iGEM 2018 — Team JMU_Wuerzburg

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# Team:JMU Wuerzburg/Notebook

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  + [Establishing an iGEM-Team](https://2018.igem.org/Team%3AJMU_Wuerzburg/Human_Practices#establishing)
  + [Collaborations for our Laboratory Work](https://2018.igem.org/Team%3AJMU_Wuerzburg/Human_Practices#collabs)
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  Judging Form

### Calendar Week 06

### 07.02.2018 Seeding of HEK293T cells

Participants: Nicole, Rick, Andreas, Maria, Corinna

Protocol: Seed cells

Oligos/Cells: HEK293T

### 09.02.2018 Transfection of HEK293T cells

Participants: Nicole, Rick, Andreas, Maria, Corinna, Bente

Protocol: Transfection with PEI

Result: a huge number of cells died.

Conclusion: This steps should be repeated.

### Calendar Week 07

### 12.02.2018 Seeding of HEK293T cells

Participants: Rick, Maria, Corinna

Protocol: Seed cells

Oligos/Cells: HEK293T

### 13.02.2018 Transfection of HEK293T cells

Participants: Rick, Andreas, Corinna, Susanna

Protocol: Transfection with PEI

Result: Two days after transfection half of the cells were alive.

Conclusion: A harvest of GFP-RNA is possible

### 15.02.2018 Harvest of GFP-RNA

Participants: Rick, Maria, Andreas, Corinna

Protocol: E.Z.N.A. Total RNA Kit I (Omega)

Result: Just a small amount of RNA was isolated

Conclusion: The Experiment should be repeated

### Calendar Week 08

### 22.02.2018 Production of GFP/PUG-RNA

Participants: Rick, Susanna, Frederik

Protocol: Transformation

### Calendar Week 09

### 26.02.2018 Seeding of HEK293T cells

Participants: Nicole, Rick, Frederik

Protocol: Seed cells

Oligos/Cells: HEK293T

### 27.02.2018 Transfection of HEK293T cells

Participants: Nicole, Rick, Frederik

Protocol: Transfection with PEI

### 01.03.2018 Harvest of GFP-RNA

Participants: Nicole, Rick, Susanna

Protocol: E.Z.N.A. Total RNA Kit I (Omega)

Result: Just a small amount of RNA was isolated

Conclusion: The experiment should be repeated

### Calendar Week 16

### 17.04.2018 Seed HEK293T cells

Participants: Nicole, Annabel

Protocol: Seed cells

Oligos/Cells: HEK293T

### 18.04.2018 Transfection of HEK293T cells

Participants: Nicole

Protocol: Transfection with PEI

### 18.04.2018 RTqPCR with GFP-RNA

Participants: Nicole, Annabel, Rick

Protocol: LightCycler® 480 RNA Master Hydrolysis Probes von Roche

### 18.04.2018 SYBRGreen qPCR Norovirus

Participants: Nicole

Protocol: LightCycler® FastStart DNA Master SYBR Green I Kit (Roche))

Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer)

Result: [20180418.pdf](https://static.igem.org/mediawiki/2018/a/aa/T--JMU_Wuerzburg--20180418.pdf)

Conclusion: No result.

### 19.04.2018 Repeat SYBRGreen qPCR Norovirus

Participants: Nicole, Rick

Protocol: LightCycler® FastStart DNA Master SYBR Green I Kit (Roche))

Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer)

Conclusion: No result.

### 20.04.2018 Control of qPCR with 1,8% Agarosegel

Participants: Nicole, Rick

Protocol: Agarosegel

Conclusion: Only marker visible.

### 20.04.2018 Noro PCR with Taq- and Q-Polymerase (Noro) like qPCR from 18.04.2018

Participants: Nicole, Rick

Protocol: PCR with Taq- and Q-Polymerase

Conclusion: We repeated this experiment twice, because we used too much Primer for the first part and did not dilute it 1:10 before use.

### 20.04.2018 1,8% Agarosegel for PCR with Taq- and Q-Polymerase (Noro)

Participants: Nicole, Rick

Protocol: Agarosegel

Conclusion: Taq and Q-Polymerase give a product. For the Taq control we only see Primer and Q control was not visible.

### Calendar Week 17

### 24.04.2018 Repeat Noro qPCR from 18.04.2018

Participants: Nicole

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: template2 (template), Noros2 (sense Primer), Noroa2 (antisense Primer)

Result: [20180424\_1.pdf](https://static.igem.org/mediawiki/2018/b/b2/T--JMU_Wuerzburg--20180424_1.pdf)

Conclusion: No result.

### Calendar Week 18

### 03.05.2018 Noro RTqPCR

Participants: Nicole, Rick

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: template3, Primer: Noro1s, Noro2.2a, annealing temperature 54°C

Conclusion: Positive results, but the negative water control is also positive.

### 04.05.2018 Repeat Noro RTqPCR from03.05.2018

Participants: Nicole

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: template3, Primer: Noro1s, Noro2.2a, annealing temperature 54°C

Conclusion: Positive results, but the negative water control is also positive.

### Calendar Week 19

### 07.05.2018 Repeat Noro RTqPCR from 03.05.2018 with 57°C annealing temperature

Participants: Nicole, Annabel, Rick

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: template3, Primer: Noro1s, Noro2.2a

Result: [20180507\_1.pdf](https://static.igem.org/mediawiki/2018/f/f1/T--JMU_Wuerzburg--20180507_1.pdf)

Conclusion: Positive results, but the negative water control is also positive.

### 07.05.2018 Repeat Noro RTqPCR from 03.05.2018 with 58°C annealing temperature

Participants: Nicole, Annabel

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: template3, Primer: Noro1s, Noro2.2a

Result: [20180507\_2.pdf](https://static.igem.org/mediawiki/2018/4/40/T--JMU_Wuerzburg--20180507_2.pdf)

Conclusion: Positive results, but the negative water control is also positive.

### 07.05.2018 Repeat Noro RTqPCR from 03.05.2018 with 59°C annealing temperature

Participants: Nicole

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: template3, Primer: Noro1s, Noro2.2a

Result: [20180507\_3.pdf](https://static.igem.org/mediawiki/2018/7/74/T--JMU_Wuerzburg--20180507_3.pdf)

Conclusion: Positive results, but the negative water control is also positive.

### 08.05.2018 Repeat Noro RTqPCR from 03.05.2018 with 62°C annealing temperature

Participants: Nicole, Rick

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: template3, Primer: Noro1s, Noro2.2a

Result: [20180508\_1.pdf](https://static.igem.org/mediawiki/2018/b/b8/T--JMU_Wuerzburg--20180508_1.pdf)

Conclusion: No results.

### 08.05.2018 Plasmodium qPCR from 03.05.2018 with 62°C annealing temperature

Participants: Nicole, Rick, Annabel

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: Plasmodiumtemplate, primer: P1s P1a

Result: [20180508\_2.pdf](https://static.igem.org/mediawiki/2018/e/ec/T--JMU_Wuerzburg--20180508_2.pdf)

Conclusion: Positive results, but the negative water control is also positive.

### Calendar Week 20

### 14.05.2018 Noro RTqPCR with Probe (FAM)

Participants: Nicole, Rick, Annabel

Protocol: Quanti Nova Probe PCR Kit (Quiagen)

Oligos/Cells: template3, Primer: Noro1s, Noro2.2a, annealing temperature 59° C

Result: [20180514.pdf](https://static.igem.org/mediawiki/2018/0/0d/T--JMU_Wuerzburg--20180514.pdf)

Conclusion: Positive results, but the negative water control is also positive. Maybe contaminated primers.

### 15.05.2018 Plasmodium gradient qPCR (annealing temperature 60.6, 63, 65, 66 °C)

Participants: Nicole, Rick

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: Plasmodium template, primer: P1s P1a

Result: [20180515.pdf](https://static.igem.org/mediawiki/2018/f/f3/T--JMU_Wuerzburg--20180515.pdf)

Conclusion: Positive results, the negative water control is also positive, but with a clear difference between the cq value of water and template. Cover of 63 °C and 65 °C were open, so these results are wrong.

### 16.05.2018 Plasmodium gradient qPCR (annealing temperature 60.6, 63 °C)

Participants: Nicole, Annabel

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: Plasmodiumtemplate, primer: P1s P1a

Result: [20180516.pdf](https://static.igem.org/mediawiki/2018/8/82/T--JMU_Wuerzburg--20180516.pdf)

Conclusion: 63 °C temperature too high, 60,6 °C positive result, H2O negative.

### 18.05.2018 Noro probe gradient RTqPCR (annealing 58.2, 59.3, 60.4, 61.3 °C)

Participants: Nicole, Rick

Protocol: Quanti Nova Probe PCR Kit (Quiagen)

Oligos/Cells: template3, Primer: Noro1s, Noro2.2a

Result: [20180518.pdf](https://static.igem.org/mediawiki/2018/5/5d/T--JMU_Wuerzburg--20180518.pdf)

Conclusion: 58 °C best temperature, because of the highest cq difference between template and H2O (5,35).

### Calendar Week 22

### 01.06.2018 Noro cDNA Synthese, PCR, Agarosegel

Participants: Nicole

Protocol: cDNA Synthese with M-MulV RT Quick Protocol from NEB, PCR with Taq, 1,8 % Agarosegel

Conclusion: no result.

### 01.06.2018 Noro Probe RTqPCR

Participants: Nicole, Rick

Protocol: LightCycler® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 0,4 µM, 0,2 µM, 0,1 µM, template 10^4 copies per PCR reaction

Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58°C

Result: [20180601.pdf](https://static.igem.org/mediawiki/2018/e/eb/T--JMU_Wuerzburg--20180601.pdf)

Conclusion: No results, we have to use the enhancer and activator.

### Calendar Week 23

### 04.06.2018 Noro RTqPCR

Participants: Nicole

Protocol: LightCycler® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 0,4 µM, 0,2 µM, 0,1 µM, template 10^4 copies per PCR reaction

Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C

Result: [20180604.pdf](https://static.igem.org/mediawiki/2018/8/8f/T--JMU_Wuerzburg--20180604.pdf)

Conclusion: 0,4 µM primer has got the best cq-difference. In the next PCR we will test from 1 mM primer.

### 04.06.2018 Noro RTqPCR

Participants: Nicole, Annabel, Rick

Protocol: LightCycler® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 1 µM, 0,5 µM, 0,25 µM, 0,125 µM, template 10^4 copies per PCR reaction

Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C

### 08.06.2018 Repeat Noro RTqPCR

Participants: Nicole, Rick, Annabel

Protocol: LightCycler® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 1 µM, 0,8 µM, 0,6 µM, 0,4 µM template 10^4 copies per PCR reaction

Oligos/Cells: template3, Primer: Noro1sneu, Noro2.2aneu, anneling temperature 58 °C

Result: : [20180608.pdf](https://static.igem.org/mediawiki/2018/1/1c/T--JMU_Wuerzburg--20180608.pdf)

Conclusion: 0,5 µM primer has got the best cq difference.

### Calendar Week 24

### 11.06.2018 Plasmodium qPCR with genomic DNA cultured Plasmodium

Participants: Nicole, Rick, Annabel

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: Plasmodiumtemplate, primer: P1sneu P1aneu, annealing temperature 61 °C

Result: [20180611.pdf](https://static.igem.org/mediawiki/2018/6/61/T--JMU_Wuerzburg--20180611.pdf)

Conclusion: Positive results, the negative water control is also positive, but with a clear difference between the cq value. We can detect the genomic DNA of cultured Plasmodium very well.

### 15.06.2018 Plasmodium standard curve

Participants: Nicole, Rick, Annabel

Protocol: RTqPCR mit Lightcycler EvoScript RNA SYBR Green I Master Kit (Roche)

Oligos/Cells: Plasmodiumtemplate, primer: P1sneu P1aneu, annealing temperature 61 °C, 100000, 20000, 4000, 800, 160, 32 particles each approach and DNA of cultured Plasmodium

Result: [20180615.pdf](https://static.igem.org/mediawiki/2018/2/2e/T--JMU_Wuerzburg--20180615.pdf)

Conclusion: Positive results, the negative water control is also positive, but with a clear difference between the cq value. We can detect the genomic DNA of cultured Plasmodium very well. There were some pipetting mistakes.

### Calendar Week 39

### 28.09.2018 gradient qPCR Plasmodium falciparum

Participants: Nicole, Rick

Protocol: LightCycler® EvoScript RNA SYBR® Green I Master (Roche), primer concentration 0,4 µM template 10^5copies per PCR reaction, annealing temperature 58,5°C, 61°C, 63,7°C

Oligos/Cells: templatePf, Primer: Pf1s, Pf1a

Result: [20180928.pdf](https://static.igem.org/mediawiki/2018/b/be/T--JMU_Wuerzburg--20180928.pdf)

Conclusion: Positive results, but H2O control is near the template cq for 58,5°C. For the higher temperature we did not got a result.

### 28.09.2018 Biobrick method 1

Participants: Nicole, Rick

Protocol: PCR with taq and Q5 polymerase, annealing temperature 59°C; double approach, one approach digested, the other approach purified with the innuPREP Gel Extraction Kit (Analytic Jena); digest with EcoRI-HF and PSTI-HF

Oligos/Cells: templateP, primer: b1a, b1s

### 30.09.2018 Biobrick method 1

Participants: Nicole, Rick

Protocol: purified with the innuPREP Gel Extraction Kit (Analytic Jena); measure DNA concentration at nano drop; ligation with T4 ligase

Oligos/Cells: templateb, primer: b1a, b1s

Result: nanodrop: vector: 19,8 ng/µl, taq-1: 8,9 ng/µl, taq-2: 9,9 ng/µl, Q5-1: 14,3 ng/µl, Q5-2: 7,2 ng/µl

### 30.09.2018 gradient qPCR Plasmodium falciparum

Participants: Nicole, Rick

Protocol: LightCycler® EvoScript RNA SYBR® Green I Master (Roche), primer concentration 0,4 µM template 10^5copies per PCR reaction, annealing temperature 57,3°C, 59,3°C, 61°C

Oligos/Cells: templatePf, Primer: Pf1s, Pf1a

Result: [20180930.pdf](https://static.igem.org/mediawiki/2018/a/a3/T--JMU_Wuerzburg--20180930.pdf)

Conclusion: Positive results, but H2O control is near the template cq for 57,3°C. For the higher temperature we did not got a result.

### Calendar Week 40

### 01.10.2018 Biobrick method 1

Participants: Nicole, Rick

Protocol: transformation

Oligos/Cells: E. coli DH5α

Result: no colonies

Conclusion: The bacteria were not competent, but maybe the digest or ligation were also false.

### 01.10.2018 qPCR probe (FAM) Plasmodium

Participants: Nicole, Rick

Protocol: LightCycler® 480 RNA Master Hydrolysis Probes (Roche), primer concentration 0,4 µM, probe concentration 0,2 µM, template 10^6, 10^2 copies per PCR reaction, annealing temperature 61 °C

Oligos/Cells: templateP, primer: P1s, P1a

### 01.10.2018 gradient qPCR SyBr Green P. falciparum and Plasmodium

Participants: Nicole, Rick

Protocol: LightCycler® EvoScript RNA SYBR® Green I Master (Roche), primer concentration 0,4 µM, , template 10^5 copies per PCR reaction, annealing temperature 56 °C, 56,7 °C, 58 °C, 61 °C

Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf1s, Pf1a

Result: [20181001.pdf](https://static.igem.org/mediawiki/2018/a/aa/T--JMU_Wuerzburg--20181001.pdf)

Conclusion: Kit is ok, positive results for Plasmodium, for P. falciparum the H2O control is also positive.

### 02.10.2018 Biobrick repeat 1 of method 1

Participants: Nicole, Rick

Protocol: PCR with Taq polymerase, annealing temperature 77 °C; purified with the innuPREP Gel Extraction Kit (Analytic Jena), before take 5µl for a control on a agarose gel; 2 % agarose gel

Oligos/Cells: template (10^4 copies each approach), templateb (20 ng), primer: b1a, b1s

Result: [20181002\_gel.pdf](https://static.igem.org/mediawiki/2018/0/06/T--JMU_Wuerzburg--20181002_gel.pdf)

Conclusion: annealing temperature too high. No band for the templateP, and a little band for template. It must be only the template amount from the beginning.

### 02.10.2018 gradient qPCR probe (FAM) Plasmodium

Participants: Nicole, Rick

Protocol: One Step RT qPCR Probe ROX L Kit (highQu), Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 57 °C, 58,6 °C, 61 °C, 63 °C

Oligos/Cells: templateP, primer: P1s, P1a

Result: [20181002.pdf](https://static.igem.org/mediawiki/2018/c/cf/T--JMU_Wuerzburg--20181002.pdf)

Conclusion: Kits from Biozym and highQu have got the same efficiency. The temperature is also good from 57 °C to 63 °C.

### 02.10.2018 Biobrick method 2

Participants: Luisa

Protocol: NEB® PCR Cloning Kit

Result: colonies on the plate

### 03.10.2018 Biobrick method 2

Participants: Nicole, Rick

Protocol: 6 Minis inoculate

### 03.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick

Protocol: PCR with taq polymerase, annealing temperature 64°C; purified with the innuPREP Gel Extraction Kit (Analytic Jena), before take 5µl for a control on a agarose gel; 2% agarose gel; digest with EcoRI-HF and PSTI-HF

Oligos/Cells: templateb (20 ng), primer: b1a, b1s

Result: [20181003\_gel.pdf](https://static.igem.org/mediawiki/2018/b/b1/T--JMU_Wuerzburg--20181003_gel.pdf)

Conclusion: band visible

### 04.10.2018 Biobrick method 2

Participants: Nicole, Rick

Protocol: 6 Minis inoculate

Result: Minis did not grow. Again 6 Minis inoculate. 05.10.2018 Also did not grow. Repeat NEB® PCR Cloning Kit

### 04.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick

Protocol: purified with the innuPREP Gel Extraction Kit (Analytic Jena); measure with the nanodrop; ligation with T4 ligase, transformation

Oligos/Cells: E. coli DH5α, two different approaches

Result: nanodrop: 1: 19,2 ng/µl, 2: 15,4 ng/µl

### 04.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick

Protocol: 6 Minis inoculate; 3 idis from positive control BBa\_J04450 in pSB1C3 inoculte

Oligos/Cells: E. coli DH5α, two different approaches

### 05.10.2018 Repeat Biobrick method 2

Participants: Luisa

Protocol: NEB® PCR Cloning Kit

Result: 03.10.2018 colonies on the plate

### 05.10.2018 qPCR SyBr Green P. falciparum with tree different primers

Participants: Nicole, Rick

Protocol: LightCycler® EvoScript RNA SYBR® Green I Master (Roche), primer concentration 0,4 µM template 10^5copies per PCR reaction, annealing temperature 58 °C

Oligos/Cells: templatePf, Primer: Pf1sneu, Pf1aneu, Pf2s, Pf2a, Pf3s, Pf3a

Result: [20181005.pdf](https://static.igem.org/mediawiki/2018/9/91/T--JMU_Wuerzburg--20181005.pdf)

Conclusion: Negative results for primer 1, positive results for primer 2, High cq for primer 3.

### 06.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick

Protocol: innuPREP Plasmid Mini Kit; nanodrop; test digest, colony PCR, 1,7 % agarose gel

Result: nanodrop: 1: 44,9 ng/ µl, 2: 47 ng/µl, 3: 51,7 ng/µl, 4: 33 ng/µl, 5: 28,2 ng/µl, 6: 55,4 ng/µl [20181006\_gel.pdf](src/files/20181006_gel.pdf)

### 06.10.2018 Repeat Biobrick method 2

Participants: Nicole, Rick

Protocol: GenElute Plasmid DNA Midiprep Kit for the vector, nanodrop, digest with PSTI-HF, inoculate 7 Minis from insert

### 06.10.2018 gradient qPCR SyBr Green P. falciparum

Participants: Nicole, Rick

Protocol: LightCycler® 480 SYBR Green I Master (Roche), primer concentration 0,4 µM template 10^5, 10^2 copies per PCR reaction, annealing temperature 57 °C, 59,1 °C, 61 °C

Oligos/Cells: templatePf, Primer: Pf2s, Pf2a

Result: [20181006.pdf](https://static.igem.org/mediawiki/2018/6/62/T--JMU_Wuerzburg--20181006.pdf)

Conclusion: All temperatures have nearly the same cq.

### 07.10.2018 Repeat Biobrick method 2

Participants: Nicole, Rick

Protocol: 1,5 % agarose gel

Result: [20181007\_gel1.pdf](https://static.igem.org/mediawiki/2018/8/83/T--JMU_Wuerzburg--20181007_gel1.pdf)

### 07.10.2018 qPCR probe Plasmodium

Participants: Nicole, Rick

Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61 °C

Oligos/Cells: templateP, primer: P1s, P1a

Result: [20181007.pdf](https://static.igem.org/mediawiki/2018/5/56/T--JMU_Wuerzburg--20181007.pdf)

Conclusion: We can detect all six samples of the biobrick plasmid.

### 07.10.2018 Repeat Biobrick method 2

Participants: Nicole, Rick

Protocol: innuPREP Plasmid Mini Kit of the insert, nanodrop, digest insert, digest vector with EcoRI-HF later PSTI-HF and PSTI-HF and later EcoRI-HF; 1,5 % agarose gel

Result: nanodrop: 1: 49,1 ng/µl, 2: 8,4 ng/µl [20181007\_gel2.pdf](https://static.igem.org/mediawiki/2018/4/42/T--JMU_Wuerzburg--20181007_gel2.pdf)

### 07.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick

Protocol: sequencing, inoculate Minis

### Calendar Week 41

### 08.10.2018 Biobrick repeat 2 of method 1

Participants: Nicole, Rick

Protocol: innuPREP Plasmid Mini Kit, nanodrop

Result: nanodrop: 1: 76,9 ng/µl, 2: 53,0 ng/µl, 3: 61,2 ng/µl, 4: 51,2 ng/µl, 5: 54,1 ng/µl, 6: 40,2 ng/µl

### 08.10.2018 Repeat Biobrick method 2

Participants: Nicole, Rick

Protocol: ligation with T4 ligase

Result: nanodrop: 1: 49,1 ng/µl, 2: 8,4 ng/µl

### 10.10.2018 qPCR probe Plasmodium

Participants: Nicole, Rick

Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61 °C, template 10^5, 1\*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction

Oligos/Cells: templateP, primer: P1s, P1a

Result: [20181010\_1.pdf](https://static.igem.org/mediawiki/2018/f/f1/T--JMU_Wuerzburg--20181010_1.pdf)

Conclusion: We detect all our five stems of cultured Plasmodia.

### 10.10.2018 qPCR SyBr Green P. falciparum

Participants: Nicole, Rick

Protocol: LightCycler® 480 SYBR Green I Master (Roche), primer concentration 0,4 µM template 10^5, 1\*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction, annealing temperature 61°C

Oligos/Cells: templatePf, Primer: Pf2s, Pf2a

Result: [20181010\_2.pdf](https://static.igem.org/mediawiki/2018/4/4f/T--JMU_Wuerzburg--20181010_2.pdf)

### 10.10.2018 qPCR probe Plasmodium

Participants: Nicole, Rick

Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61°C, template 10^5, 1\*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction

Oligos/Cells: templateP, primer: P1s, P1a

Result: [20181010\_3.pdf](https://static.igem.org/mediawiki/2018/5/53/T--JMU_Wuerzburg--20181010_3.pdf)

Conclusion: We detect all our five stems of cultured Plasmodia.

### 12.10.2018 qPCR probe (FAM) Plasmodium and probe (HEX) P. falciparum

Participants: Nicole, Rick

Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61°C, template 10^5, 1\*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction

Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf2a, Pf2s

Result: [20181012.pdf](https://static.igem.org/mediawiki/2018/a/a6/T--JMU_Wuerzburg--20181012.pdf)

Conclusion: Both probes have good results. Multiplexing must be also possible with our two primer/probe pairs.

### 13.10.2018 Multiplex qPCR probe (FAM) Plasmodium and probe (HEX) P. falciparum

Participants: Nicole, Rick

Protocol: Biozym Probe qPCR Kit, primer concentration 0,4 µM, probe concentration 0,2 µM, annealing temperature 61°C, template 10^5, 1\*10^4, 4000, 800, 160, 32, 6,4 copies per PCR reaction

Oligos/Cells: templateP, primer: P1s, P1a; templatePf, primer Pf2a, Pf2s

Result: [20181013.pdf](https://static.igem.org/mediawiki/2018/3/3c/T--JMU_Wuerzburg--20181013.pdf)

Conclusion: Multiplexing is also possible with our two primer/probe pairs. We can detect the cultured Plasmodia DNA.


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:JMU Wuerzburg/Experiments

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  Judging Form

### Dry Lab

The general *Plasmodium* primer-probe pair was created as described below.

To distinguish suitable sequences for primer/probe design we created a
FASTA file with all available mitochondrial nucleotide sequences of
Plasmodium falciparum, P. malariae, P. knowlesi, P. ovale and P. vivax from the NCBI database[5](#drylab_refs).
These 2080 Sequences were aligned with ClustalO[1](#drylab_refs). For this step we used ClustalO,
running on a computing cluster and the following bash command:

`nohup clustalo --outfmt=msf -o outputname.msf -i inputname.fasta & > nohup.log`

Afterwards, we created a frequency matrix from the resulting MSF file. The frequency matrix was created with Emboss Prophecy
[2](#drylab_refs). We used the following parameters:

`EMBOSS data file: EDNAFULL`

`Threshold reporting percentage: 75`

`Gap opening penalty: 3.0`

`Gap extension penalty: 0.3`

Subsequently, we calculated a new matrix based on the frequency matrix. Table 1 shows how we calculated this matrix.

![fehlt](https://static.igem.org/mediawiki/2018/d/d6/T--JMU_Wuerzburg--project-experiments_drylabmatrix.png)
Table 1:  We calculated a matrix
with eight columns and as many rows as positions in the frequency matrix. In the columns,
B - F are the nucleotides Adenin (A), Cytosin (C), Guanin (G) and Thymin (T).
The first row and column are only shown for a better orientation in the matrix.

In this matrix, we searched for positions with a high value in column I
(near or equal 1) and a nucleotide summation as high as possible.
The sequence of Plasmodium is often Thymin/Adenin rich,
so we had also to look for a high Guanin/Cytosin-content.
If a suitable sequence is long enough for a primer and probe design,
we designed primers and a probe with OligoArchitectTM[3](#drylab_refs).
Afterwards, we calculated the secondary structure of the primers and the probe with RNAstructure[4](#drylab_refs).
Therefore we used the following parameters:

`Temperature [K]: 310.15`

`Maximum Loop Size: 30`

`Maximum % Energy Difference (MFE, MEA): 10`

`Maximum Number of Structures (MFE, MEA): 20`

`Window Size (MFE, MEA): 3`

`Gamma (MEA): 1`

`Iterations (Pseudoknot Prediction): 1`

`Minimum Helix Length (Pseudoknot Prediction): 3`

If the calculated secondary structure is moderate or low, we started testing the primers and the probe in the wet lab.

Our primer/probe pair for *P. falciparum* was designed as written below.
We created five alignments with ClustalO[1](#drylab_refs).
One alignment is for P. falciparum with 1012
mitochondrial sequences of *P. falciparum* from NCBI
and four alignments are for  *P. malariae, P. knowlesi,
P. ovale* and *P. vivax*[5](#drylab_refs).
These four alignments were calculated with mitochondrial nucleotide
sequences from NCBI of each *Plasmodium* species[5](#drylab_refs).
The alignments were calculated with ClustalO with the following bash command:

 `nohup clustalo --outfmt=msf -o outputname.msf -i inputname.fasta & > nohup.log`

We used the resulting consensus sequences and aligned these
five sequences with ClustalO.
Subsequently, we searched in the resulting MSF
file after differences between the consensus sequence of
*P. falciparum* and the other four consensus sequences.
Afterwards, we created a frequency matrix as described
in the part for *Plasmodium* in general and checked if our regions
of interest are highly conserved as described in the part of *Plasmodium* in general.
The primer and probe design were also carried out as described
in the upper part for *Plasmodium*.

Furthermore, we aligned over 40 000 nucleotide sequences from
NCBI[5](#drylab_refs), DDBJ[6](#drylab_refs)
and Embl[7](#drylab_refs) of Norovirus with ClustalO.
We searched in the resulting alignment for highly conserved
regions, as described in the part for Plasmodium in general
and designed a primer/probe pair for Norovirus.

---

##### List of References

1 Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7:539 doi:10.1038/msb.2011.75

2 Rice P., Longden I. and Bleasby A. EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics. 2000 16(6):276-277

3 <https://www.sigmaaldrich.com/technical-documents/articles/biology/oligoarchitect-online.html>

4 J.S. Reuter and D.H. Mathews. "RNAstructure: software for RNA secondary structure prediction and analysis." BMC Bioinformatics, 11:129. (2010).

5 <https://www.ncbi.nlm.nih.gov/>

6 <https://www.ddbj.nig.ac.jp/index-e.html>

7 <https://www.ebi.ac.uk/ena>

---

### Hardware

Having a qPCR test system is fine. But it is only useful, if you have the specific hardware device for it. Our cycler is constructed very simply: a drawer for the test itself has two clamps to fixate the tube, so it won’t move. Underneath the drawer-slot are the operators for our test system. These operators are heat-elements, thermo sensors and relapsing magnets. To get the blood from one chamber of the tube to the next, we use a pump-lever. This pump-lever is montaged above the test-tube and runs by a small stepper-motor. The lever drives on a gear shaft that rotates by the stepper motor. At the end of this lever there is a roller, which pulls the liquid forward. At the end of the device there are LED-diodes and photocells wired to an ARDUINO computer. The diodes radiate light with 495 nm wavelength at the fluorescent probe. The photocells measure the emitted light from the probe and give the data to the ARDUINO computer. This data makes it possible to derive the presence of *Plasmodium* types.

![fehlt](https://static.igem.org/mediawiki/2018/6/6d/T--JMU_Wuerzburg--project-experiments-hardware.png)
 Scheme of our hardware device.

---

### Oligonucleotides

We designed the following oligonucleotieds as either Primer, Probe or our BioBrick:

![fehlt](https://static.igem.org/mediawiki/2018/1/1c/T--JMU_Wuerzburg--project-experiments-oligonu.png)

---

### Protocols

We used the following kits and protocols to conduct our experiments:

#### (RT)qPCR Probe-Kis

*Biozym Probe qPCR Kit* (Biozym)[1](#experiment_refs)

*One Step RT qPCR Probe ROX L Kit* (highQu)[2](#experiment_refs)

*Quanti Nova Probe PCR Kit* (Quiagen)[3](#experiment_refs)

*LightCycler ® 480 RNA Master Hydrolysis Probes* (Roche)[4](#experiment_refs)

#### (RT)qPCR Sybr Green Kits

*LightCycler® FastStart DNA Master SYBR Green I* (Roche)[5](#experiment_refs)

*LightCycler® EvoScript RNA SYBR® Green I Master* (Roche)[6](#experiment_refs)

*LightCycler® 480 SYBR Green I Master* (Roche)[7](#experiment_refs)

#### DNA/Plasmid Extaction Kit

*innuPREP Plasmid Mini Kit* (Analytic Jena)[8](#experiment_refs)

*innuPREP Gel Extraction Kit* (Analytic Jena)[9](#experiment_refs)

*GenElute Plasmid DNA Midiprep Kit* (Analytic Jena)[10](#experiment_refs)

E.Z.N.A. Total RNA Kit I (Omega)[11](#experiment_refs)

#### Agarose gel

1,8 % (0,9 g Agarose, 50 ml TAE buffer), 2 µl Ethidiumbromid
Voltage: 100 V

#### *NEB® PCR Cloning Kit* (NEB)[12](#experiment_refs)

#### cDNA Synthese with M-MuLV RT Quick Protocol (NEB)[13](#experiment_refs)

Template RNA 4 µl, Primer 1 µl, 10x M-MulV buffer 2 µl, M-MulVRT 1 µl, 10 mM dNTP 1 µl, H2O 11 µl
1h incubation at 42 °C

![fehlt](https://static.igem.org/mediawiki/2018/f/f4/T--JMU_Wuerzburg--project-experiments_protocols.png)

#### Digest with EcoRI-HF and PSTI-HF[14](#experiment_refs) [15](#experiment_refs)

DNA 1 µg, CutSmart 10 x buffer 10 µl, EcoRI-HF 2 µl, PSTI-HF 2 µl, H2O to 100 µl

#### Ligation with T4 DNA ligase[16](#experiment_refs)

#### Agarplates with chloramphenicol resistance

10 g tryptone, 5 g yeast extract, 5 g NaCl, 10 g Agar, H2O to 1 l; autoclave; 10 mg/ml Chloramphenicol

#### Transformation (Changed from NEB)[17](#experiment_refs)

Thaw chemical competent cells on ice

Put 15 ng of the ligation mixture to 50 µl of competent cells

Place the mixture 10 minutes on ice.

Heat shock at 42°C for 45 seconds

Add 1 ml LB-Medium

Place tube at 37°C for 60 minutes and shake 300 rpm.

Centrifuge 2 min at 11000 rpm

Take 850 µl supernatant und resuspend the 200 µl.

Spread it onto the plates.

Incubate overnight at 37°C

#### Sequencing[18](#experiment_refs)

1 µl DNA, 1 µl BigDye 1.1, 1 µl Primer (10 µM), 2 µl H2O

```
    Thermocycler program:
    1. denaturation 	96°C 	1min
    2. denaturation 	96°C 	20 s
    3. annealing            50°C	15 s
    4. polymerisation 	60°C 	4 min
    5. cooling 		4°C	∞
```

#### Seed cells

1. Aspirate medium from cell-culture (two 75 cm2)

2. Trypsinize cells with 2 ml ATV

3. Check cell-detachment under microscope

4. Add 2 ml FCS-medium to stop trypsinization

5. Resuspend cells by pipetting up and down

6. Transfer 3 ml to fresh falcon tube

7. Add 9 ml medium to the flask

8. Transfer 10 µl to the Neubauer chamber,

   Count cells in a four to four square (Count 16 mini squares);

   *we counted 182 cells, so we had 1,82 \* 10 ^6 cells per ml;*

   *seed 1,5 \* 10^5 cells per well in a 6-well plate, 1,5 \* 10^5 / 1,82 \* 10^6 = 82,4 µl*

9. Dilute cells;

   *We had 6 ml in a falcon, so we add 6 ml medium*

10. Prepare 2\* 917,5 µl = 1,835 ml of cell-culture medium per well in a 6-well plate

11. Add 165 µl cell suspension to each well

12. Shake 6-well plate (3 pieces)

#### Transfection with PEI

For each well of a 6-well plate

1. 0,3 - 0,5 µg GFP

2. 2,5 - 3,5 µg PUC

3. 100µl DMEM

4. Mix

5. 6 - 9 µl PEI

5. Mix

6. Incubate 15 min at room temperature (RT)

7. Drop briefly at the cells

---

##### List of References

1 <https://www.biozym.com/desktopmodules/webshop/downloads/331455_170220_Biozym-Probe-qPCR-Kit.pdf>

2 <https://www.highqu.com/1step-rt-qpcr-probe-rox-l-kit.html>

3 <https://www.qiagen.com/us/resources/resourcedetail?id=5167d782-9fef-4202-bc79-95f358be7d8c&lang=en>

4 <https://lifescience.roche.com/en_de/products/lightcycler14301-480-rna-master-hydrolysis-probes.html#documents>

5 <https://lifescience.roche.com/en_de/products/lightcycler-faststart-dna-master-sybr-green-i.html#documents>

6 <https://www.lifescience.roche.com/en_de/products/lightcycler-evoscript-rna-sybr-green-i-master.html#documents>

7 <https://biochimie.umontreal.ca/wp-content/uploads/sites/37/2015/11/LC480SYBRMasterguide.pdf>

8 <https://www.analytik-jena.de/fileadmin/content/pdf_life_science/Manual/Manual_innuPREP_Plasmid_Mini_Kit_2.0.pdf>

9 <https://www.analytik-jena.de/fileadmin/content/pdf_life_science/Manual/Manual_innuPREP_Gel_Extraction_Kit.pdf>

10 <https://www.sigmaaldrich.com/catalog/product/sigma/pld35?lang=de&region=DE>

11 <http://omegabiotek.com/store/wp-content/uploads/2013/04/R6834-Total-RNA-Mini-Kit-I-Combo-Online.pdf>

12 <https://international.neb.com/-/media/catalog/datacards-or-manuals/manuale1202.pdf>

13 <https://international.neb.com/protocols/2016/04/26/first-strand-cdna-synthesis-quick-protocol-neb-m0253>

14 <https://international.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions>

15 <https://nebcloner.neb.com/#!/redigest>

16 <https://international.neb.com/Protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202>

17 <https://international.neb.com/protocols/2012/05/21/transformation-protocol>

18 <http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_041330.pdf>

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:JMU Wuerzburg/Results

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  Human Practices

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  + [Education](https://2018.igem.org/Team%3AJMU_Wuerzburg/Human_Practices#education)
  + [Establishing an iGEM-Team](https://2018.igem.org/Team%3AJMU_Wuerzburg/Human_Practices#establishing)
  + [Collaborations for our Laboratory Work](https://2018.igem.org/Team%3AJMU_Wuerzburg/Human_Practices#collabs)
* [![](https://static.igem.org/mediawiki/2018/2/29/T--JMU_Wuerzburg--src-menu_png-judging_form.png)](https://igem.org/2018_Judging_Form?team=JMU_Wuerzburg)

  Judging Form

### Bioinformatics

To detect *Plasmodium* in general we aligned 2080 sequences from NCBI.
Our primers and the probe has a consensus of more than 98% and in one case 94,9%.
The oligos are located in the mitochondria in a regulatory region.

In the case of *Plasmodium falciparum*, we aligned 1012 sequences of
*Plasmodium falciparum* and our primer set and the probe has a higher
consensus than 97,5%. The location of the oligos is in a regulatory region of the mitochondria.

---

### Wet Lab

In general, we can detect *Plasmodium* in all five cultured *Plasmodium* strains of *Plasmodium falciparum*
(FCR3, Mali2K, HB3, Dd2 and 3D7 from Tuebingen and Heidelberg).
In addition, we can detect our Biobrick (BBa\_K2614000)
that includes a synthetic template as a positive control for the
qPCR that detects *Plasmodium* in general (figure 1).
We can detect less than 1000 copies of our synthetic template with a probe (FAM)
and less than 50 copies with SybrGreen.

![fehlt](https://static.igem.org/mediawiki/2018/3/3f/T--JMU_Wuerzburg--project-result_fig1.png)
Figure 1: qPCR with cultured *Plasmodium* stems and our BioBrick as a control for *Plasmodium* general

Moreover, we are able to detect *P. falciparum* in all five cultured *Plasmodium* strains.
Our BioBrick is a negative control for our qPCR that detects *P. falciparum*.
With the probe (HEX) we detect less than ten copies of our synthetic template. (figure 2)

![fehlt](https://static.igem.org/mediawiki/2018/d/db/T--JMU_Wuerzburg--project-result_fig2.png)
Figure 2: qPCR with cultured *Plasmodium stems* and our BioBrick as a control for *P. falciparum*.

In a multiplex system the two probes (FAM and HEX) also
detect the five cultured *Plasmodium* strains.
They detect them with nearly the same cq-mean which means that
both qPCRs can detect *Plasmodium*. With this result we showed that
multiplexing is also possible with the two primer/probe pairs (figure 3).

![fehlt](https://static.igem.org/mediawiki/2018/7/7e/T--JMU_Wuerzburg--project-result_fig3.png)
Figure 3: Multiplex qPCR with cultured *Plasmodium* stems and our BioBrick as a control for *Plasmodium* general and specific for *P. falciparum*

---

### Human practices

We organised different workshops from scientists about
malaria or primer/probe design which were open for
all interested students. There was also a pipetting test where
more experienced students could give their knowledge to pipetting beginners.

At our “cake and science” events we presented our project to the public.

---

### Interlab

We participated successfully at the
*fifth International InterLaboratory Measurement Study*
in synthetic biology. At the iGEM 2018 Vibrigens InterLab Study
from Marburg we also take place.

---

### Collaboration

We were able to establish a very pleasant and close
collaboration with team Munich. They have mentored us with great
care and reliability and answered all our questions.
So, it was much easier to start a new iGEM team in Wuerzburg.
We also received advice from different members of team Tuebingen
who were so kind to exchange some valuable information and experience with us.