Zurich 1crosstalkPrhl.png|400px|thumb|center| '''Figure 1 Overview of possible crosstalk of the [https://parts.igem.org/Part:BBa_C0171 RhlR]/[https://parts.igem.org/Part:BBa_I14017 pRhl] system with three different [[AHL|AHLs]].''' Usually, [[AHL|C4-HSL]] binds to its corresponding regulator, [https://parts.igem.org/Part:BBa_C0171 RhlR], and activates the [https://parts.igem.org/Part:BBa_I14017 pRhl] promoter (green). However, [https://parts.igem.org/Part:BBa_C0171 RhlR] may also bind [[AHL|3OC12-HSL]] (red) or [[3OC6HSL|3OC6-HSL]] (light blue) and then unintentionally activate [https://parts.igem.org/Part:BBa_I14017 pRhl].]]
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| − | ==== Second Level crosstalk: other regulatory proteins, like LuxR and LasR, bind to their natural AHL substrate and activate the pRhl promoter ====
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| − | In the conventional system [[AHL|C4-HSL]] binds to its corresponding regulator, [https://parts.igem.org/Part:BBa_C0171 RhlR], and activates the [https://parts.igem.org/Part:BBa_I14017 pRhl] promoter (Figure 2, green). However, [https://parts.igem.org/Part:BBa_I14017 pRhl] can potentially be activate by other regulators ([https://parts.igem.org/Part:BBa_R0062 LuxR], [https://parts.igem.org/Part:BBa_C0179 LasR]), binding their corresponding regulator (figure 2, [[3OC6HSL|3OC6-HSL]] in light blue, [[AHL|3OC12-HSL]] in red). This leads then to unwanted gene expression (crosstalk).
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| − | [[File:ETH_Zurich_2crosstalkPrhl.png|thumb|400px|center|'''Figure 2 Overview of possible crosstalk of the [https://parts.igem.org/Part:BBa_C0171 RhlR]/[https://parts.igem.org/Part:BBa_i14017 pRhl] system with two additional regulators ([https://parts.igem.org/Part:BBa_R0062 LuxR] and [https://parts.igem.org/Part:BBa_C0179 LasR]).''' Usually, [https://parts.igem.org/Part:BBa_C0171 RhlR] together with inducer [[AHL|C4-HSL]] activate their corresponding promoter [https://parts.igem.org/Part:BBa_I14017 pRhl] (green). However, [https://parts.igem.org/Part:BBa_I14017 pRhl] may also be activated by the [https://parts.igem.org/Part:BBa_C0062 LuxR] regulator together with [[3OC6HSL|3OC6-HSL]] (light blue) or by the [https://parts.igem.org/Part:BBa_C0179 LasR] regulator together with [[AHL|3OC12-HSL]] (red).]]
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| − | == Second order crosstalk: Combination of both cross-talk levels ==
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| − | The second order crosstalk describes unintended activation of [https://parts.igem.org/Part:BBa_I14017 pRhl] by a mixture of both the levels described above. The regulator and inducer are being different from [https://parts.igem.org/Part:BBa_C0171 RhlR] and [[AHL|C4-HSL]], respectively, and at the same time they do not belong to the same module. For example, the inducer [[3OC6HSL|3OC6-HSL]] (light blue), usually binding to the regulator [https://parts.igem.org/Part:BBa_R0062 LuxR], could potentially interact with [https://parts.igem.org/Part:BBa_C0179 LasR] regulator (red) and together activate [https://parts.igem.org/Part:BBa_I14017 pRhl] (green). This kind of crosstalk is explained in Figure 3.
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| − | [[File:ETH Zurich 2014 2nd order rhl.png|400px|thumb|center| '''Figure 3 Overview of possible crosstalk of the [https://parts.igem.org/Part:BBa_I14017 pRhl] promoter with both the regulator and inducer being unrelated to the promoter and each other.''' Usually, [https://parts.igem.org/Part:BBa_C0171 RhlR] together with inducer [[AHL|C4-HSL]] activate their corresponding promoter [https://parts.igem.org/Part:BBa_I14017 pRhl] (green). However, [https://parts.igem.org/Part:BBa_I14017 pRhl] may also be activated by another regulator together with an unrelated inducer. For example, the inducer [[3OC6HSL|3OC6-HSL]] (light blue) may interact with the [https://parts.igem.org/Part:BBa_C0179 LasR] regulator (red) and together activate [https://parts.igem.org/Part:BBa_I14017 pRhl] (green).]]
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| − | == Results ==
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| − | {|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"
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| − | |+'''Table 1''' Crosstalk matrix for the promoter prhl ([https://parts.igem.org/Part:BBa_I14017:Experience BBa_I14017])
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| − | |colspan="4" style='font-size:10pt';text-align:left|
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| − | In this set of experiments the promoter [https://parts.igem.org/Part:BBa_I14017 pRhl] was tested for potential crosstalk. In the top left position we observe the induction of [https://parts.igem.org/Part:BBa_I14017 pRhl] by C4-HSL bound to the [https://parts.igem.org/Part:BBa_C0171 regulator RhlR]. The switching behaviour was observed at a C4-HSL concentration of 1 μM.
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| − | In the case of 3OC12-HSL binding the [https://parts.igem.org/Part:BBa_C0171 RhlR regulator] and subsequently the [https://parts.igem.org/Part:BBa_I14017 promoter pRhl]insignificant crosstalk has been observed.
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| − | Severe crosstalk was observed in the case of 3OC6-HSL binding the [https://parts.igem.org/Part:BBa_C0171 RhlR regulator] followed by induction of [https://parts.igem.org/Part:BBa_R0071 pRhl]. The transition occurred at a concentration of the inducer molecule of 1 μM but compared to the reference curve a lower value of fluorescence per OD was observed (1000 a.u.). Another case of crosstalk with the [https://parts.igem.org/Part:BBa_I14017 pRhl] was detected with 3OC12-HSL binding to the corresponding [https://parts.igem.org/Part:BBa_C0179 LasR regulator] followed by inducing the promoter [https://parts.igem.org/Part:BBa_I14017 pRhl]. Here switching occurred at a concentration 1 nM of 3OC12-HSL and reached fluorescence per OD of 750 a.u.. This is approximately 0.5 fold the value of the fluorescence per OD shown by the reference curve indicated in green.
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| − | |-
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| − | |style="width:100px"|[[File:ETH_Zurich_2014_qs-table_CornerRhl.png|100px|link=https://parts.igem.org/Part:BBa_I14017:Experience]]
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| − | |[[File:ETH_Zurich_2014_qs-table_C4-HSL.png|100px|link=https://parts.igem.org/Part:BBa_I14017:Experience]]
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| − | |[[File:ETH_Zurich_2014_qs-table_3OC6-HSL.png|100px|link=https://parts.igem.org/Part:BBa_I14017:Experience]]
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| − | |[[File:ETH_Zurich_2014_qs-table_3OC12-HSL.png|100px|link=https://parts.igem.org/Part:BBa_I14017:Experience]]
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| − | |-
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| − | |[[File:ETH_Zurich_2014_qs-table_RhlR.png|100px|link=https://parts.igem.org/Part:BBa_C0171:Experience]]
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| − | |[[File:ETH_Zurich_2014_qs-table_PrhlRef.png|200px]]
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| − | |[[File:ETH_Zurich_2014_qs-table_PrhlRhlRLuxAHL.png|200px]]
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| − | |[[File:ETH_Zurich_2014_qs-table_PrhlRhlRLasAHL.png|200px]]
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| − | |-
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| − | |[[File:ETH_Zurich_2014_qs-table_LuxR.png|100px|link=https://parts.igem.org/Part:BBa_C0062:Experience]]
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| − | |[[File:ETH_Zurich_2014_qs-table_PrhlLuxRRhlAHL.png|200px]]
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| − | |[[File:ETH_Zurich_2014_qs-table_PrhlLuxRLuxAHL.png|200px]]
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| − | |[[File:ETH_Zurich_2014_qs-table_PrhlLuxRLasAHL.png|200px]]
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| − | |-
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| − | |[[File:ETH_Zurich_2014_qs-table_LasR.png|100px|link=https://parts.igem.org/Part:BBa_C0179:Experience]]
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| − | |[[File:ETH_Zurich_2014_qs-table_PrhlLasRRhlAHL.png|200px]]
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| − | |[[File:ETH_Zurich_2014_qs-table_PrhlLasRLuxAHL.png|200px]]
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| − | |[[File:ETH_Zurich_2014_qs-table_PrhlLasRLasAHL.png|200px]]
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| − | |}
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| − | === Modeling crosstalk ===
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| − | Each experimental data set was fitted to an Hill function using the Least Absolute Residual method.
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| − | [[File:ETHZ_HillEq.png|center|200px]]
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| − | <p>The fitting of the graphs was performed using the following equation :<br><br>
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| − | rFluo = the relative fluorescence (absolute measured fluorescence value over OD)[a.u.]<br>
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| − | a = basal expression rate [a.u.](“leakiness”)<br>
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| − | b = maximum expression rate [a.u.]("full induction")<br>
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| − | n = Hill coefficient (“cooperativity”)<br>
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| − | K<sub>m</sub> = Half-maximal effective concentration (“sensitivity”)<br>
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| − | [AHL] = AHL concentration [nM]</p>
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| − | <br clear="all"/>
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| − | {| border="1" class="wikitable" style="margin: 1em auto 1em auto;text-align:center;"
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| − | |+ Parameters of HillFunction for crosstalk with Prhl (with 95% confidence bounds)
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| − | !
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| − | ! [[AHL|C4-HSL]]
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| − | ! [[3OC6HSL|3OC6-HSL]]
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| − | ! [[AHL|3OC12-HSL]]
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| − | |-
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| − | ! [https://parts.igem.org/Part:BBa_C0171 RhlR]
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| − | | a = 178.4 (174.9, 182) [a.u.]<br> n = 1.053 (0.9489, 1.157)<br> Km = 1969 (1625, 2313) [nM]<br>b = 1736 (1629, 1842) [a.u.]<br>
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| − | | a = 169.1 (155.2, 182.9) [a.u.]<br> n = 0.507 (0.2303, 0.7837) <br> Km = 1.08e8(0, 2.681e10) [nM]<br> b = 9.708e4 (0, 1.192e7) [a.u.]<br>
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| − | | a = 162.8 (150.4, 175.1) [a.u.]<br> n = 0.404 (0, 0.998)<br> Km = 9.627e8 (0, 7.824e11) [nM]<br>b = 2.537e4 (0, 8.109e6) [a.u.]<br>
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| − | |-
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| − | ! [https://parts.igem.org/Part:BBa_C0062 LuxR]
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| − | | No crosstalk
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| − | | No crosstalk
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| − | | No crosstalk
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| − | |-
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| − | ! [https://parts.igem.org/Part:BBa_C0179 LasR]
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| − | | No crosstalk
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| − | | No crosstalk
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| − | | a = 149.3 (140.6, 158.1) [a.u.]<br>n = 1.366 (0.808, 1.923) <br> Km = 1.674 (1.259, 2.09) [nM]<br> b = 628.9 (599, 658.7) [a.u.]<br>
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| − | |}
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| − | |}
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| − | {|width='80%' style='border:1px solid gray'
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| − | <partinfo>BBa_I14017 AddReview 0</partinfo>
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| − | <I>Antiquity</I>
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| − | This review comes from the old result system and indicates that this part did not work in some test.
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| − | |}
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Lambert_GA 2019 used this part in experiments to characterize the effect of different strengths of promoters and ribosomal binding sites on expression. The data obtained can aid in the design of constructs by giving information about controlling expression.
