![](https://static.igem.org/mediawiki/2018/c/c4/T--Bielefeld-CeBiTec--colorful_tubes_vk.png)
Composite Parts
Short summary
This Composite Part Our new TACE system consists of an expression vector which transcribes siRNAs, and a target vector which transcribes a target mRNA as well as a reporter protein which enables the user to quantify the silencing ability of a tested siRNA.
The first generation of target vectors featured a direct fusion of a target sequence to a Reporter Protein (Blue fluorescent Protein BFP or AmilCP). This assures the shortest reaction time possible to the degradation of the target mRNA, as the destruction of the target mRNA also leads to the degradation of the marker proteins mRNA. However this has a problem, as a non-functional reporter protein can lead to false positives. This led to the development of the second generation of vectors. This Generation also features an inverter as well as additional control mechanisms. The vector is shown in Figure 1:
![](https://static.igem.org/mediawiki/2018/1/10/T--Bielefeld-CeBiTec--ALE-pTale2_B_GS_V1_for_Best_composite_Part.png)
The part features a Golden Gate Assembly (GGA) Cassette between an ATG and an GGGGS Linker. This allows the user to seamlessly insert a target sequence. Short target sequences can be ordered as oligo nucleotides, annealed and inserted via Golden Gate Assembly. Long Targets can be inserted the same way, if the needed overlaps can be added. Alternatively the Part can be amplified using the Primers in Table 2, and the target sequence can be inserted via Gibson Assembly after adding matching overlaps. Through the start codon upstream of the GGA cassette the LacI inhibitor will still be produced when a target sequence without an own start codon is inserted. However, the user still has to keep the LacI inhibitor in frame with a start codon. The used terminator B0015has a high terminating ability to prevent random transcription of the reporter protein. Because of the pLac promotor, the time in an experiment when the reporter protein shall be expressed can be chosen freely. BFP was chosen as a reporter protein because it has a low excitation wavelength at 399 nm and emission wavelength at about 450 nm. This recommends the BFP for a FRET system, making further measurements possible.
We designed this system to test as many siRNAs as possible with a high sample throughput. To achieve this, we constructed expression vectors which allow comparable expressions of different siRNAs, as well as a target vector which grants easy measurement of the silencing effectivity of a given siRNA.
It is important to make all expressed siRNAs comparable to each other. Therefore we designed four Biobricks featuring the same promoter to establish the same expression rate for all siRNAs. As a first Generation, we designed the four Biobricks BBa_K2638701, BBa_K2638702, BBa_K2638758 and BBa_K2638759 (Figure 1-4) for our expression vectors. They contain a Golden Gate Assembly (GGA) cassette which can be cut out using the restriction enzyme BbsI. So it is possible to replace the whole GGA cassette with a specific siRNA with the usage of our GGA protocol. The transcription of the siRNA is terminated by the strong Terminator BBa_B0015 to make sure that all expression processes are completely terminated.
While the Biobrick BBa_K2638701(Figure 1) is supposed to transcribe an siRNA without further modifications, the Biobrick BBa_K2638702 (Figure 2) also includes the Hfq binding sequence originating of the MicC-siRNA (Chen et al., 2004). The Biobrick BBa_K2638758 (Figure 3) contains the 5’-UTR from ompA as a protective sequence upstream of the siRNA insertion site and the Biobrick BBa_K2638759 (Figure 4) with both features surrounding the insertion site as well as sequences combined to add further functions to the siRNA.
![](https://static.igem.org/mediawiki/2018/3/3e/T--Bielefeld-CeBiTec--ALE-pGuide_V2_Genkarte.png)
![](https://static.igem.org/mediawiki/2018/b/b4/T--Bielefeld-CeBiTec--ALE-pGuide-Hfq_V2.png)
![](https://static.igem.org/mediawiki/2018/7/77/T--Bielefeld-CeBiTec--ALE-pGuide_OmpA_V2.png)
![](https://static.igem.org/mediawiki/2018/7/7d/T--Bielefeld-CeBiTec--ALE-pGuide_OmpA_Hfq_V2.png)
Therefore, we can choose one of these explained expression vectors to be the first part of the complete TACE-system. The second part of our TACE system is a target vector, pTale, which transcribes one specific mRNA. The chosen mRNA should be silenced by the constructed siRNAs using one of the described Biobricks above. To get the optimal conditions for measuring the silencing effect of our siRNA we scheduled and constructed two Generations of target vectors. For the first generation of the target vectors we used two different Reporter Proteins: the chromoprotein AmilCp (BBa_K592009) and the blue fluorescent protein BFP (BBa_K592100). Additionaly feature the target Biobrick a linker between the GGA cassette and the reporter protein. This way, the inserted target mRNA forms a CDS fusion with the reporter protein without losing any function. If the mRNA is destroyed, no reporter protein is formed. This results in no measurement of the BFP fluorescents which is proportional to the silencing strength of the siRNA. The Target Vectors were cloned with 4 different linkers (GGGGS, BBa_K2638721; EAAAK, BBa_K2638722<(a>; XP, BBa_K2638723; cMyc, BBa_K2638724), so users of this System can choose the perfect linker for their own system. The structure of the first generation of pTale is shown in Figure 5.
![](https://static.igem.org/mediawiki/2018/0/0a/T--Bielefeld-CeBiTec--ALE-pTale_V1.png)
![](https://static.igem.org/mediawiki/2018/7/73/T--Bielefeld-CeBiTec--ALE-pTale2_V1.png)
Biobrick number | Tag | Linker | Reporter Protein | Other features |
---|---|---|---|---|
BBa_K2638711 | pTale_A_GS | GGGGS | AmilCP | - |
BBa_K2638712 | pTale_A_EA | EAAAK | AmilCP | - |
BBa_K2638713 | pTale_A_CM | cMyc | AmilCP | - |
BBa_K2638714 | pTale_A_XP | XP | AmilCP | - |
BBa_K2638707 | pTale_B_GS | GGGGS | BFP | - |
BBa_K2638708 | pTale_B_EA | EAAAK | BFP | - |
BBa_K2638709 | pTale_B_CM | cMyc | BFP | - |
BBa_K2638710 | pTale_B_XP | XP | BFP | - |
BBa_K2638707 | pTale2_B_GS | GGGGS | BFP | 2. Generation |
BBa_K2638708 | pTale2_B_EA | EAAAK | BFP | 2. Generation |
BBa_K2638709 | pTale2_B_CM | cMyc | BFP | 2. Generation |
BBa_K2638710 | pTale2_B_XP | XP | BFP | 2. Generation |
BBa_K2638701 | pGuide | - | - | - |
BBa_K2638702 | pGuide_Hfq | - | - | Hfq scaffold |
BBa_K2638758 | pGuide_Omp | - | - | OmpA 5’-UTR |
BBa_K2638759 | pGuide_Hfq_Omp | - | - | Hfq Scaffold, Hfq Scaffold |
Testing the System
![](https://static.igem.org/mediawiki/2018/d/d4/T--Bielefeld-CeBiTec--ALE-Vectortest_T1B_BFP.png)
![](https://static.igem.org/mediawiki/2018/5/5d/T--Bielefeld-CeBiTec--ALE-Vectortest_T1B_GFP_V1.png)
The results presented in figures 7 and 8 show that the cells were growing very slowly in the 12 h of measurement time owing to the oxygen limited culture conditions in the 96 well plate used. In Figure 7 a significant increase in the Fluorescence of BFP was visible, but no noteworthy increase in the Fluorescence signal of GFP was detectable. Sequencing showed that only 50 BP of the GFP were inserted into the Target vector, explaining the missing fluorescence signal. Though it could not be shown that our system works with larger protein fusions, we were able to show that our vector works as expected with short target sequences. Vice versa, in Figure 8 a strong GFP signal was detectable, while no BFP was produced. Possibly the larger GFP protein keeps the BFP from folding correctly or inhibits the fluorophore formation in another way. Both figures show a strong increase in Fluorescence upon induction with ahTc. This confirms that the strain is producing the TetR repressor, but not on a level that is high enough to repress the promotor. Possibly the copy number of pSb1C3 proved to be too high, so that not enough TetR was abundant.
Next, we tried to clone the Guide vectors into competent cells containing the target vectors shown above. As the cells were constantly expressing GFP, we were not able to perform the blue white screening following the Golden Gate Assembly, and therefore were not able to test the pTale vectors in combination with the pGuide vectors. We were however able to clone the pGuide Biobricks into a pSB1K3 plasmid which had its native origin of replication (ori) replaced with a synthetic one (BBa_K2638751) made from parts of Team Vilnius 2017. We were able to grow cells containing that vector and sequence the ori.
As we ran out of time, we were not able to perform further tests on these vectors.
Next, we tried to clone the Guide vectors into competent cells containing the target vectors shown above. As the cells were constantly expressing GFP, we were not able to perform the blue white screening following the Golden Gate Assembly, and therefore were not able to test the pTale vectors in combination with the pGuide vectors. We were however able to clone the pGuide Biobricks into a pSB1K3 plasmid which had its native origin of replication (ori) replaced with a synthetic one (BBa_K2638751) made from parts of Team Vilnius 2017. We were able to grow cells containing that vector and sequence the ori.
As we ran out of time, we were not able to perform further tests on these vectors.
Usage protocol
![](https://static.igem.org/mediawiki/2018/0/01/T--Bielefeld-CeBiTec--ALE-siRNA_Testsystem_Übersichtsgrafik_2.png)
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Prepare Biobricks
To use this system it is important that a stable integration of two different vectors in the E. coli cell is possible. Therefore, the usage of two selection markers like antibiotic resistances, and optimally the usage of different Origins of Replications with about equal strength is necessary. The parts of iGEM Vilnius 2017 are extremely useful for this purpose. -
Preparation of the target sequences
Prior to the experiments, the target sequences and siRNAs need to be designed. First decide for a target vector to use. It might be useful to try cloning the target sequence into vectors with different linkers and test which works best for a given target sequence.-
There are two ways to clone a target sequence into a target vector:
- By Gibson Assembly: Linearize the vector using BbsI or amplification with PCR using the primers in Table 2 corresponding to the chosen vector.
- By Golden Gate assembly: Specially suitable for short target sequences which can be ordered as oligonucleotides, as overlaps of 4 nucleotides are needed to assemble the vector. Dimerize the oligos by Oligo Annealing and perform the Golden Gate assembly as described in our protocols. This might be useful when only a fragment of a coding sequence is used as a target sequence.
- To screen several vectors for the best compartibility with a target sequence transform all vectors with the Target gene inserted.
- For pTale vectors: induce with anhydrotetracycline (ahTc) and compare the levels of formed protein markers.
- For pTale2: Induce with IPTG. Let the cells grow until the BFP can be detected. Induce with ahTc and measure and compare the decline of the BFPs fluorescence to find out if a correctly folded LacI fusion is expressed.
- Once a target vector is chosen, prepare competent cells harboring this vector.
Design siRNAs
- Prior to the siRNA design, a mechanism for the silencing should be chosen. Our vector system can be used with any siRNA that has overlaps to the pGuide expression vector. At present, our system also provides three vectors with already integrated sequences to give an siRNA further features. If an siRNA shall be used to prevent the translation of the mRNA, it is advantageous to use the pGuide_Omp vector ( BBa_K2638758), as the OmpA 5-UTR significantly increases the half-life of the siRNA. The Hfq adapting scaffold present in the Expression vectors pGuide_Hfq and pGuide_OmpA_Hfq is recommendable in most cases, as it protects the siRNA and strengthens the bond to the target sequence. The vector used determines which overlaps to the vector need to be included into the siRNAs. The overlaps for the different vectors can be seen in Table 3.
- Design the siRNAs. Either by using our siRCon tool to predict siRNAs, or by adapting externally designed siRNAs to our system.
- Order siRNAs as Oligonucleotides
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Transformation and measurement
- Perform an oligoannealing and a Golden Gate assembly as described in our protocols to insert the siRNAs into the expression vector.
- Transform the Golden Gate Assembly into the competent cells containing the target vector.
- Measure the reporter protein and the OD600. BFP has an excitation peak at 399 nm and an emission peak at 456 nm. AmilCP has a maximum absorption at 588 nm.
Vektor | Linker | Forward Primer | Reverse Primer | Tm |
---|---|---|---|---|
pGuide | - | gattatttgcacggcgtcac | gaggaagcctgcataacgc | 57°C |
pTale1_BFP | all | gtgatagagattgacatccctatcagtg | ccctgagtatggttaatgaacgttttg | 57°C |
pTale1_AmilCP | all | gtgatagagattgacatccctatcagtg | cagtgagctttaccgtctgc | 57°C |
pTale2 | all | gtgatagagattgacatccctatcagtg | gtggcaacgccaatcagc | 57°C |
pTale2_amplification | GGGGS | gggggtggaggttcgg | catctttcctgtgtgagtgctcag | 57°C |
pTale2_amplification | EAAAK | gaggcggctgcaaaagag | catctttcctgtgtgagtgctcag | 57°C |
pTale2_amplification | cMyc | gaacagaagctgattagcgaagaag | catctttcctgtgtgagtgctcag | 57°C |
pTale2_amplification | XP | gctcccgctccgaagc | catctttcctgtgtgagtgctcag | 57°C |
pTale1_ amplification | GGGGS | gggggtggaggttcgg | catctttcctgtgtgagtgctcag | 57°C |
pTale1_ amplification | EAAAK | gaggcggctgcaaaagag | catctttcctgtgtgagtgctcag | 57°C |
pTale1_ amplification | xMyc | gaacagaagctgattagcgaagaag | catctttcctgtgtgagtgctcag | 57°C |
pTale1_ amplification | XP | gctcccgctccgaagc | catctttcctgtgtgagtgctcag | 57°C |
Vektor | Linker | Forward overlap | Reverse overlap |
---|---|---|---|
pTale1, pTale2 | GGGGS | GATG | CCCC |
pTale1, pTale2 | EAAAK | GATG | CCTC |
pTale1, pTale2 | cMyc | GATG | GTTC |
pTale1, pTale2 | XP | GATG | GAGC |
All Composite Parts
Identifier | Components | Description | Designer | Length |
---|---|---|---|---|
BBa_K2638003 | BBa_K525998, BBa_K2638001 | T7 + RBS + CopC | Erika Schneider | 425 |
BBa_K2638004 | BBa_K525998, BBa_K2638002 | T7 + RBS + CopD | Erika Schneider | 950 |
BBa_K2638005 | BBa_I0500, BBa_B0030, BBa_K2638001 | T7 + RBS + CopC | Erika Schneider | 1626 |
BBa_K2638006 | BBa_I0500, BBa_B0030, BBa_K2638002 | T7 + RBS + CopD | Erika Schneider | 2151 |
BBa_K2638109 | BBa_R0040, BBa_K1460002 | PTetR + CRS5 | Johannes Ruhnau | 1032 |
BBa_K2638110 | BBa_J61101, BBa_K2638103, BBa_J61101, BBa_K2638150 | PTetR + gshA + gshB + Phyto | Johannes Ruhnau | 2450 |
BBa_K2638112 | BBa_I0500, BBa_B0030 BBa_B0034, BBa_K2638121, BBa_B0034, BBa_K2638103, BBa_B0034, BBa_K2638120 | PTetR + gshA + gshB + GSR | Johannes Ruhnau | 4167 |
BBa_K2638113 | BBa_I0500, BBa_B0030, BBa_B0034, BBa_K2638100 | PTetR + ahpC + ahpF | Johannes Ruhnau | 1800 |
BBa_K2638114 | BBa_R0040, BBa_K554003, BBa_K1104200 | PTetR + SoxR + RBS + OxyR | Johannes Ruhnau | 1489 |
BBa_K2638117 | BBa_R0040, BBa_J61101, BBa_K2638106 | PTetR + RBS + sodA + KatE | Johannes Ruhnau | 701 |
BBa_K2638118 | BBa_R0040, BBa_B0034, BBa_K2638406, BBa_B0034, BBa_K2638105 | PTetR + RBS + sodA + KatG | Johannes Ruhnau | 2908 |
BBa_K2638201 | BBa_K525998, BBa_K2638200 | OprC (TonB dependent copper transport porin, BBa_K2638200) with T7 promotor and RBS (BBa_K525998) | Jakob Zubek | 2165 |
BBa_K2638204 | BBa_I0500, BBa_B0030, BBa_K2638200 | OprC (TonB dependent copper transport porin, BBa_K2638200) with AraC/pBad | Jakob Zubek | 3366 |
BBa_K2638400 | BBa_K2638500 | Combination of BBa_K2638500 + BBa_K2638560 | Levin Joe Klages | 889 |
BBa_K2638401 | BBa_K2638502, BBa_K2638426 | 13 | Levin Joe Klages | 889 |
BBa_K2638402 | BBa_K2638503, BBa_K2638426 | 14 | Levin Joe Klages | 889 |
BBa_K2638403 | BBa_K2638504, BBa_K2638426 | 15 | Levin Joe Klages | 889 |
BBa_K2638404 | BBa_K2638506, BBa_K2638426 | 17 | Levin Joe Klages | 889 |
BBa_K2638405 | BBa_K2638507, BBa_K2638426 | 18 | Levin Joe Klages | 889 |
BBa_K2638406 | BBa_K2638509, BBa_K2638426 | 110 | Levin Joe Klages | 889 |
BBa_K2638407 | BBa_K2638510, BBa_K2638426 | 111 | Levin Joe Klages | 889 |
BBa_K2638408 | BBa_K2638511, BBa_K2638426 | 112 | Levin Joe Klages | 889 |
BBa_K2638409 | BBa_K2638517, BBa_K2638426 | 118 | Levin Joe Klages | 903 |
BBa_K2638410 | BBa_K2638520, BBa_K2638426 | 21 | Levin Joe Klages | 890 |
BBa_K2638411 | BBa_K26358522, BBa_K2638426 | 23 | Levin Joe Klages | 890 |
BBa_K2638412 | BBa_K2638525, BBa_K2638426 | 26 | Levin Joe Klages | 890 |
BBa_K2638413 | BBa_K2638526, BBa_K2638426 | 27 | Levin Joe Klages | 890 |
BBa_K2638414 | BBa_K2638528, BBa_K2638426 | 29 | Levin Joe Klages | 890 |
BBa_K2638415 | BBa_K2638531, BBa_K2638426 | 212 | Levin Joe Klages | 890 |
BBa_K2638416 | BBa_K2638532, BBa_K2638426 | 213 | Levin Joe Klages | 889 |
BBa_K2638417 | BBa_K2638534, BBa_K2638426 | 215 | Levin Joe Klages | 900 |
BBa_K2638418 | BBa_K2638537, BBa_K2638426 | 218 | Levin Joe Klages | 904 |
BBa_K2638419 | BBa_K2638542, BBa_K2638426 | 33 | Levin Joe Klages | 889 |
BBa_K2638420 | BBa_K2638545, BBa_K2638426 | 36 | Levin Joe Klages | 889 |
BBa_K2638421 | BBa_K2638548, BBa_K2638426 | 39 | Levin Joe Klages | 889 |
BBa_K2638422 | BBa_K2638551, BBa2638426 | 312 | Levin Joe Klages | 889 |
BBa_K2638423 | BBa_K2638554, BBa_K2638426 | 315 | Levin Joe Klages | 899 |
BBa_K2638424 | BBa_K26358556, BBa_2638426 | 317 | Levin Joe Klages | 909 |
BBa_K2638425 | BBa_K2638557, BBa_K2638426 | 318 | Levin Joe Klages | 903 |
BBa_K2638703 | BBa_K2638716, BBa_B0010, BBa_B0012, BBa_R0010, BBa_B0032, BBa_K592100 | siRNA Target Vector 2 with BFP and GGGGS Linker | Antonin Lenzen | 2787 |
BBa_K2638704 | BBa_K2638717, BBa_B0010, BBa_B0012, BBa_R0010, BBa_B0032, BBa_K592100 | siRNA Target Vector 2 with BFP and GGGGS Linker | Antonin Lenzen | 2787 |
BBa_K2638705 | BBa_K2638718, BBa_B0010, BBa_B0012, BBa_R0010, BBa_B0032, BBa_K592100 | siRNA Target Vector 2 with BFP and cMyc Linker | Antonin Lenzen | 2787 |
BBa_K2638706 | BBa_K2638719, BBa_B0010, BBa_B0012, BBa_R0010, BBa_B0032, BBa_K592100 | siRNA Target Vector 2 with BFP and XP Linker | Antonin Lenzen | 2787 |
BBa_K2638991 | BBa_K2638999 | araBAD and RBS and Mutated Human Ferritin Heavy Chain (without Stop) | Vanessa Krämer | 1765 |
BBa_K2638997 | BBa_K118902, BBa_B0030, BBa_I0500 | araBAD and RBS and Human Ferritin Heavy Chain | Vanessa Krämer | 1793 |
Chen, S., Zhang, A., Blyn, L. B., & Storz, G. (2004). MicC, a second small-RNA regulator of Omp protein expression in Escherichia coli. Journal of bacteriology, 186(20), 6689-6697.
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