iGEM 2024 — Team radboud-university

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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|  |  |  |  |
| --- | --- | --- | --- |
| 2024 | mRNA | LNP's | Cell Line Experiments |
| May | PCR | Ion. Lipids |  |
| June | PCR |  |  |
| July | |  |  |  | | --- | --- | --- | | PCR | Plasmid | IVT | |  |  |
| Aug. | |  |  | | --- | --- | | Purification | IVT | | Yeast mRNA |  |
| Sept. | Purification | |  |  | | --- | --- | | Yeast mRNA | F8 GFP mRNA | | HEK |

# Task

Time: xx:xx xx/xx/xx

## All Participants:

* Scientist 1
* Researcher 2
* Data collector 3

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Sed risus ultricies tristique nulla aliquet enim tortor. Felis eget nunc lobortis mattis aliquam faucibus. Feugiat nisl pretium fusce id velit ut. Ut tortor pretium viverra suspendisse potenti nullam ac tortor vitae. Morbi tristique senectus et netus et malesuada fames ac turpis. Duis at tellus at urna condimentum mattis pellentesque id nibh. Sed velit dignissim sodales ut eu sem. At ultrices mi tempus imperdiet nulla malesuada pellentesque elit. Molestie ac feugiat sed lectus vestibulum mattis ullamcorper velit sed. Dis parturient montes nascetur ridiculus mus mauris. Ornare lectus sit amet est placerat in egestas. Nunc faucibus a pellentesque sit amet. Scelerisque varius morbi enim nunc faucibus a pellentesque sit amet. Neque ornare aenean euismod elementum nisi quis eleifend quam adipiscing.

Name of Notebook Task/Page

01/01/1970 - 21/12/12

* Participant 1
* ...
* Participant n

|  |  |  |
| --- | --- | --- |
| Title 1 **Background:** Hmm.. You're not supposed to be seeing this.. || 1st Jan 1970 Example loaded inParticipants:  * Tim  Protocols:  * Testing protocol  Experimental: Procedure:   1. Put example in 2. See if something works 3. Forget to remove it |
| 2nd Jan 1970 Example getting removedParticipants:  * Tim  Protocols:  * Undoing my mistakes protocol  Experimental: Procedure:   1. Realise you forgot to remove example 2. Remove it 3. Commit, push, merge |

DNA Amplification via PCR

16/05/24 - 04/07/24

* Henriete
* Kate
* Lea
* Maks
* Thijmen
* Vasilis

|  |
| --- |
| RAD101: New pcr with forward primer (T7 terminator) and reverse primer (F8 primer) with gel electrophoresis and its extraction. |
| 16th of May 2024 Participants:  * Henriete * Vasilis  Experimental: (1st part) First PCR:   1. The reverse primer was diluted to 100 uM by adding 235 ul of miliq and made stock solution. 2. 100 ul of the stock solution was diluted to 10 uM by adding 90 ul of Miliq to an epp and added 10 ul of F8. 3. 83.25 ul of MiliQ water was added to 3 epps. 4. 11.25 ul of 10x PCR buffer was added to each of the 3 epps. 5. 11.25 ul of 2 mM dNTPs was added to each of the 3 epps. 6. 2.35 ul of 10 mM forward (T7 terminator) and 2.25 ul of 10 mM reverse primer (F8 primer) was added. 7. 1 ul of F8 DNA template was added into the reaction epp. 8. 1 ul of positive control (PET 28 P450 BM3 No2). 9. 1 ul of Mili Q was added to the negative control epp. 10. 2.25 ul of Pfu polymerase was added to the 3 epps. |
| 24th of May 2024 Participants:  * Henriete * Vasilis  Experimental: (2nd part) gel electrophoresis:   1. We dissolved the stock solution of 10x TBE to 0.5x TBE by adding 50mL to a measuring cylinder and then adding 950mL demi water to a 1L measuring cylinder. 2. We dissolved 0.9g of agarose into 100 mL of 0.5x TBE. 3. The agarose solution was dissolved until clear. 4. We added 5μl of ethidium bromide to the agarose solution. 5. We poured the agarose solution to the casting tray. 6. We prepared our samples by adding 10μL of the positive, negative and reaction. 7. 2 ul of loading dye (6X) was added to the samples for a total volume of 12 ul. 8. We added the gel to the apparatus and filled it with the buffer until submerged. 9. We added 5 ul of DNA ruler in the 1st and 5th well, and then 12ul of positive, reaction and negative control in 2nd, 3rd and 4th well, respectively. 10. We ran the gel at 110 V for 50 min. 11. We inspected the gel |
| 3rd of June 2024 Participants:  * Vasilis * Thijmen  Experimental: (3rd part) DNA extraction from gel:   1. We prepared the reaction sample to run another gel, by adding 20 ul of the 6x dye to the 100 ul sample. 2. We loaded 5 ul of the DNA ladder to the 6 th well, and 2 wells of 40 ul aliquot of the sample to the 7th and 8th wells respectively. We also filled the remaining 20 ul of the sample to the 9th well. 3. We run the gel at 110 V for 1 hour. 4. We cut 4 bands of the factor 8. 5. We weighted the gel with the DNA with a weight of 0.276 (sample 1) and 0.238 g (sample 2). 6. We added 828 ul of Buffer QG and 714 ul to the 2 Epps respectively. 7. The gel and the buffer was vortexed until dissolved. 8. 1 volume of isopropanol was added to the mixture in both epps. 9. The mixtures we added to spin columns and centrifuged for 1 min. 10. The flow-through was discarded and more mixture was added. 11. This was repeated until all of the mixture was centrifuged. 12. 750 ul of buffer PE was added to the column and was centrifuged. 13. The DNA was eluted with 50 ul of mili q water.  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad101-1.webp) Figure 1. Gel, 24/05/24  We can see little smears and some nice bands, it’s not perfect but based on these results we decided to extract the DNA out of the gel and amplify it in order to get a lot of ‘pure’ factor 8 DNA. ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad101-2.webp) Figure 2. 1st DNA ladder, 2nd negative control, 3rd positive, 4th PCR reaction with new primers (RAD102), 5th eluted DNA, 6st another DNA ladder. 03/06/24  The purified DNA gives a clear band at the expected height, but the concentration is too low to continue with it. In addition, the controls don’t look right: the negative control shows a smear while the positive control does not show anything.  **Conclusion:** New attempts of DNA amplification are needed, new set of primers should be used and the results should be compared. |
| RAD102: New pcr with T7 promoter and T7 terminator and gel electrophoresis with eluted and the new DNA samples. **Background:** New attempt of the PCR reaction with old and new set of primers |
| 3rd of June 2024 Participants:  * Henriete * Maks * Vasilis  Experimental: PCR:   1. The forward primer was dissolved. 2. 83.25 ul of miliQ water was added to all samples 3. The current samples are 1 positive, 2 negative, 4 reactions (2 were made using the first f8 template and 2 using the eluted DNA from the gel). Both primers were used this time. 4. 11.25 ul of 10 x PCR buffer was added to all samples. 5. 11.25 ul of dNTPs was added to all. 6. 2.25 ul of positive control (T7 promoter and T7 terminator) was added. 7. For the negative control (1) T7 promoter and F8 negative primer was added and for negative control (2) f8 reverse and forward. 8. For reaction samples for P1 (first set of primers) T7 promoter and F8 reverse were added. 9. For reaction samples for P2 (second set of primers ) F8 reverse and forward were added. 10. 1 ul of PET 28 to the positive control was added. 11. 1ul of Miliq were added to the negative (1) and negative (2) was added. 12. 1ul of eluted F8 into the P1 and P2 was added. 13. 1ul of pcDNA F8 into P1 and P2 was added.   Gel Electrophoresis:   1. The stock solution of 10x TBE was dissolved to 0.5x TBE by adding 50ml of 10x TBE to 950 ml demi water. 2. 0.7 g of agarose were dissolved in 100ml of 0.5x TBE. 3. The agarose solution was dissolved until clear. 4. 5 ul of ethidium bromide was added to the agarose solution. 5. The solution was poured into the tray. 6. The samples were prepared by adding 10ul of the negative, positive, reaction and the eluted DNA and 2ul of 6x loading dye (total volume 12ul). 7. The gel slots were filled like this: 1st DNA ladder, 2nd negative control, 3rd positive, 4th reaction, 5th eluted DNA, 6th another DNA ladder. 8. Note: the tray was filled after we added the first 2 samples. 9. (Discussion: 10. Question - These samples are the 1st DNA ladder and negative sample? Or the negative sample and positive sample? 11. Answer: I believe 1st DNA ladder and negative sample, not entirely sure. We forgot to fill up the tray before adding the ladder/sample to the gel.) 12. The gel was run for 50 min at 110 V. 13. The gel was inspected. 14. To the positive and negative control we added the new primer. |
| 6th of June 2024 Participants:  * Maks * Thijmen  Experimental: Electrophoresis:   * 2uL loading dye and 10uL sample (vortexed) were added to a new clean tube. Each sample was briefly vortexed and added to the gel in the following order: ladder, p2 eluted, p1 eluted, positive control, p1 cdna, p2 negative control, p2 cdna, p1 negative and at last again a ladder. * The gel was run at 110 volts, 50 min  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad102-1.webp) Figure: 1st DNA ladder, 2nd negative control, 3rd positive, 4th reaction, 5th eluted DNA (RAD101), 6st another DNA ladder. 03/06/24  There was no band in the positive control as not the correct primer was used. The negative control had a smear. The reaction had a smear and a small band. The eluted DNA was not amplified, thus there was a small band as expected. ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad102-2.webp) Figure 2. Left to right: 1st DNA ladder, p2 eluted, p1 eluted, positive control, p1 cdna, p2 negative control, p2 cdna, p1 negative and at last again a ladder. 06/06/2024  Again very big smears, molecular ladder was not good visible, only good well was the positive control. For some unknown reason, these samples have not run far on the gel, so the results are difficult to interpret. Advice: Column purifies sample 4 and 5. Another suggestion is to restart and make all samples again. Samples 4 and 5 are the 2 samples with good bands on the right site of the gel (you can also count the slots/bands from left to right and then you will see that those are the 4th and 5th). Conclusion: We decided to remove the smeared DNA by the means of purification in the next experiment, RAD103. The insight after the purification will provide understanding of such behavior of the DNA. |
| RAD103: PCR purification of the DNA product from experiment RAD102, and its analysis. **Background:** The smeared DNA from the experiment RAD102 was decided to be removed by the means of purification in the next experiment. The insight after the purification will provide understanding of such behavior of the DNA. |
| 10th of June 2024 Participants:  * Henriete * Thijmen * Vasilis  Experimental: Purification of PCR product:   1. For the PCR purification, the protocol QIA PCR purification was followed ( “QIAquick® PCR Purification Kit”) 2. For the QI quick purification samples were noted as + con (positive control), P1 cDNA (Plasmid DNA from the frst primers) and P2 -con (Negative control using second set of primers). P2 - con was used since there was a band. 3. 60 microliters of the negative control was pippeted into the QIAquick column after this 300 microliter of PB buffer was added. For the P1 cDNA and positive control samples 90 microliters was pippeted into the QIAquick collumn and after this 450 microliters of PB buffer was added. 4. Next the samples were centrifuged for 60 seconds on 13.000 RPM. 5. The flow-through was discarded. 6. 750 uL PE buffer was added and the samples were centrifuged for 60 seconds at 13000 RPM. 7. The flow-through was discarded and the samples were centrifuged again for 60 seconds. 8. the QIAquick tubes were put into 1.5 ml epps. 9. Next, there was 50 microliters of miliQ water added to the QIAquick membrane for each sample. 10. Samples are centrifuged again for 1 minute.  Used protocol:![Protocol](https://static.igem.wiki/teams/5342/images/notebook/rad-103-1.webp) PCR of purified PCR product:  3 samples were prepared: positive control (not the purified PCR ‘+’ control) and 2 samples of plasmid cDNA from the purified PCR product (see, 10/06/2024 purification of PCR product).   1. PCR mix was prepared (per sample):    * 83.25 μL MilliQ water    * 11.25 μL 10x PCR buffer    * 11.25 μL 2 mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP)    * 2.25 μL 10 μM forward primer (T7 promoter sequence)    * 2.25 μL 10 μM reverse primer react (1st reverse primer prepared) 2. 1 uL of the PCR purified sample DNA were added: PET 28 as positive control and plasmid cDNA for reaction vials. 3. 2.25 Pfu polymerase was added per sample. 4. the thermocycler was set 51 degrees for 20 cycles. 5. the samples were collected the next day (11.06) at 15:10.   Making of the gel & electrophoresis:  Agarose gel:   1. The stock solution 10x TBE was dissolved to 0.5x TBE by adding 50ml of 10x TBE to 950 ml demi water. 2. 0.7 g of agarose were dissolved in 100ml of 0.5 xTBE. 3. The agarose solution was dissolved until clear. 4. 5 ul of ethidium bromide was added to the agarose solution. 5. The solution was poured into the tray. 6. The samples were prepared by adding 1ul of loading dye to 5 ul of the 3 samples which are positive control, p2 negative control and P1 cDNA. P2 negative control contained DNA band at approximately 7.5 kB and that’s why it was used for purification. 7. For negative control, 5 ul of milliQ was added with 1 ul loading dye. 8. Samples were pipetted in the following way: 1st slot: 5ul DNA ladder, 2nd: 5 ul of p2 negative control (containing DNA), 3rd: 6ul of positive control, 4th: 6 ul p1 cDNA, 5th: 6ul of negative control (5ul of MiliQ +1ul loading dye). 9. Note: The first 2 slots were punctured. 10. The gel was run at 110V for 50 min. 11. The gel was inspected. |
| 28th of June 2024 Participants:  * Thijmen * Vasilis  Experimental: Gel electrophoresis of 2 cdna:   1. The stock solution of 10x TBE was dissolved to 0.5x TBE by adding 50ml of 10x TBE to 950 ml demi water. 2. 0.7 g of agarose were dissolved in 100ml of 0.5 xTBE . 3. The agarose solution was dissolved until clear. 4. 5 ul of ethidium bromide was added to the agarose solution. 5. The solution was poured into the tray. 6. The gene ruler 1kb was used and it was prepared by mixing 100 ul of gene ruler 1kb (25-10000) with 100 ul 6x tritrack loading dye and 400 ul of miliq. 7. The samples were prepared by adding 1ul of loading dye to 5 ul of the 3 samples which are positive control, p1 cDNA and p2 CDNA( it should have no elongation) and a negative control. 8. The gel was filled the following way: 1st was punctured with ladder 5ul DNA ladder, 2nd 5ul of DNA ladder, 3rd 6ul positive control, 4th 6ul p1 cDNA, 5th 6ul p2 CDNA, 6th 6ul negative control and 7th 5ul DNA ladder. 9. The gel was run at 110V for 50 min. 10. The gel was inspected.   NOTE: This note describes how the gel is made on this day. On the same day when the gel has been made the PCR products of RAD103 (5th part) have been loaded onto the gel. Later the PCR product of RAD104 (1st part) has been loaded on the same gel. Results of that can be seen in RAD104 (3rd part). Results and Discussion:![Figure 3](https://static.igem.wiki/teams/5342/images/notebook/rad-103-2.webp) Figure 3. 28/06/24  In the picture above we can see that our results still have smears which is not what we want, the next time we will perform PCR and look at the product on a gel we will use new primers in the hope we can fix the issue with the smears. Nothing has happened which could potentially influence the results.  Indication of 4 slots (from left to right): gene ladder, negative control P2, positive control, cDNA with primers P1. The 4th slot contains a very slight band indicating the amount of amplified DNA was very scarce. if the picture is zoomed in, a slight band at around 7.5 kB can be seen. Conclusion: DNA amplification was unsuccessful as no clear band is visible in the expected range (ca 8000 bp). The smears likely indicate bonding between the primers and the formation of large structures of various sizes. Another primer combination should be tried. |
| RAD104: Analysis of FVIII from RAD103 and GFP DNA PCR & analysis **Background:** We made combinations of the different Factor 8 primers using both old and new primers and we also wanted to perform PCR on GFP DNA by using the standard GFP primers. So there are no new primers for the GFP but only the ones that are always used. |
| 28th of June 2024 Participants:  * Lea * Thijmen * Vasilis  Experimental: GFP; new pcr with new primers:   * We first hydrated the new Factor 8 primers (which are coded as FVIII Fwd new2, FVIII Rvd-PA and FVIII Rvd no PA) and the GFP primers to 100 uM. * Then made 10uM dilutions with the same label as on the primer vials. * We then made different Factor 8 primer combinations with old and new primers. For the GFP we used the standard used primers. * We first made a PCR master mix per primer combination (see table below) as follows: * GFP DNA dilution: 20ng/uL * 25 microliters of mastermix 1 (PCR M1) are added to PCR tube 1, after this 1 microliter of F8 DNA (or for PCR tube 6, 10 microliters of 20 ng/uL GFP DNA) was added to the PCR tube after which 2,25 microliters of Pfu polymerase is added to the PCR tube. Now the PCR tube can be placed into the PCR machine. Repeat for all remaining master mixes and PCR tubes and then select the program named f8 comb + GFP and start the PCR.  |  |  |  | | --- | --- | --- | | **Combination (names derived from tube)** | **Coding on the real-life tubes** | **Master mix ingredients** | | FVIII Fwd new2 + FVIII Rvd | PCR M1 | * 83,25 uL of miliQ water * 11,25 uL of 10x PCR buffer * 11,25 uL of 2mM dNTPs * 2,25 uL of forward primer * 2,25 uL of backward primer | | FVIII Fwd new2 + FVIII Rvd-PA | PCR M2 | * 83,25 microliters of miliQ water * 11,25 microliters of 10x PCR buffer * 11,25 microliters of 2mM dNTPs * 2,25 microliters of forward primer * 2,25 microliters of backward primer | | FVIII Fwd new2 + FVIII Rvd no PA | PCR M3 | * 83,25 microliters of miliQ water * 11,25 microliters of 10x PCR buffer * 11,25 microliters of 2mM dNTPs * 2,25 microliters of forward primer * 2,25 microliters of backward primer | | FVIII Fwd + FVIII Rvd-PA | PCR M4 | * 83,25 microliters of miliQ water * 11,25 microliters of 10x PCR buffer * 11,25 microliters of 2mM dNTPs * 2,25 microliters of forward primer * 2,25 microliters of backward primer | | FVIII Fwd + FVIII Rvd no PA | PCR M5 | * 83,25 microliters of miliQ water * 11,25 microliters of 10x PCR buffer * 11,25 microliters of 2mM dNTPs * 2,25 microliters of forward primer * 2,25 microliters of backward primer | | GFP Fwd + GFP Rvd | PCR M6 (GFP) | * 83,25 microliters of miliQ water * 11,25 microliters of 10x PCR buffer * 11,25 microliters of 2mM dNTPs * 2,25 microliters of forward primer * 2,25 microliters of backward primer | |
| 1st of July 2024 Participants:  * Lea * Thijmen * Vasilis  Experimental: Gel electrophoresis:   1. The samples were prepared, by adding 1ul of loading dye to 5ul of the samples.    * 1st sample: FVIII Fwd new2 + FVIII Rvd,    * 2nd: FVIII Fwd new2 + FVIII Rvd-PA,    * 3rd: FVIII Fwd new2 + FVIII Rvd no PA,    * 4th : FVIII Fwd + FVIII Rvd-PA,    * 5th:FVIII Fwd + FVIII Rvd no PA,    * 6th:GFP Fwd + GFP Rvd(see 28/06 new PCR with new primers for more info). 2. The gel was filled the following way:    * 8th slot: 5ul of DNA ladder    * 9th: 6ul 1st sample    * 10th: 6ul 2nd sample    * 11th: 6ul 3rd sample    * 12th: 6 ul 4rd sample    * 13th: 6ul 5th sample    * 14th: 6ul 6th sample    * 15th: 5ul of DNA ladder.Something went wrong with the 4th one - it disappeared. (Discussion needs to be done.) 3. After this, the gel electrophoresis was performed where it was run for 50 minutes on 110 V.  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad-104-1.webp) Figure 1. Gel electrophoresis  As seen in the picture above we again got smears on our gel. Another strange anomaly is that in the GFP well (second to last) we could see a very vague band all the way at the bottom of the gel and a very vague band somewhere at the top. This of course shouldn’t be happening since the gel should show 1 band somewhere low in the gel.  For the 5 Factor 8 samples the same could have gone wrong as with all the other PCR reactions that have been performed (we still don’t know what it is), however it is very strange that also the GFP (second to last slot) has also failed to give a good result. We have used the primers that normally work well so it can’t be the primers fault, this might indicate that something is wrong with the procedure which causes the smears. Conclusion: We will have to meetup with Frank in order to discuss these results. The reliability of our results is probably good enough, however for the 4th sample the pipetting went a bit wrong which led to the loss of some of the sample (it disappeared in the gel) furthermore during the transfer of the gel from the gel electrophoresis machine onto the UV machine, the gel broke. We reassembled it and we could still see things like the molecular ladder therefore we think it hasn’t impacted our results by a lot.  After consultation with Frank, it was decided to try the multiplying F8 DNA with a different method (RAD107) and to try again with the GFP because it had worked before. |
| RAD105: GFP DNA - second attempt **Background:** The purpose of the experiment is to produce a placeholder GFP mRNA for model experiments. PCR mix was made following [“PCR procedure Radboud-University Team”](https://static.igem.wiki/teams/5342/documents/protocols/pcr-procedure-radboud-university-team.pdf). Since primers are already known, the negative and positive controls are not going to be used.  **Materials:** For the production of the GFP mRNA 20ng/ul PUC-18 GFP template was used. The primers to be used were the following: Forward primer used: EF-eGFP fwd for cloning, Tm = 65.2 C. Reverse primer used: EF-eGFP rvd for cloning, Tm = 65.3 C. Both primers were deluted to 10 uM in the previous experiment - RAD104. The gel electrophoresis procedure in the reference indicated to use 10x TBE (also for the buffer) 100 ml and 1mg agarose. However, our method will use 0.5x TBE (also for the buffer) 100 ml and 0.7 agarose gel. It is safe to dispose of this buffer in the sink, but the gel needs to be thrown out in the waste.  **Calculations:** Amounts of GFP template to be added: 2.5 ng (needed DNA)/ 20 ng/ul (given concentration) = 0.125 ul - to add. Amounts of DNA poly template to be added: (need to be calculated, but were over-added compared to “PCR procedure Radboud-University Team”). |
| 3rd of July 2024 Participants:  * Kate * Lea * Thijmen  Experimental: PCR:   1. GFP template was diluted 10x times to concentration of 2ng/ul. 2. In 1 epp.: 3. 25 ul PCR mix. (prepared by the procedure) 4. 1 ul GFP template. (=2 ng) 5. 1 ul DNA poly. (More that required by the procedure, but assumed to be not significant.) 6. The PCR mixture was added to the PCR machine for 10 cycles. 1 cycle: 95C, 95C, 62C, 72C, 72C, 10C.   Gel electrophoresis:   1. 0.7 g of agarose were dissolved in 100ml of 0.5 xTBE. 2. The mixture with the agarose was heated in the microvawe for 5 min. 3. 5 ul ethidium bromide was added. 4. The gel was poured into the tray and solidified in 20 min. 5. 0.5x TBE buffer was prepared. 6. The buffer was added onto the gel, and afterwards the comb was removed. 7. The analysis samples were prepared, by adding 1ul of loading dye to 5ul of the GFP samples. (3 in total) 8. The samples were loaded in the gel: 9. 1st well: DNA ladder (Kate) 10. 2nd well: GFP sample 1 (Lea) 11. 3rd well: GFP sample 2 (Lea) 12. 4th well: GFP sample 3 (Kate) 13. 5th well: DNA ladder (Thijmen)  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad-105-1.webp) Figure 1. 1st well: DNA ladder (Kate); 2nd well: GFP sample 1 (Lea); 3rd well: GFP sample 2 (Lea); 4th well: GFP sample 3 (Kate); 5th well: DNA ladder (Thijmen).  A GFP mRNA was correctly received at the 1000 bp region (see the ladder). However, the spot is very dim, so this means that not many DNA was produced. Conclusion: The decision was made to redo the PCR to make more DNA. Then, 3 to 4 reactions of 100 ug with 20 ng of the template per 100 ul will need to be prepared, and they will be run for 25 PCR cycles.  After consultation with Frank, it was decided to try the multiplying F8 DNA with a different method (RAD107) and to try again with the GFP because it had worked before. |
| RAD106: GFP DNA - Upscaled PCR reaction **Background:** The purpose of the experiment is to increase of the amounts of production of GFP mRNA for model experiments compared to the experiment attempt RAD105. PCR mix was made following [“PCR procedure Radboud-University Team”](https://static.igem.wiki/teams/5342/documents/protocols/pcr-procedure-radboud-university-team.pdf). Since primers are already known, the negative and positive controls are not going to be used.  **Materials:** For the production of the GFP mRNA 20ng/ul PUC-18 GFP template was used. The primers to be used were the following: Forward primer used: EF-eGFP fwd for cloning, Tm = 65.2 C. Reverse primer used: EF-eGFP rvd for cloning, Tm = 65.3 C. Both primers were deluted to 10 uM in the previous experiment - RAD104. The gel electrophoresis procedure in the reference indicated to use 10x TBE (also for the buffer) 100 ml and 1mg agarose. However, our method will use 0.5x TBE (also for the buffer) 100 ml and 0.7 agarose gel. It is safe to dispose of this buffer in the sink, but the gel needs to be thrown out in the waste.  **Calculations:** The amount of mastermix was increased 4x: 333 μL MilliQ water, 45 μL 10x PCR buffer, 45 μL 2 mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP), 9 μL 10 μM forward primer, 9 μL 10 μM reverse primer, 9 μL Pfu polymerase (2ul per 100 g) 450 μL final volume. |
| 3rd of July 2024 Participants:  * Lea * Maks * Thijmen  Experimental: PCR:   1. Mastermix was prepared (see calculations). 2. In 1 PCR epp.: 3. 100 ul PCR Mastermix. 4. 1 ul GFP template. (=20 ng) - 1st: 2ng, 2nd: 2ng (pipette tip was not changed), 3rd: 1 ng, 4th: 1 ng (potentially, less mastermix: bubbles/ not correct ammounts/ was not well visible?). 5. 2 ul DNA poly. - was added in the end. - 1st: 2ng, 2nd: 2ng, 3rd: 2 ng (spill), 4th: 2 ng. 6. The PCR mixture was added to the PCR machine for 35 cycles. 1 cycle: 95C, 62C, 72C. 7. The samples were left in the PCR machine at 4C overnight.   Gel electrophoresis:   1. 0.7 g of agarose were dissolved in 100ml of 0.5 xTBE. 2. The mixture with the agarose was heated in the microvawe for 5 min. 3. 5 ul ethidium bromide was added. 4. The gel was poured into the tray and solidified in 20 min. 5. 0.5x TBE buffer was prepared. 6. The buffer was added onto the gel, and afterwards the comb was removed. 7. The analysis samples were prepared, by adding 1ul of loading dye to 5ul of the GFP samples. (3 in total) 8. The samples were loaded in the gel: 9. 1st well: DNA ladder (Kate) 10. 2nd well: GFP sample 1 (Lea) 11. 3rd well: GFP sample 2 (Lea) 12. 4th well: GFP sample 3 (Kate) 13. 5th well: DNA ladder (Thijmen)  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad-105-1.webp) Figure 1. 1st well: DNA ladder (Kate); 2nd well: GFP sample 1 (Lea); 3rd well: GFP sample 2 (Lea); 4th well: GFP sample 3 (Kate); 5th well: DNA ladder (Thijmen).  A GFP mRNA was correctly received at the 1000 bp region (see the ladder). However, the spot is very dim, so this means that not many DNA was produced. Conclusion: The decision was made to redo the PCR to make more DNA. Then, 3 to 4 reactions of 100 ug with 20 ng of the template per 100 ul will need to be prepared, and they will be run for 25 PCR cycles.  After consultation with Frank, it was decided to try the multiplying F8 DNA with a different method (RAD107) and to try again with the GFP because it had worked before. |
| 4th of July 2024 Participants:  * Lea * Maks * Thijmen  Experimental: Gel electrophoresis:   1. samples were prepaired with 1uL loading dye and 5uL PCR product. 2. 5uL of the samples was loaded on the gel in the following order: DNA ladder, SPILL, normal, normal, X and another DNA ladder. 3. The gel was run at 110V for 50 min. 4. Since no compound was seen, the PCR samples were mixed with ethidium bromide. Observation: the flask shined under the UV light.   Gel electrophoresis 2:   1. Same gel as above was used and the samples are remade (so again 1 ul of loading dye, 5 ul of PCR product) 2. The wells were loaded as follows: DNA ladder, 2, 2, SPIL, 1. 3. The gel was run at 110V for 50 min.  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad-106-1.webp) Figure 1. 1st gel of our product: DNA ladder, SPILL, normal, normal, X and another DNA ladder. ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad-106-1.webp) Figure 2. 2nd gel of our product: DNA ladder, 2, 2, SPIL, 1.  In the first gell, no DNA was seen, but we wanted to check the presence of DNA, and added the ethidium bromide to one PCR sample. Since the flask shined, this indicated that there is some DNA. Thus, the new PCR was prepared, as well as the samples were loaded onto a new gel again for second analysis. Still, in the second gel no results were seen which indicated that the DNA was indeed not present. Thus, the new experiment was decided to be conducted - RAD108. Conclusion: No DNA apparently was present in the sample. It is likely that an error was made and one of the ingredients was not added correctly. This meant that the procedure would need to be repeated. |
| RAD108: GFP mRNA - increase the amount 2 **Background:** The purpose of the experiment is to upscale production of GFP mRNA for model experiments compared to the experiment attempt RAD106. PCR mix was made following [“PCR procedure Radboud-University Team”](https://static.igem.wiki/teams/5342/documents/protocols/pcr-procedure-radboud-university-team.pdf). Since primers are already known, the negative and positive controls are not going to be used.  **Materials:** For the production of the GFP mRNA 20ng/ul PUC-18 GFP template was used. The primers to be used were the following: Forward primer used: EF-eGFP fwd for cloning, Tm = 65.2 C. Reverse primer used: EF-eGFP rvd for cloning, Tm = 65.3 C. Both primers were diluted to 10 uM in the previous experiment - RAD104. The gel electrophoresis procedure in the reference indicated to use 10x TBE (also for the buffer) 100 ml and 1 mg agarose. However, our method will use 0.5x TBE (also for the buffer) 100 ml and 0.7 agarose gel. It is safe to dispose of this buffer in the sink, but the gel needs to be thrown out in the waste.  **Calculations:** The amount of mastermix was increased 4x: 333 μL MilliQ water, 45 μL 10x PCR buffer, 45 μL 2 mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP), 9 μL 10 μM forward primer, 9 μL 10 μM reverse primer, 9 μL Pfu polymerase (2ul per 100 g) 450 μL final volume. |
| 3rd of July 2024 - 4th of July 2024 Participants:  * Lea * Maks * Thijmen  Experimental: PCR:   1. Mastermix was prepared (see calculations). 2. In 1 PCR epp.: 3. 100 ul PCR Master Mix. 4. 1 ul GFP template. (=20 ng) - 1st: 2ng, 2nd: 2ng (pipette tip was not changed), 3rd: 1 ng, 4th: 1 ng (potentially, less mastermix: bubbles/ not correct amounts/ was not well visible?). 5. 2 ul DNA poly. - was added in the end. - 1st: 2ng, 2nd: 2ng, 3rd: 2 ng (spill), 4th: 2 ng. 6. The samples were left for 25 cycles PCR. 1 cycle: 95C, 62C, 72C.   Gel electrophoresis:   1. 0.7 g of agarose was dissolved in 100ml of 0.5 xTBE. 2. The mixture with the agarose was heated in the microwave for 5 min. 3. 5 ul ethidium bromide was added. 4. The gel was poured into the tray and solidified in 20 min. 5. The 0.5x TBE buffer was added onto the gel, and afterwards the comb was removed. 6. The analysis samples were prepared, by adding 1ul of loading dye to 5ul of the GFP samples. (3 in total)   The samples were loaded in the gel run on 110V for 50 min. Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad-108-1.webp) Figure 1. Gel electrophoresis (0.7% w/v agarose) of the PCR samples  All samples showed a clear band at 1000kb, where it is expected. Conclusion: It was decided to continue with the purification. |
| RAD109: PCR purification of the GFP DNA product from experiment RAD108, and its analysis. **Background:** The DNA from the experiment RAD108 had smears, so it was decided to purify it. For the PCR purification, the protocol QIA PCR purification was followed. ([“QIAquick® PCR Purification Kit”](https://static.igem.wiki/teams/5342/documents/protocols/qiaquick-pcr-purification-kit.pdf)). |
| 4th of July 2024 Participants:  * Lea * Maks * Thijmen  Experimental: Purification of PCR product:   1. 500 ul buffer for 100 ul PCR sample. 2. The color of the solution did not change. 3. Some acid and base were added to the buffer. However, no change in color was observed. 4. 600 ul of each sample were added to a separate QIAquick column. 5. Next the samples were centrifuged for 60 seconds at 13.000 RPM. 6. The flow-through was discarded. 7. 750 uL PE buffer was added and the samples were centrifuged for 60 seconds at 13000 RPM. 8. The flow-through was discarded. 9. The samples were centrifuged again for 60 seconds. 10. The QIAquick tubes were put into 1.5 ml epps. 11. Next, there were 50 microliters of milliQ water added to the QIAquick membrane for each sample. 12. Samples are centrifuged again for 60 seconds. 13. The sample was labeled GFP cDNA.   Gel electrophoresis of purified product:   1. 3 ul of dye were added to the 15 ul of the sample. 2. The gel was loaded: 5 ul of the loading die; sample. 3. Gel was run for 50 min at 110 V.  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad-109-1.webp) Figure 1. Agarose gel of purified product: ladder; sample.  The result of the Gel is good - a little smear at the top probably occurred due to too much DNA concentration. The band is around 1000 bp, which is where you would expect EGFP. ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad-109-2.webp) Figure 2. Analysis with nanodrop:  Measured samples of the GFP cDNA showed a concentration of 60.2 ng/ul. Conclusion: The pure GFP cDNA sample of 185 ul with concentration 60.2 ng/ul was received. The application of it is to produce mRNA for the further experiments and testing of the vesicles. |

DNA Amplification via Bacterial Plasmids

03/07/24 - 08/07/24

* Lea
* Kate

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| RAD107: Transformed bacteria colony preparation **Background:** The purpose of the experiment is to make the transfected bacteria with an inserted FVIII vector plasmid. The bacteria are cultured on antibiotic full media, so only the ones that have absorbed this plasmid can survive. The bacterial cultures used are Top10 and XL1Blue (both E.coli strains optimized for plasmid amplification) |
| 3rd of July 2024 - 4th of July 2024 Participants:  * Lea  Experimental: Procedure:   1. Top10Transf colony was prepared the following way: 1ul FVIII plasmid solution was mixed with 200ul Top10 bacteria suspension. 2. XL1BlueTransf colony was prepared the following way: 1ul FVIII plasmid solution was mixed with 200ul XL1Blue bacteria suspension. 3. Both of the cultures were left on ice for 0.5 h. 4. Both bacteria were heat shocked for 54 seconds at 42C. 5. 800 ul of LB medium. 6. Left in shaker for 0.5h 37C. 7. Bacteria were spun down with a centrifuge and 900uL medium was removed 8. The pallet was resuspended in the remaining 100uL medium and plated on agar plates containing ampicillin. 9. The bacteria were left in the incubator (37 C)overnight. 10. (the next day) 11. after 24 h incubation, the Top10 plate had many miniscule colonies, while Xl1Blue had less, but larger colonies. 12. 6 colonies were picked from the XL1Blue plate and 4 from the Top10 plate. 13. in total 10 5mL liquid cultures were prepared and incubated overnight at 37C.   **Conclusion:** These successful bacteria synthesis allows for further FVIII isolation and production (RAD110) |
| RAD110: Miniprep of cp FVIII DNA and digestion **Background:** Extraction of the DNA samples from the bacteria cultures, prepared in the RAD107 experiment: Top10 and XL1 Blue. Plasmid used is linked [here](https://www.addgene.org/41036/). |
| 5th of July 2024 Participants:  * Kate  Experimental: Cultures used:   * E. Coli XL1 Blue: 1, 2, 3, 4, 5, 6 cultures with pc DNA Factor VIII * E. Coli: Top 10 1, 2, 3, 4 cultures with pc DNA Factor VIII   Procedure:   1. 2ml of each bacterial culture was added. Labeled respectively: XL1, XL2, XL3, XL4, XL5, XL6; Top1, Top2, Top3, Top4 2. I centrifuged the pipetted cultures for 3 minutes at 1000 rpm, RT 3. Supernatant was collected leaving pellets dry 4. 2 ml more of of cultures were added to each sample 5. Samples were centrifuged again, same way 6. Supernatant was collected leaving pellets dry 7. 250 ul of P1 buffer was added to all samples 8. To fractions XL1-5 250 ul of P2 buffer were added 9. Samples XL1-5 were inverted 6 times (kept in the buffer P2 less than 5 min) 10. 350 ul of N3 buffer was added to solutions XL1-5, samples were rotated 4-6 times [NOTE: XL2 had too much liquid (~1.25 ml), but pH paper showed that it is okay, so probably N3 buffer was added 2 times)] 11. Same as to XL1-5 was doe to XL6 and Top1-4 12. The samples were put to centrifuge 10 min at 13000 rpm, 20C 13. 800 ul supernatants was collected to QIAprep spin column 14. This supernatant in QIA prep was centrifuged for 30-60 seconds at 13000 rpm, and the flow-though was discarded 15. 250 ul PE buffer were added 16. Centrifuged 60 sec 17. flow-through was discarded 18. 250 ul PE buffer were added 19. Centrifuged 60 sec 20. flow-through was discarded 21. 50 ul H2O MiliQ added 22. Centrifuged 60 sec 23. flow-through was discarded 24. 50 ul H2O MiliQ added   Digestion:   * Combine EcoRI with plasmid for 10 digestions * Combine XhoI with plasmid for 10 digestions * From pc DNA FVIII (12086 bp) * XL1 Blue: 2.5 ul to EcoRI and 2.5 ul to XhoI & Top10 2.5 ul to EcoRI and 2.5 ul to XhoI   Linearisation:   * EcoRI: 4 expected bands to be seen on gel: cuts at 347bp, 1728bp, 4688bp, 5323bp * XhoI: 1 expected bands to be seen on gel: cuts at 12.1 kbp * Composition for linearization mixture per one linearization: 2.5 ul buffer, 0.5 ul enzyme, 5 ul DNA, 17 ul MQ * Sample with enzymes is linearized at 37 C for 2 hours  ![](https://static.igem.wiki/teams/5342/images/notebook/rad110-1.webp) Analysis:   * Prepare gel * After linearization, mix with 6ul 6xLB * Put on gel  Results and Discussion:![ECORI digest](https://static.igem.wiki/teams/5342/images/notebook/rad110-2.webp) Figure 1. EcoRI digest  The EcoRI digestion showed 4 bands, as expected. ![Xhol digest](https://static.igem.wiki/teams/5342/images/notebook/rad110-3.webp) Figure 2. XhoI digest.  The Xhol digestion shows one band, as expected. ![Results scheme](https://static.igem.wiki/teams/5342/images/notebook/rad110-4.webp) Figure 3. Expected results scheme  **Conclusion:** FVIII pcDNA was extracted from the bacterial cultures Top10 and XLBlue. The gel analysis of enzymatically linearized DNA confirmed its proper structure. The further steps of the FVIII DNA processing is the linearization of more plasmid, and then purifying it. This DNA then should be transcribed to FVIII mRNA. |
| RAD111: Large Xho1 digestion **Background:** Scaled up linearisation of plasmids for the use in in vitro transcription. The XL1Blue plasmids are used because they were isolated in a higher concentration. The Xho1 digestion will linearise the plasmid right after the coding sequence, allowing for run off transcription. |
| 8th of July 2024 Participants:  * Lea  Experimental: Cultures used:   * E. Coli XL1 Blue: 1, 2, 3, 4, 5, 6 cultures with pc DNA Factor VIII * E. Coli: Top 10 1, 2, 3, 4 cultures with pc DNA Factor VIII   Procedure:   1. the concentration of all isolated plasmid samples was determined 2. 4 enzyme digestions with a volume of 150 uL were prepared using plasmids isolated from XL1Blue following the protocol. The DNA concentration of the plasmids used was 758 and 756 ng/uL,so 11.9 uL was used to reach 9ng pcDNA. 3. digestion was incubated at 37 C for 3 hours 4. The digested plasmids were purified using a spin column (following the same protocol as before): 5. 600uL PB buffer to 120uL digestion mix, centrifuge for 1 min at 13000 rpm 6. the concentrations of the purified product were measured to be 79.9, 96.6, 94.7 and 88.8 ng/uL in that order 7. the purified products were put on gel (110V 50 min) to check the quality 8. the samples were floating out of the wells, so we prepared them again with a 5:2 sample to dye (double the amount of dye) 9. samples were loaded: ladder-unpurified———middle wells empty———1-2-ladder-3-4  Results and Discussion:![Digested plasmids](https://static.igem.wiki/teams/5342/images/notebook/rad111-1.webp) The digested plasmids show clear bands at the length where we expected them.  **Conclusion:** A large amount of linear FVIII pcDNA was produced and purified. This can now be used to perform in vitro transcription and produce FVIII mRNA. |

IVT and mRNA Purification

09/07/24 - 30/08/24

* Adrian
* Henriete
* Kate
* Lea
* Thijmen

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| RAD112: IVT of the GFP and Factor VIII RNA **Background:** The "[In vitro synthesis of RNA using T7 RNA polymerase](https://static.igem.wiki/teams/5342/documents/protocols/in-vitro-synthesis-of-rna-using-t7-rna-polymerase.pdf)” protocol was used to translate mRNA from the cpFVIII DNA samples received in the experiment RAD110, as well as the GFP DNA received in the experiment RAD109. |
| 9th of July 2024 Participants:  * Lea  Experimental:  1. a mastermix for 10 reactions of 20uL was prepared following the protocol for IVT. → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping.    mastermix amounts:    * Tris (1M): 8uL    * MgCl2 (1M): 5uL    * DTT(0.1M): 10uL    * spermidine (25mM): 8uL    * rNTPs(20mM): 40uL    * MilliQ: 129uL    * total: 200uL 2. prepare 10 epps, with each 20uL mastermix 3. 1uL dsDNA of the five different DNA samples (4 FVIII DNA, 1 GFP DNA) was added to epps (for FVIII linearised plasmid, 75 ng of DNA is needed for 20uL reaction, for GFP it is 26ng. The concentration of the FVIII DNA was close enough that 1uL could be taken from the tube. The GFP DNA was diluted 1:1 to a concentration of 30ng/uL and 1uL was used from that ) 4. For every different DNA sample, one epp got 2uL T7 polymerase and the other 4uL. → 2uL is the amount recommended by the protocol, but because of the length of the FVIII gene, we wanted to see if a larger amount of T7 would be beneficial. 5. The reaction was incubated at 37 C for 3 hours. 6. 5uL of all samples was mixed with 2uL loading dye and run on an agarose gel (110V 40 min)  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad112-1.webp) Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.  It is weird that 1, 2 and 4 with 2uL T7 did not show a lot of transcription while 3 seems to have a lot, because they should practically be the same. Overall, the reaction seems to work better with 4uL T7. The FVIII RNA was expected to be longer than 7000 instead of 1200. This could be the result of RNA degradation or self splicing, but it could also be because of the properties of the gel and because RNA is not linear. In addition, the GFP RNA did not work out that well in both cases. it could have something to do with the template. maybe the calculations on the amount of template needed was incorrect.  → I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.  **Conclusion:** Experiment needs to be repeated with proposed changes:   * different version of T7 poly. * Add RNAse inhibitor. * Reduce temperature to prevent autocatalytic breakdown. |
| RAD113: 2nd IVT iteration **Background:** Mutated T7 (polymerase) is the T7 used last time, now we are also testing the wild type. We are redoing the EGFP with the new calculation and trying an RNAse inhibitor to try and stop RNA degradation.  Calculations:   |  |  |  |  | | --- | --- | --- | --- | | EFGP + mutT7 = 9 | EGFP + mutT7 + RNAse inh. = 10 | EGFP + WT T7 = 4 | EGFP + WT T7 + RNAse inh. = 5 | |  | F8 -1 + mut T7 + RNAse inh. = 3 | F8 - 1 + WT T7 = 1 | F8 - 1 + WT T7 + RNAse inh. = 2 | |  | F8- 4 +mut T7 + RNAse inh. = 8 | F8 - 4 + WT T7 = 6 | F8 - 4 + WT T7 + RNAse inh. = 7 | |
| 10th of July 2024 Participants:  * Lea * Thijmen  Experimental: IVT:   1. a 200 uL mastermix was prepared the same as the one in RAD112 2. The mix was divided over 10 epps labeled the same as the descriptions in the table. 3. samples were prepared following the table:    * 4uL of T7 was used for both versions    * 0.5 uL of RiboLock RNase inhibitor was used    * 1 uL of F8 DNA template was used from both template samples, 1.5 uL of the diluted GFP template was used to reach the right amount of template. 4. The reactions were incubated for 3 hours.   Gel electrophoresis:   1. Samples with dye were prepared - 5 ul sample + 2 ul dye. 2. The samples were loaded as follows:  * 1st well: Sample 1, * 2nt: Sample 2, * 3th: Sample 3 (see discussion), * 4th: Sample 4, * 5th: Sample 5, * 6th: DNA ladder, * 7th: Sample 6, * 8th: Sample 7, * 9th: Sample 8, * 10th: Sample 9, * 11th: Sample 10.  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad113-1.webp) Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.  During the pipetting of the sample 3 the pipette tip fell off, which may be the cause of the absent band. Samples 4 and 5 seem pure with only minor smearing. Samples 2 and 7 have a significant smear, which could be perhaps resolved by using denaturing conditions that would eliminate the RNA’s autocatalytic activity.  **Conclusion:** Adding an RNase inhibitor improves the outcome of IVT. Sample analysis on denaturing gel should be carried out in order to eliminate autocatalytic activity of the RNA molecule. GFP samples give more clear bands on the native gel than the factor VIII samples. |
| RAD114: IVT of GFP + FVIII **Background:** For the IVT, the In vitro synthesis of RNA using T7 RNA polymerase protocol was used. For IVT we need between 5 and 50 nM of dsDNA. We want 10 nM, for FVIII linearised plasmid, 75ng of DNA is needed for 20uL reaction, for GFP it is 26ng. The concentrations of the FVIII DNA was close enough that 1uL could be taken from the tube. The GFP DNA was diluted 1:1 to a concentration of 30ng/uL and 1uL was used from that (from RAD112).  Calculations:   |  |  |  |  | | --- | --- | --- | --- | | EFGP + mutT7 = 9 | EGFP + mutT7 + RNAse inh. = 10 | EGFP + WT T7 = 4 | EGFP + WT T7 + RNAse inh. = 5 | |  | F8 -1 + mut T7 + RNAse inh. = 3 | F8 - 1 + WT T7 = 1 | F8 - 1 + WT T7 + RNAse inh. = 2 | |  | F8- 4 +mut T7 + RNAse inh. = 8 | F8 - 4 + WT T7 = 6 | F8 - 4 + WT T7 + RNAse inh. = 7 | |
| 15th of July 2024 Participants:  * Adrian * Thijmen  Experimental:  1. a mastermix for 5 reactions of 40uL was prepared following the protocol for IVT.    → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping. mastermix amounts:    * Tris (1M): 8uL    * MgCl2 (1M): 5uL    * DTT(0.1M): 10uL    * spermidine (25mM): 8uL    * rNTPs(20mM): 40uL    * MilliQ: 129uL    * total: 200uL 2. prepare 5 epps, with each 40uL mastermix, labeled with numbers 1 - 5. Note - epp number 5 had an air droplet in the pipette tip, so it's doubtful whether it has the correct amount 3. Factor 8 DNA - 2, Factor 8 DNA 3, GFP diluted were measured on nanodrop for concentration: 4. ![](https://static.igem.wiki/teams/5342/images/notebook/rad114-1.webp) 5. The following were added to epps 1 - 5 in order from left to right:    * 1 = 1.78 ul Factor 8 - 2 + 8 ul polymerase    * 2 = 1.78 ul Factor 8 - 3 + 8 ul polymerase    * 3 = 1.78 ul Factor 8 - 2 + 1 ul RNase inhibitor + 8 ul polymerase    * 4 = 1.78 ul Factor 8 - 3 + 1 ul RNase inhibitor + 8ul polymerase    * 5 = 2.14 ul GFP DNA + 1 ul RNase inhibitor + 8 ul polymerase Note: From calculating the concentration of DNA specified in the manual (10nM) considering that our DNA is roughly 8000 bp which are all roughly 618g/mol each we calculated that the final concentration that manual uses is 49 ng/ul which is way higher than what we have used in all experiments so far.  Note: Use the mutated polymerase frank gave us (green label)  Solutions transferred to PCR tubes labeled 1-5 with roman numbers  Solutions stored overnight in PCR machine at 4 degrees centigrade 6. All 5 products analyzed on a denaturing agarose gel:    * Added 0.35g agarose in 5 ml 10xMOPS solution and 36 mL MilliQ    * Heat in microwave for about 15 seconds (until complete dissolution)    * Stir continuously while slowly adding 9ml 37% formaldehyde    * Stir for 5 seconds more and pour in casting tray    * Wait 50 minutes    * Load samples on gel    * Run for 50 minutes    * Analyze under UV  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad114-2.webp) Figure 1.  The samples with inhibitors show more clearly visible bands on the gel. There are significant smears on the gel. Further purification and modification is required in order to obtain a clearer result. There is only one visible band per sample.  **Conclusion:** The procedure used in this experiment is effective at synthesizing and analyzing mrna. Rnase inhibitors should always be used for better results. |
| RAD115: IVT. RNA synthesis scale up of FVIII & GFP |
| 17th of July 2024 Participants:  * Adrian * Lea * Thijmen  Experimental:  1. 200uL of master mix was prepared:    * Tris (1M): 8uL    * MgCl2 (1M): 5uL    * DTT(0.1M): 10uL    * spermidine (25mM): 8uL    * rNTPs(20mM): 40uL    * MilliQ: 129uL    * total: 200uL Note: the GFP DNA template made an unexpected sound of ice cracking when taken out of the fridge. 2. The mastermix was distributed over 5 epps giving them 40 uL each. 3. 1uL RiboLock RNase inhibitor was added to all epps 4. 2.14 uL of diluted GFP DNA template was added to epps z (labled GFP RNA on the side) 5. 1.78 uL (for a final concentration of 49ng/uL) of FVIII - 3 DNA template was added to epps 4\* and 5\* (labled FVIII RNA on the side) 6. 8uL of mutated T7 RNA polymerase was added to every epp. 7. The reaction was incubated at 37C for 3 hours. 8. The samples were checked on a denaturating agarose gel (40min 110V).  Results and Discussion:![](https://static.igem.wiki/teams/5342/images/notebook/rad115-1.webp) The bands all show up at the same hight, while a significant difference in length was expected. the first 3 samples were made with GFP template DNA and the last 2 with F8, so the first 3 should be way lower compared to the last 2. Although we were quite confident in our pipetting, this could be due to a pipetting mistake while adding the template DNA or when putting the samples on the gel. RiboLock RNase inhibitor should always be addded in this procedure.  **Conclusion:** Only GFP mRNA was rececived & analysed on the gel. Samples 1\*, 2\*, 3\*, 4\*, 5\*.  **Next steps:**  purification with phenol chlorophorm extraction.  optimise for GFP, then move on to FVIII. |
| RAD116: 1st trial RNA purification using phenol:chloroform **Background:** protocol based on: [RNA Clean-up by phenol:chloroform v2](https://www.protocols.io/view/rna-clean-up-by-phenol-chloroform-yxmvmw46g3pe/v2?step=null). With the mRNA purification, we are trying to get samples which would only contain the mRNA that we want, this will be done by following a protocol which will lead to the removal of any IVT ‘ingredients’ that are present in the sample. By removing these and keeping the mRNA in tact, we will get a pure mRNA for further experiments. |
| 18th of July 2024 Participants:  * Adrian * Lea * Maks * Thijmen  Experimental:  1. 20uL of eGFP RNA sample 1\* (from RAD115) was combined with 20uL Phenol/Chloroform/Isoamyl alcohol (https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1) 2. shaken vigorously inside fume hood for 15 seconds. 3. centrifuged at 12000G at 4C for 2 min 4. the top aqueous layer was transferred to a new epp and combined with a new 20uL of phenol/chloroform/isoamyl alcohol 5. shaken vigorously inside fume hood for 15 seconds 6. centrifuged at 12000G at 4C for 2 min 7. aqueous layer was transferred to a new epp 8. 50uL of isopropanol was added and shaken 9. centrifuged at 20000G at 4C for 10min 10. the isopropanol was removed and the pellet was washed with 40 uL ice cold isopropanol 11. centrifuged at 25000G at 4C for 5 min 12. The isopropanol was removed and the palate was dried to air. 13. the pellet was resuspended in 10 uL milliQ. sample labeled: RNA pure GFP 18-07-24 14. the concentration of the purified RNA was measured with nanodrop to be 2562 ng/uL   22nd of July 2024 Participants:  * Lea * Maks  1. To prevent high concentrations of RNA on the gel, the 10 uL sample was diluted with 850uL MilliQ 2. 5uL of the diluted sample was combined with 15uL of denaturing RNA loading buffer, incubated at 65C for 5min 3. the quality of the RNA was investigated by a denaturing agarose gel (110V, 50 min).   the gel showed nothing. possibly because the sample was too diluted. Results and Discussion:  * Gel on purified product showed no product; problem not in running buffer. * Maybe, a mistake was made (someone pipetted the wrong fraction), maybe, the sample did not have the mRNA - only single nucleotides. * Should try to run the gel for 5 minutes. * Maybe, the reason is that mRNA doesn’t have a cap, it might denature. RNAse might break it down. * Maybe, we should first modify mRNA, and then purify. - look it up/ ask Frank. 3’-5’ mRNA   Endo or exonuclease which might affect the mRNA.   * Before that - try to work with chemicals from Frank’s lab. * Maybe, Afka can help you to troubleshoot this purification. (Maks)   **Conclusion:**The purification procedure does not seem to yield the desired results. Procedure optimisation is required prior to further attempts. |
| RAD117: 2nd trial purification **Background:** Further attempts of the purification of the IVT samples |
| 22nd of July 2024 Participants:  * Lea * Maks  Experimental:  1. we tried to reconcentrate the diluted RNA sample (see RAD116) by precipitating it with 200uL ice cold isopropanol and centrifuging at 27500 rfc at 4C for 5 min → did not do anything 2. to the remaining 20 uL of sample 1\* (see RAD115), 20 uL phenol/chloroform was added and shaken. 3. the sample was centrifuged at 12000 rfc, 4C for 2 min 4. the top (aqueous) layer was transported to a new epp together with 20 uL new phenol chloroform and shaken 5. centrifuged at 12000 rfc, 4C for 2 min 6. top layer was added to 50 uL ethanol, shaken 7. centrifuged at 29000 rfc, 4C for 10 min 8. supernatant was removed and the pellet was washed with 40 uL ice cold ethanol. 9. centrifuged at 29000 rfc, 4C for 5 min 10. the supernatant was removed and the pellet was air dried. 11. a small bit of the pellet was removed, and resuspended in 10uL milliQ for analysis 12. the rest of the pellet is stored in the freezer, before use it should be resuspended in milliQ 13. The fragment of the pellet was analyzed on a denaturating agarose gel (110V, 50min) 14. absolutely no bands were observed on the gel  Discussion: The problem is most likely not caused by a too dilute sample, but it could be the result of the buffer being multiple days old. It might have picked up RNases that have degraded the sample while in the gel.  23rd of July 2024 Participants:  * Maks * Thijmen  1. Fresh buffer was prepaired 2. the palete was resuspended in 15uL milliQ and the concentration was measured to be 1881 ng/uL. 3. 5uL of the sample was run on a denaturing agarose gel (110V 50min) 4. No bands were discovered.  Discussion:![](https://static.igem.wiki/teams/5342/images/notebook/rad117-1.webp) The problems are not caused by a diluted sample or by an old buffer. It might be a more fundamental problem with the purification method.  **Conclusion:** Optimization of the purification procedure is needed before the continuation of the experiments as the current procedure seems to yield suboptimal results. |
| RAD118: repeat upscale IVT FVIII RNA **Background:** Preparation of the new F8 mRNA as the previous samples have been stored in the freezer for a long time |
| 22nd of July 2024 Participants:  * Lea * Maks  Experimental:  1. 200 uL mastermix was prepared, mixed with 10 uL FVIII - 2 template DNA. the IVT mix was divided over 2 PCR epps and after incubating for 10 minutes, we realized we forgot to add T7 2. 5 uL of WT T7 was added to each PCR tube. 3. the reaction was run in the PCR machine at a constant 37C for 3 hours, then kept at 4C overnight.   23rd of July 2024 Participants:  * Maks * Thijmen  1. The IVT reactions were removed from the PCR machine and the RNA concentration was measured. 2. The tube with the red marking had a concentration of 7446ng/uL and the one without marking had a concentration of -6389ng/uL even after cleaning and re-blanking. 3. The two samples were run on a denaturing agarose gel (110V 50 min)  Results and Discussion: The gel showed no bands, likely indicating either a complete samples’ degradation or a mistake made during the purification. The latter idea is supported by the seemingly negative RNA concentration in the sample  **Conclusion:** New mRNA samples have to be prepared, care should be taken to avoid any potential mistakes during IVT setup or mRNA purification. |
| RAD119: 5-minute gel test **Background:** Previous experiments with purification led to results that were not favourable. This checkup is done in order to check whether there truly is no mRNA. |
| 29th of July 2024 Participants:  * Lea  Experimental: The purified GFP mRNA sample was taken out of the fridge after which it was quickly vortexed. In order to have enough sample to be able to pipet it 5ul of miliQ water was added to the sample. After this 5uL of the purified mRNA GFP sample was added to an epp and 2ul of loading dye was added to the same epp. This epp was then vortexed to mix the 2 ingredients. Next, the sample + loading dye mixture was pipetted into the gel after which the gel was run for 5 minutes at 110 volts. Result![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad119-1.webp) Figure 1. Attempt 1 gel of purified GFP mRNA. 5 minute gel check. Discussion As can be seen on the photo, even after 5 minutes of running the gel there is no band visible. This would indicate that the current purification method is not working properly. A new purification method is used in RAD120.  **Conclusion:** Purification method should be revised, and the new method repeated and performed. |
| RAD120: Purification of GFP mRNA trial 3 **Background:** GFP samples 2\* and 3\* from the experiment RAD115. |
| 29th of July 2024 Participants:  * Maks * Thijmen   Note: all the materials that were used during the purification (like epps and pipet points) were sterile and all the work was done with gloves. This all will ensure a RNase free environment.   1. 20uL of a GFP mRNA sample 1 was combined with 20uL [Phenol/Chloroform/Isoamyl alcohol](https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1) 2. shaken vigorously inside the fume hood for 15 seconds. 3. centrifuged at 12000 G at 4C for 2 min 4. the top aqueous layer was transferred to a new epp and combined with a new 20uL of phenol/chloroform/isoamyl alcohol 5. shaken vigorously inside fume hood for 15 seconds 6. centrifuged at 12000 G at 4C for 2 min 7. aqueous layer was transferred to a new epp 8. 50uL of 96% ethanol was added and shaken 9. centrifuged at 25000G at 4C for 2min (this was a mistake and should have been 10 minutes) 10. The ethanol was removed and 50 ul of 70% ethanol solution was added to the sample. 11. centrifuged at 25000G at 4C for 5 min 12. The ethanol was removed and the palate was dried to air. 13. the pellet was resuspended in 10 uL milliQ. sample labeled: Pure GFP 29/07/2024   29th of July 2024 Participants:  * Maks * Thijmen   The purified GFP mRNA sample named Pure GFP 29/07/2024 was taken. After this 5uL of the purified GFP sample was added to an epp and 2ul of loading dye was added to the same epp. This epp was then vortexed to mix the 2 ingredients. Next, the sample + loading dye mixture was pipetted into the gel after which the gel was run for 5 minutes at 110 volts.  30th of July 2024 Participants:  * Thijmen   The purified GFP mRNA sample named Pure GFP 29/07/2024 was taken. After this 5uL of the purified GFP sample was added to an epp containing 2ul of loading dye. This epp was then vortexed to mix the 2 ingredients. Next, the gel was loaded. In the first well 5 ul of a molecular ladder was pipetted and in the second well 5 ul of the sample + loading dye was pipetted. After this the gel was run for 50 minutes at 110 volts.  30th of July 2024 Participants:  * Thijmen   A new gel was made by using the following protocol:   1. 100 ml of 0.5 xTBE was put in a flask 2. 0.7 g of agarose was dissolved in the 100ml of 0.5 xTBE flask. 3. The solution was then mixed 4. The mixture with the agarose was heated in the microwave until it became clear 5. 5 ul ethidium bromide was added to the mixture. 6. The gel was poured into the tray and solidified by waiting.   After the new gel was made the sample was prepared. The purified GFP mRNA sample named Pure GFP 29/07/2024 was taken. After this 5uL of the purified GFP sample was added to an epp containing 2ul of loading dye. This epp was then vortexed to mix the 2 ingredients. Next, the gel was loaded. In the first well 5 ul of a molecular ladder was pipetted and in the second well 5 ul of the sample + loading dye was pipetted. After this the gel was run for 50 minutes at 110 volts. Results and Discussion: We hope that by replacing the isopropanol with ethanol and by working with sterile equipment that the mRNA will survive the purification step and that we will receive pure GFP mRNA. In order to check if the purification really worked we have to run the samples on a gel. ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad120-1.webp) Figure 1. Attempt 2 gel of purified mRNA. 5 minute gel check. Date: 29-07-2024  As can be seen on the figure 1, there is mRNA present in the sample. In order to check if the RNA is pure and if it is the right RNA a complete gel electrophoresis must be done including a molecular ladder. ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad120-2.webp) Figure 2. Gel electrophoresis of pure GFP RNA sample part 1. Date: 30-07-2024  As you can see on the photo the sample looks pure as far as we can see. However both the sample and the ladder are very vague. This could be due to an error, a too low concentration (which would be bad in the case of the sample) or that the gel is too old. It was hypothesized that the gel is too old since it has been made 1 or 2 weeks prior. Next step is to refresh the gel and rerun the sample. |
| RAD121: Purification of mRNA trial 4, scaling upA **Background:** Same phenol-chloroform procedure as in the RAD120 extraction with larger quantities of GFP and FVIII mRNA. |
| 1st of August 2024 Participants:  * Maks  Experimental:  1. 40 uL of a GFP mRNA sample 1 was combined with 40 uL [Phenol/Chloroform/Isoamyl alcohol](https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1) 2. shaken vigorously inside fume hood for 15 seconds. 3. centrifuged at 12000G at 4C for 2 min 4. the top aqueous layer was transferred to a new epp and combined with a new 40 uL of phenol/chloroform/isoamyl alcohol 5. shaken vigorously inside fumehood for 15 seconds 6. centrifuged at 12000G at 4C for 2 min 7. aqueous layer was transferred to a new epp 8. 100 uL of 96% ethanol was added and shaken 9. centrifuged at 21300G at 4C for 2min (this was a mistake and should have been 10 minutes) 10. The ethanol was removed and 100 ul of 70% ethanol solution was added to the sample. 11. centrifuged at 21300G at 4C for 5 min 12. The ethanol was removed and the palate was dried to air. 13. The pellet was resuspended in 50 uL milliQ. sample labeled: GFP, white box, freezer C1 in frank’s lab 14. Steps 1-13 were repeated with 30 uL FVIII mRNA taken from epps 2, 3, 4 (10 uL each) 15. The samples were analyzed on the regular agarose gel (see below)  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad121-1.webp) Figure 1. Gel electrophoresis of the purified samples  A small band is visible for the GFP, however, with a large smear above and below it. Factor 8 did not appear on the gel, however, as it was taken from an old epp, it may have been degraded prior to purification. The likely explanation for the weak gel signal is the unsterile conditions of the pipette tips and epps as they have not been sterilized after previous use, or the old buffer used to run the gel.  **Conclusion:** More proper sterilization of the equipment is needed prior to every purification procedure. More mRNA of both GFP and factor 8 should be produced. |
| RAD122: Making more FVIII & GFP mRNA **Background:**We try to make some more mRNA for both GFP and F8. The F8 has gone bad, and more GFP mRNA was decided to be produced. |
| 1st of August 2024 Participants:  * Maks * Thijmen  Experimental: We labeled epps 1 to 4 and we used the same protocol as was described in RAD114. Only difference is that we used the WT polymerase instead of the mutated polymerase and also the amount of DNA added to the samples is different due to a lower DNA concentration.   * 1= F8 (4,5 ul) + mut poly (8 ul) * 2= GFP (5 ul) + mut poly (8 ul) * 3= GFP (5 ul) + WT poly (8 ul) * 4= GFP (5 ul) + WT poly (8 ul) * F8 pcdna 1 t/m 4 (pcdna = plasmid cloning DNA).  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad122-1.webp) Figure 1. Gel electrophoresis of the samples obtained from the IVT  As can be seen, the gel shows no bands, likely indicating a mistake during the preparation of the IVT mixture.  **Conclusion:** Since there likely was a mistake in the IVT setup, the experiment should be repeated. |
| RAD123: 2\* and 3\* sample purification **Background:** We try to purify a previously (week or 2/3 ago) made sample, to purify it we used the updated purification method described in RAD120, but we used different amounts of certain ingredients. We used 35 ul of sample (both pipetted in different epps), 35 ul of the phenol chloroform or something (real name is in RAD120), 75ul of ethanol (both 96% and 70%) and last but not least 50 ul of the Mili Q water outside that we followed the normal protocol. |
| 2nd of August 2024 Participants:  * Maks * Thijmen  Experimental: 2\* and 3\* sample for purification. Same protocol as for RAD116 In both cases, 35 ul of sample and 35 ul phenol/chloroform/isoamyl alcohol mixture were initially combined, 75 uL ethanol were used and in the last step the sample was dissolved in 50 uL Milli-Q water. Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad123-1.webp) Figure 1. Gel electrophoresis of the purified samples  As can be seen, only 2 small bands corresponding to the eGFP mRNA are visible, both containing a smear; and no bands are observed that would correspond to factor 8. The likely explanation for that would be mRNA degradation as a result of using non-sterile equipment when handling the samples.  **Conclusion:** The mRNA purification was unsuccessful and should be repeated with sterile equipment and solutions. |
| RAD124: IVT of factor 8 **Background:** The factor 8 from previous entries has degraded so a new batch has to be prepared. DNA stored at -19.7 C is used. Procedure for “RAD 114” is used.   * mRNA storage: * Short-term storage: * 4°C: mRNA can be stored at 4°C for a short period (hours to a few days) if it’s in a buffer containing RNase inhibitors. * Medium-term storage: * -20°C to -80°C: For longer periods (weeks to months), mRNA should be stored at -20°C (for months) or preferably at -80°C (for years). Use a buffer like TE (Tris-EDTA) or other RNA-stabilizing solutions to help protect the mRNA. * Long-term storage: * -80°C: For the longest storage duration (several years), store mRNA at -80°C. Aliquoting the mRNA into small volumes before freezing can help avoid repeated freeze-thaw cycles, which can degrade the RNA. * DNA storage: * Short-term storage: * 4°C: DNA can be stored at 4°C for a short period (days to a few weeks). This is suitable for DNA that will be used frequently. * Medium-term storage: * -20°C: For medium-term storage (weeks to months), DNA should be stored at -20°C. It’s recommended to store DNA in TE buffer at this temperature. * Long-term storage: * -80°C: For long-term storage (months to years), DNA should be stored at -80°C. This is particularly important for DNA that needs to be preserved for extended periods. |
| 13th of August 2024 Participants:  * Adrian * Kate * Thijmen  Experimental:  * Procedure for purification followed from RAD120 * NOTE: samples vortexed as well as vigorously shaked * NOTE: Second and third extractions are performed with 80ul phenol/chloroform/alcohol instead of 20ul * NOTE: 385 ul ethanol in 165 ul milliq to make 67.2% ethanol solution * NOTE: sample with red dot on label was touched without glove * NOTE: When the tray with the samples was being put down after the last centrifuge step, it was knocked over and the step had to be repeated * NOTE: The samples were airdried 30 min * NOTE: results show only phenol/chloroform present; Further washing with 70% ethanol or extraction with just chloroform can help concentrate the sample  Results DNA concentration of the samples in ng/uL   * F81 - 225.5 * F82 - 325.7 * F83 - 32 * F84 - 1144.6 * GFP 4\* - 27.6 * GFP 5\* - 381.2 * Factor 8 unknown - 42.8  Discussion: It seems that the machine showed the phenol contamination, as well as no mRNA present in the samples. This leads us to the conclusion that we will not test these mRNAs on the gel, since no mRNA was left.  **Conclusion:** The experiment needs to be repeated in RAD126. |
| RAD125: Purification of the samples 1, 2, 3, 4 from RAD124 and 4\*, 5\* from RAD115; 1, 2, 5 from RAD114; F8 mRNA (unkown); unlabellled sample (7000 ng/ul [c]) **Background:** In order to conclusively analyse the GFP and FVIII mrna samples from experiments RAD114 , RAD115, and RAD124, the samples will be first purified using phenol chloroform extraction, following the procedure from experiment RAD120. |
| 9th of July 2024 Participants:  * Lea  Experimental:  1. a mastermix for 10 reactions of 20uL was prepared following the protocol for IVT. → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping.    mastermix amounts:    * Tris (1M): 8uL    * MgCl2 (1M): 5uL    * DTT(0.1M): 10uL    * spermidine (25mM): 8uL    * rNTPs(20mM): 40uL    * MilliQ: 129uL    * total: 200uL 2. prepare 10 epps, with each 20uL mastermix 3. 1uL dsDNA of the five different DNA samples (4 FVIII DNA, 1 GFP DNA) was added to epps (for FVIII linearised plasmid, 75 ng of DNA is needed for 20uL reaction, for GFP it is 26ng. The concentration of the FVIII DNA was close enough that 1uL could be taken from the tube. The GFP DNA was diluted 1:1 to a concentration of 30ng/uL and 1uL was used from that ) 4. For every different DNA sample, one epp got 2uL T7 polymerase and the other 4uL. → 2uL is the amount recommended by the protocol, but because of the length of the FVIII gene, we wanted to see if a larger amount of T7 would be beneficial. 5. The reaction was incubated at 37 C for 3 hours. 6. 5uL of all samples was mixed with 2uL loading dye and run on an agarose gel (110V 40 min)  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad112-1.webp) Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.  It is weird that 1, 2 and 4 with 2uL T7 did not show a lot of transcription while 3 seems to have a lot, because they should practically be the same. Overall, the reaction seems to work better with 4uL T7. The FVIII RNA was expected to be longer than 7000 instead of 1200. This could be the result of RNA degradation or self splicing, but it could also be because of the properties of the gel and because RNA is not linear. In addition, the GFP RNA did not work out that well in both cases. it could have something to do with the template. maybe the calculations on the amount of template needed was incorrect.  → I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.  **Conclusion:** Experiment needs to be repeated with proposed changes:   * different version of T7 poly. * Add RNAse inhibitor. * Reduce temperature to prevent autocatalytic breakdown. |
| RAD126: Attempt 2. Purification of the samples 1, 2, 3, 4 from RAD124 and 4\*, 5\* from RAD115; 1, 2, 5 from RAD114; F8 mRNA (unknown); unlabellled sample (7000 ng/ul [c]) **Background:** Experiment RAD125 failed. We need to modify the procedure, and add a step of chloroform purification to prevent phenol contamination. |
| 13th of August 2024 Participants:  * Kate * Thijmen  Experimental:  * Procedure RAD120. * NOTE: For epps GFP 5\*, F8 4, F8 II, F8 1, GFP V there was not enough material. * NOTE: Sample 4\* opened during the first centrifuge, we think that nothing spilled. * NOTE: We added to the regular procedure: 20 ul aqueous/ 20ul organic. + 80 ul MiliQ & + 80 uL Phenol/Chloroform/Isoamyl in all of the steps. So, 100 ml aqueous & 100 ml organic - for better separation. * NOTE: Phenol/Chloroform/Isoamyl mixture was whitish and colloidal. Associated either with how it looks or it went bad. * NOTE: F8 mRNA (unknown), F8 II, F8 4, 5\* had too little of an organic phase. * NOTE: After 2 steps of Phenol/Chloroform/Isoamyl extraction, we have also done an extraction step with chloroform. * Note: roman 2 fell on the centrifuge. * NOTE: Step with 70% ethanol was repeated 2x times. * No pellet was seen after the centrifuge. * Samples GFP 4\*, 5\*, F8 3, F8 1 and unlabelled sample (red dot) dried & 10 ul of MilliQ was added to each. * Not fully dried samples were left in the freezer overnight. * NOTE: Sample GFP 4\* and F8 3 were accidentally pipetted out. * NOTE: No liquid was seen in GFP sample 4\*.  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad126-1.webp) Figure 1. Gel of all the samples except for 5\* GFP, FVIII 1, FVIII 2.  No samples were seen. Since the procedure was repeated twice, the likely reason for no visible bands on the gel is mRNA degradation  → I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.  **Conclusion:** The new samples need to be made in the following experiment. |
| RAD127: Synthesis of factor 8 and GFP mRNA **Background:** The quality of the RNA samples previously made is under question, so a new batch is produced following the procedure from RAD 114 |
| 14th of August 2024 Participants:  * Adrian  Experimental: Mastermix prepared:   * Tris - 24 ul * MgCl2 - 15ul * DTT - 30ul * Spermidine - 24ul * rNTPs - 120ul * Milliq - 387 ul   NOTE: 120ul left after experiment and labelled MM  Samples prepared in following manner added from right to left:   * A: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * B: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * C: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * D: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * E: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * F: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * G: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * H: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * I: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * J: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * K: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * L: 40 ul mastermix; 1 ul Ribulock RNAse inhibitor; * Stored in freezer (-20C) overnight * The next day, the T7 poly was added to all samples, and the mRNA in the following manner: A, B - F8 1, C, D - F8 2, E, F - F8 3, G, H - F8 4; I, J, K, L - GFP cDNA (linearized). * The samples were left for incubation for 4h at 37C, and then left at 4C overnight.  Results and Discussion: RNA concentration in the samples (ng/ul)   * A - 2937.9 * B - 3080.2 * C - 2760.7 * D - 3565.7 * E - 3172.1 * F - 3296.7 * G - 2965.1 * H - 3077.8 * I - 4896.8 * J - 3059.2 * K - 0.3; 4722.5 * L - 3134.5   Low ratio of A230/A260 indicates contamination of the samples, hence sample purification is needed in the following experiment.  **Conclusion:** RNA samples show relatively high levels of contamination. Next step is to purify & analyze this RNA. |
| RAD128: Purification of GFP and FVIII mRNA. Samples from RAD127 **Background:** It was understood that 3 volumes of ethanol for 100 ul aqueous phase and 10 ul 3 mol sodium acetate need to be added since we have increased the amount of aqueous phase from previous experiments. For the step with 70%, it doesn’t matter how much ethanol you add because it is used to collect the remaining contamination.  The gel without formaldehyde - the regular agarose gel (TBE, EtBr) can be |
| 16th of August 2024 Participants:  * Henriete * Thijmen  Experimental:  1. 20uL of a GFP mRNA and F8 mRNA samples (A, L, J, H, F, E) was combined with 100uL Phenol/Chloroform/Isoamyl alcohol (https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1) and 80 ul MilliQ. NOTE: The samples where left on ice before any shaking around XXX minutes due to absence of gloves. samples were shaken vigorously in the fume hood for 15 seconds. 2. centrifuged at 12000 G at 4C for 2 min. NOTE: J was pipetted in F2 (denoted as J2) and F was pipetted in J2 (denoted as F2) 3. another 100 ul of Phenol/Chloroform/Isoamyl alcohol was added to the aqueous samples, shaken and centrifuged for 2 min at 4C 12000G. NOTE: J2 and F2 had no phase separation. they were vortexes again, still nothing. 4. 100 ul of J2 and F2 were taken and the aqueous layers of the other samples. 100 ul of chloroform was added.  Participants:  * Kate * Thijmen  1. 100 ul of chloroform was added and shaken vigorously 2. samples were shaken vigorously in the fume hood for 15 seconds. 3. centrifuged at 12000 G at 4C for 2 min 4. the aqueous phase was transferred to another epp 5. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol 6. samples were shaken vigorously in the fume hood for 15 seconds. 7. centrifuged at 25000G at 4C for 10 min 8. 50 ul of 70% ethanol was added 9. samples were shaken vigorously in the fume hood for 15 seconds. 10. centrifuged at 25000G at 4C for 5 min 11. 50 ul of 70% ethanol was added 12. samples were shaken vigorously in the fume hood for 15 seconds. 13. centrifuged at 25000G at 4C for 5 min 14. 10 ul of milliQ were added to the samples.  Participants:  * Kate  * Denaturing agarose gel was prepared: * 0.35 g of agarose gel was added to 36 ml of MilliQ & 5 ml of 10xMOPS. * It was heated in the microwave for 15 seconds. * In fume hood, 9ml 36.5% formaldehyde was added to solution while continuously stirring. After that, it was stirred for 5 sec more & added to the casting tray. * Waited for gel to solidify - 50 min. |
| 20th of August 2024 Participants:  * Henriete * Thijmen   The samples A-L now are analyzed.  All phases taken were aqueous.   1. 20uL of all GFP mRNA and F8 mRNA samples was combined with 100uL Phenol/Chloroform/Isoamyl alcohol (https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1) and 80 ul MilliQ. 2. centrifuged at 12000 G at 4C for 2 min 3. C2, A2, I2, G2, D2 and H2 did not have phase separation 4. 100 ul chlorophenol was added to the C1, A1, I1, G1, D1 and H1 and vortexed H1 showed phase separation, A1 did not, C, I, G, D did show. These are supposed to be trash. The “organic layer” from the first step. 5. Another step of chlorophenol was added and samples were centrifuged. Also the trash samples were accidentally centrifuged with chlorophenol only once. The original samples – twice. 6. 100 ul of samples (aqueous layer) were taken from the samples and added 300 ul 100% ethanol.    * NOTE: Due to labeling inconsistencies, aqueous & organic layers have to be analyzed by adding 10 ul of miliq to all samples - no separation.    * NOTE: Storage container with all of the materials has been left 15 minutes out of the freezer. (enzymes might go bad).    * The labels that were labeled similarly initially are labeled with a star (C, D, H, G, J). 7. 10 ul of 3 mol sodium acetate was added & the all were centrifuged at 25000G at 4C for 10 min. 8. The ethanol was taken out.  Experimental:  1. a mastermix for 10 reactions of 20uL was prepared following the protocol for IVT. → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping.    mastermix amounts:    * Tris (1M): 8uL    * MgCl2 (1M): 5uL    * DTT(0.1M): 10uL    * spermidine (25mM): 8uL    * rNTPs(20mM): 40uL    * MilliQ: 129uL    * total: 200uL 2. prepare 10 epps, with each 20uL mastermix 3. 1uL dsDNA of the five different DNA samples (4 FVIII DNA, 1 GFP DNA) was added to epps (for FVIII linearised plasmid, 75 ng of DNA is needed for 20uL reaction, for GFP it is 26ng. The concentration of the FVIII DNA was close enough that 1uL could be taken from the tube. The GFP DNA was diluted 1:1 to a concentration of 30ng/uL and 1uL was used from that ) 4. For every different DNA sample, one epp got 2uL T7 polymerase and the other 4uL. → 2uL is the amount recommended by the protocol, but because of the length of the FVIII gene, we wanted to see if a larger amount of T7 would be beneficial. 5. The reaction was incubated at 37 C for 3 hours. 6. 5uL of all samples was mixed with 2uL loading dye and run on an agarose gel (110V 40 min)  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad112-1.webp) Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.  It is weird that 1, 2 and 4 with 2uL T7 did not show a lot of transcription while 3 seems to have a lot, because they should practically be the same. Overall, the reaction seems to work better with 4uL T7. The FVIII RNA was expected to be longer than 7000 instead of 1200. This could be the result of RNA degradation or self splicing, but it could also be because of the properties of the gel and because RNA is not linear. In addition, the GFP RNA did not work out that well in both cases. it could have something to do with the template. maybe the calculations on the amount of template needed was incorrect.  → I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.  **Conclusion:** Experiment needs to be repeated with proposed changes:   * different version of T7 poly. * Add RNAse inhibitor. * Reduce temperature to prevent autocatalytic breakdown.  Participants:  * Kate   Denaturing agarose gel was prepared:   * 0.35 g of agarose gel was added to 36 ml of Demi water * It was heated in the microwave for ~ 15 seconds. * In fume hood, 5 ml of 10xMOPS were added, and then 9ml 36.5% formaldehyde was added to solution while continuously stirring. After that, it was stirred for 5 sec more & added in casting tray. * Waited for gel to solidify - 50 min. |
| 21st of August 2024 Participants:  * Henriete * Thijmen   Samples from Friday were noted with 5 at the end of the letter (A, E, H, J, L, F) (ex. A5)  Samples from Tuesday 20.08 were labeled with 2d.   1. Total 13 samples including positive control eGFP 2. 4.5 ul loading dye was added to 1.5 ul of sample. 3. The samples were incubated at 70 degrees for 10 minutes 4. Gel was set to 110 V, 400 A, 20 min (double welled gel) 5. Samples were run in this direction:  * 1st row: F5 A5 L5 H5 E5 J5 eGFP * 2nd row: B2d L2d J2d F2d K2d E2d |
| 21st of August 2024 Participants:  * Henriete * Thijmen   Note for agarose gel:  Instead of 0,7 g of agarose, there was 0,82 g of agarose added. This will probably not influence the results but in the future it should be 0,7 g of agarose.  Instead of the normal 0,5x TBE buffer we used in this gel a 1x TBE buffer. This was done by mixing 10 ml of 10x TBE with 90 ml of demi-water.   1. A 1x TBE buffer was made (see notes), to this 0,82 g (see notes) of agarose was added. 2. After this the mixture was swirled and placed in the microwave. Here it was microwaved until the mixture became clear/see through. 3. After this 5 ul of ethidium bromide was added to the mixture after which it was swirled. Now the gel was poured into the tray and the comb was placed in the liquid gel. Now the gel was left to stoll (is this a word?). When the gel was solidified. 4. Gel solidified for 50 minutes. 5. Gel was cut in half. 6. The gel was loaded with a GFP test sample.   Another gel was prepared:  all amounts halved: 45 ml demi H20 + 5 ml TBE 10x. + 2.5 ul EtBr.  This gel was loaded left to right: H5, I2d, F5, K2d, E5, I2d\*, J2d\*, A5, C2d\*, H2d\*, B2d, D2d\*, F2d. (Figure 6) |
| 23rd of August 2024 Participants:  * Kate * Thjmen   The new gel was prepared: 100 ml + 0.7 g agarose was heated in the microwave, but the solution boiled, and ~ 25 ml went out. I only took 50 from the solution. 2.5 ul EtBr was added and continuously swirled before added to the tray. The gel was poured in the tray, and cooled for 50 min.  The samples were prepared for loading: 9ul of loading die (2x LB RNA) + 1 ul of sample (14 samples in total) Incubated for 10 min at 70 C. Results and Discussion:![labeling and order](https://static.igem.wiki/teams/5342/images/notebook/rad128-1.webp) Labeling: A, B - F8 1, C, D - F8 2, E, F - F8 3, G, H - F8 4; I, J, K, L - GFP ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad128-2.webp) Figure 1. Data lost. Bright - GFP, Dim - FVIII. Most probably: A, E, F, H, J, L ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad128-3.webp) Figure 2. Mutated poly FVIII, wild poly FVIII, GFP test ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad128-4.webp) Figure 1. Loading order: 1st row: F5 (F8) A5 (F8) L5 (eGFP) H5 (F8) E5 (F8) J5 (eGFP) eGFP (test); 2nd row: B2d (F8) L2d (GFP) J2d (GFP) F2d (F8) K2d (GFP) E2d (F8)  Nanodrop:  Sample L and E (showed empty) from Tuesday had errors and also I\* and G\* had errors. K4 has very low concentration. F2d had very high concentration, but didn’t show on the gel.  Friday (5):   * A5 - 688.9 ng/ul * E5 - 908.1 ng/ul * H5 - 2374.7 ng/ul * J5 - 728.5 ng/ul * L5 - 2220.9 ng/ul * F5 - 254 ng/ul   Tuesday: ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad128-5.webp) Figure 2. Concentrations tuesday 1 ![Figure 3](https://static.igem.wiki/teams/5342/images/notebook/rad128-6.webp) Figure 3. Concentrations tuesday 2  The nanodrop showed the concentrations of following samples to be faulty: L2d, E2d, I2d\* and G2d\*. E2d got empty. The analysis was repeated.  They were remeasured with nanodrop: L2d 28.2 ng/ul, others did not give any results (probably, since they had no RNA (the faulty ones due to mislabelling), or due to the presence of ethanol - quick evaporation (probably, ethanol is also present in the L2d). ![Figure 4](https://static.igem.wiki/teams/5342/images/notebook/rad128-7.webp) Figure 4. Masses of the RNAs and their concentrations  The following total masses of RNAs were received. It is weird that F5 has very big concentration, but is not visible on the gel. Same for the B2d, F2d. The gel results are hampered by Ethidium Bromide, and were repeated, but it can be concluded that the samples loaded seem to show the presence of valid compounds, so with that assumption experiments are decided to be continued with these samples. ![Figure 5](https://static.igem.wiki/teams/5342/images/notebook/rad128-8.webp) Figure 5. Test GFP on the cut gel  The new gel made was too thick, so the cutting procedure was done to make it thin, which was successful. The gel made afterwards was made by the techniques described in the background. ![Figure 6](https://static.igem.wiki/teams/5342/images/notebook/rad128-9.webp) Figure 6. This gel was loaded left to right: H5, I2d, F5, K2d, E5, I2d\*, J2d\*, A5, C2d\*, H2d\*, B2d, D2d\*, F2d.  The GFP mRNA has left the gel, so the gel should be run for less than 50 min (blues line in the middle) ![](https://static.igem.wiki/teams/5342/images/notebook/rad128-10.webp) ![Figure 7](https://static.igem.wiki/teams/5342/images/notebook/rad128-11.webp) Figure 7. F8 mRNA G2d sample visible, row 10. Visible: 1 (band), 3, kinda 4, 8, 10, 12; Loading order: Frank’s F8; A2d; A5; B2d; C2d; D2d\*; E5; F4; F5; G2d; H2d; H5; I2d; Frank GFP  Only F8 G2d was seen, and no GFP - probably ran away.  **Conclusion:**The FVIII mRNA (G2d) was received in the sufficient amount for further modification - capping and tailing, 7.5 ul, 1428 ng/ul, 10.71 ug.  **Next steps:**   * Tailing and then capping and analyze with CE * New IVT of both GFP and FVIII * Combine all F8 (KEEP G2d PURE!!!) and GFP samples for capping and tailing * Analyze combined F8 on the gel again |
| RAD131: IVT and Purification of F8 and GFP samplesA **Background:** Preparation of new GFP and factor 8 mRNA, purification and analysis on 0.7% and 2% agarose gel |
| 26th of August 2024 Participants:  * Henriete * Thijmen   IVT was performed by following the following protocol:   1. a mastermix for 10 reactions of 40 ul was prepared by adding the following compounds to an empty epp. mastermix amounts:    * Tris (1M): 16uL    * MgCl2 (1M): 10uL    * DTT(0.1M): 20uL    * spermidine (25mM): 16uL    * rNTPs(20mM): 80uL    * MilliQ: 258uL    * total: 400uL 2. The master mix was then very shortly vortexed and divided over 10 PCR tubes labelled A to J. 3. After this 1ul RiboLock RNase inhibitor, 1.78 ul sample (F8-1, F8-2, F8-3, F8-4 and GFP) and 8ul polymerase was added to each tube. 4. After this the samples were incubated for 3 hours at 37 degrees and stored in the freezer overnight.   What DNA each tube contains (I have to check the freezer for this):   * A = F8 * B = F8 * C = GFP * D = F8 * E = F8 * F = F8 * G = GFP * H = F8 * I = F8 * J = F8 |
| 27th of August 2024 Participants:  * Thijmen   This day I wanted to start with purification of the samples from 26-08-2024, but due to phenol oxidation and lack of time, it couldn’t be done. What was done today is as follows:   * 20 ul of sample was pipetted out of the PCR tubes and pipetted into epps labelled A to J. * To each epp 80 ul of mili Q water was added. |
| 28th of August 2024 Participants:  * Thijmen   This day the purification was performed and analyzed. The purification was done in the following way:   1. 100uL Phenol/Chloroform/Isoamyl alcohol was added to each epp. 2. The epps were shaken vigorously for 15 seconds in the fume hood. 3. The epps were centrifuged for 2 minutes at 12000 rpm at 4C. 4. The aqueous phase was transferred to another epp. 5. 100uL Phenol/Chloroform/Isoamyl alcohol was added to each epp. 6. The epps were shaken vigorously for 15 seconds in the fume hood. 7. The epps were centrifuged for 2 minutes at 12000 rpm at 4C. 8. The aqueous phase was transferred to another epp. 9. 100uL chloroform was added to each epp. 10. The epps were shaken vigorously for 15 seconds in the fume hood. 11. The epps were centrifuged for 2 minutes at 12000 rpm at 4C. 12. The aqueous phase was transferred to another epp. 13. 10 ul of 3 mol sodium acetate was added to each sample 14. 300 ul of 96% (or higher) ethanol was added to each epp. 15. Samples were centrifuged for 10 minutes at 25000 rpm at 4C 16. The ethanol was pipetted out of the epps with care to not disturb the pallet (not every epp had a pallet but most did). 17. 50 ul of 70% ethanol was added. 18. Samples were shaken vigorously in the fume hood for 15 seconds. 19. Centrifuged at 25000G at 4C for 5 min. 20. The ethanol was pipetted out of the epps with care to not disturb the pallet (not every epp had a pallet but most did). 21. 50 ul of 70% ethanol was added to the epps. 22. Samples were shaken vigorously in the fume hood for 15 seconds. 23. Centrifuged at 25000G at 4C for 5 min. 24. The ethanol was pipetted out of the epps with care to not disturb the pallet (not every epp had a pallet but most did). 25. The epps were left to dry for about 20 to 30 minutes. 26. When the epps were dry 10 ul of mili Q water was added to the epps. The epps were labelled as “sample letter + AQ3” for example sample A was labelled as “A AQ3”.   After the purification was done the samples were loaded on a gel which was made the day before. To load the samples the following protocol was used:   1. In each epp 9 ul of loading dye was pipetted and 1 ul of sample. 2. Samples were then lightly vortexed. 3. The epps were then placed in a heating block for around 10 minutes at 70 degrees. 4. After this 9 ul of the mixture was pipetted out of the epp and into the gel. The wells were filled in the following order: A-B-C-D-E-F-G-H-I-J-F8,1 (capped and tailed)-F8,2 (capped and tailed)-GFP (capped and tailed). 5. The gel was then run for 40 minutes at 120 V. 6. After this the gel was placed on a UV machine (don't know the exact name) and was analysed.   Further notes: Sample A probably has a bit less volume because of a pipetting error (shouldn’t be more then 1 ul). During the loading the wells looked a bit weird from sample E onwards, it was like there was an air bubble in there. Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad131-1.webp) As can be seen, the gel looks empty which could have multiple reasons, to be sure what the actual reason is that it looks this way Frank is consulted the next day. Frank said that the gel looks very blue while it should look more purple/pink. The reason why it is blue is probably because the gel doesn’t contain any ethidium bromide.  **Conclusion:** The gel looks empty because of a lack of ethidium bromide in the gel. For future work it is advised to make a new gel and rerun the samples. During the gel running the samples have also been nano-dropped. For this 1,5 ul of each sample was put in the nanodrop machine. Results and Discussion:![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad131-2.webp) Purely based on the nanodrop results the samples look very promising, they have high concentrations with a good clean curve and no sign of contamination.  **Conclusion:** Samples look good in the nanodrop, only the gel will tell us whether the samples are truly good. For future await gel results. |
| 29th of August 2024 Participants:  * Thijmen   By the advice of Frank a new gel was prepared today and also a ‘gel making station’ was setup in lab 3 (from 30-08-24 in lab 4).  The gel was made in the following way:   1. 1L of 1x TBE buffer was made by adding 100 mL of 10X TBE to 900 mL demi-water. This Liter of 1X TBE was used to make the gel and to fill the electrophoresis basket. 2. 0,7g of agarose was weighed and placed in an Erlenmeyer containing 100 mL 1X TBE buffer. 3. This mixture was then heated in the microwave until it was clear and see-through. 4. After this 5 ul of ethidium bromide was added to the mixture. 5. The mixture was poured into the gel tray where is was solidified over a 50 minute period. 6. Next, the gel tray was placed in the electrophoresis basket and the basket was filled with 1X TBE buffer until the gel was submerged. 7. After this the gel was ready to be loaded. 8. This was again done by mixing 9 ul of loading dye with 1 ul of sample. 9. This mixture was then denatured in a heating block for 10 minutes at 70 degrees. 10. After this 9 ul of the mixture was pipetted out of the epp and into the gel. The wells were filled in the following order: A-B-C-D-E-F-G-H-I-J-F8,1 (capped and tailed)-F8,2 (capped and tailed)-GFP (capped and tailed)-Positive sample. 11. The gel was then run for 30 minutes at 120 V. 12. After this, the gel was placed on a UV machine (don't know the exact name) and was analysed.  Results and Discussion:![Figure 4](https://static.igem.wiki/teams/5342/images/notebook/rad131-3.webp) Most of the samples that were loaded didn’t show up on the gel. We did see a clear band for sample C and the modified GFP. We also see some very vague smears at samples B, D and E. We think that there is again something wrong with the gel because the gel results and the nanodrop results do not match. What is also weird is that our positive sample didn’t show up which could be both a fault in the gel or a fault in the positive sample.  **Conclusion:** After consulting with Frank, the most likely reason why the gel doesn’t show anything and the nanodrop shows high concentrations is that the mRNA probably is degraded into little mRNA pieces, which would then run out of the 0,7% agarose gel but will still show a high concentration on the nanodrop. The modified GFP looks good and can be used for vesicle experiments!! In the future, it is recommended to rerun the bad samples (so not sample C and modified GFP) on a 2% agarose gel to see whether there is degraded mRNA. Also, the modified GFP can be used in future experiments, the modified F8 can not be used until further notice. |
| 30th of August 2024 Participants:  * Thijmen   Frank advised to make a 2% agarose gel so that’s what was done as follows:   1. 100 mL 1X TBE buffer was made by adding 10 mL of 10X TBE buffer to 90 mL of demi water. This was made in an Erlenmeyer. 2. To this Erlenmeyer 2 g of agarose was added. 3. This mixture was heated in the microwave until it became clear and seethrough 4. After this 5 ul of ethidium bromide was added to the mixture. 5. The mixture was poured into the gel tray where is was solidified over a 50 minute period. 6. Next, the gel tray was placed in the electrophoresis basket. 7. After this the gel was ready to be loaded for the first time. 8. This was again done by mixing 9 ul of loading dye with 1 ul of sample. 9. This mixture was then denatured in a heating block for 10 minutes at 70 degrees. 10. After this 9 ul of the mixture was pipetted out of the epp and into the gel. The wells were filled in the following order: A-F8,1 (capped and tailed). 11. The gel was then run for 10 minutes at 120 V. 12. After this, the gel was placed on a UV machine (don't know the exact name) and was analysed.  Results and Discussion:![Figure 4](https://static.igem.wiki/teams/5342/images/notebook/rad131-4.webp) If you closely observe the gel you can see that the first loaded gel (sample A) has something in it. This might be the degraded mRNA of which Frank spoke. This is of course not certain but it is an indication.  **Conclusion:** We decided to load all the bad samples and run them for a longer period of time to see if we could spot more of these possible fragmented pieces of mRNA.   1. After this the gel was ready to be loaded for the second time. 2. This was again done by mixing 9 ul of loading dye with 1 ul of sample. 3. This mixture was then denatured in a heating block for 10 minutes at 70 degrees. 4. After this 9 ul of the mixture was pipetted out of the epp and into the gel. The wells were filled in the following order: B-D-E-F-G-H-I-J-F8,2 (capped and tailed)-Positive sample. 5. The gel was then run for 30 minutes at 120 V. 6. After this, the gel was placed on a UV machine (don't know the exact name) and was analysed.  Results and Discussion:![Figure 5](https://static.igem.wiki/teams/5342/images/notebook/rad131-5.webp) We can see in the gel that sample G looks very good, it looks bright and pure which is weird given that it didn’t show up like this on the previous gel. Why this is we don’t know and we should consult Frank about this. Furthermore, we can see that samples A, D and J are mainly a big vague smear which could indicate that these samples contain degraded mRNA fragments. Last but not least we see that the positive control sample shows up in this gel as a little pure line. This is good but also weird because why does it show up here and not on the previous gel? This is again something to discuss with Frank.  **Conclusion:** Sample G could be a good purified sample which can be used for capping and tailing but it must first be discussed with Frank because why didn’t it show up on the previous gel? As for samples A, D and J we can say that these are degraded mRNA fragments but just to check show Frank. Also, ask Frank why the positive sample shows up on this gel but not the previous one. I think these are the best steps to take for the next lab day. |

mRNA Purification and Modification

26/08/24 - 11/09/24

* Henriete
* Lea
* Maks
* Thijmen

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| RAD129: Cap-0 synthesis using Vaccinia Capping Enzyme (NEB #M2080) **Background:** We used the following [capping protocol](https://www.neb.com/en/protocols/0001/01/01/capping-protocol-m2080) for the following.  Post-transcriptional capping and Cap-1 methylation  Post-transcriptional capping is often performed using the mRNA capping enzyme from Vaccinia virus or Faustovirus. This enzyme complex converts the 5´-triphosphate ends of in vitro transcripts to m7G-cap (Cap-0) required for efficient protein translation in eukaryotes. The Fausto Virus capping enzyme ([NEB #M2081](https://www.neb.com/en/products/m2081-faustovirus-capping-enzyme)) comprises three enzymatic activities (RNA triphosphatase, guanylyltransferase, guanine N7-methyltransferase) that are necessary for the formation of the complete Cap-0 structure, m7Gppp5´N, using GTP and the methyl donor S-adenosylmethionine. As an added option, the inclusion of the mRNA Cap 2´ O-Methyltransferase (NEB #M0366) in the same reaction results in formation of the Cap-1 structure (m7Gppp5´Nm), a natural modification in many eukaryotic mRNAs responsible for evading cellular innate immune response against foreign RNA.  This enzyme-based capping approach results in a high proportion of capped message, and it is easily scalable. The resulting capped RNA can be further modified by poly(A) addition before final purification.  This protocol is designed to cap up to 10 µg of RNA (100 nt or larger) in a 20 µl reaction. Reaction size can be scaled up, as needed. The system provides enough reagents to perform 40 reactions at the 10 µg RNA/20 µl reaction scale.  Samples:   1. C1: 13.5 ul rna (A2d +C2d) + 1.5 ul MilliQ = 15 ul 2. C2: 7.5 ul rna (G2d) + 7.5 ul MilliQ = 15 ul 3. GFP: 6 ul rna (L5) + 9 ul MilliQ = 15 ul   Aliquots of 2ul of SAM. +30 ul milliq = 32 ul of SAM (2 mmol) (Because SAM degrades very easily and for every sample, new aliquot was used)  For GFP: 13.3 ug = 1.33 times more than specified in protocol, so the volumes of materials for the GFP sample were increased. |
| 26th of August 2024 Participants:  * Henriete * Thijmen  Experimental:  1. RNA and MilliQ in a 1.5 ml microfuge tube was added to a final volume of 15.0 µl. 2. Heated at 65°C for 5 minutes. 3. Samples were left on ice for 5 min. 4. the following components were added in the order specified:    * Denatured RNA (from above): 15.0 µl for F8 , 19.95 ul for GFP    * 10X Capping buffer: 2.0 µl for F8, 2.66ul for GFP    * GTP (10 mM) :1.0 µl for F8, 1.33 ul for GFP    * SAM (2 mM, dilute 32 mM stock to 2 mM): 1.0 µl for F8, 1.33ul for GFP    * Vaccinia Capping Enzyme: 1.0 µl for F8, 1.33 ul GFP. Incubate at 37°C for 30 minutes. 5. Incubate at 37°C for 30 minutes. 6. RNA is now capped and ready for use in downstream applications.  * NOTE: when adding the GTP, 1.33 ul was accidentally added to all samples. It technically should not change the proficiency of capping as it is the guanosine triphosphate. * NOTE: The samples were prepared until vaccina enzyme and left on ice for a certain time due to the heating block being too hot and took a while to cool down   Purification of the capped samples:   1. 80 ul MilliQ and 100 ul of chloroform was added and shaken vigorously 2. samples were shaken vigorously in the fume hood for 15 seconds. 3. centrifuged at 12000 G at 4C for 2 min 4. the aqueous phase was transferred to another epp 5. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol 6. samples were shaken vigorously in the fume hood for 15 seconds. 7. centrifuged at 25000G at 4C for 10 min 8. 50 ul of 70% ethanol was added 9. samples were shaken vigorously in the fume hood for 15 seconds. 10. centrifuged at 25000G at 4C for 5 min 11. Pellet was dried and 10 ul MilliQ was added.  Results and Discussion: As far as we can tell the capping was done successfully since we followed the provided protocol, however, we can’t check right now if it has been successful since we will only place the samples on the gel when the whole RNA modification is done. Tomorrow we will continue with the tailing so for now the samples are stored in the freezer (-20 degrees Celcius).  **Conclusion:** The RNA capping has been done successfully, so we can proceed with tailing the samples tomorrow. |
| RAD130: A-tailing using E. coli Poly(A) Polymerase **Background:** The tailing protocol that we used is found [here](https://static.igem.wiki/teams/5342/documents/protocols/in-vitro-synthesis-of-rna-using-t7-rna-polymerase.pdf).  The poly(A) tail confers stability to the mRNA and enhances translation efficiency. The poly(A) tail can be encoded in the DNA template by using an appropriately tailed PCR primer, or it can be added to the RNA by enzymatic treatment with E. coli Poly(A) Polymerase (NEB #M0276). The length of the added tail can be adjusted by titrating the Poly(A) Polymerase in the reaction (Figure 6).  The importance of the A-tail is demonstrated by transfection of untailed vs. tailed mRNA. When luciferase activity from cells transfected with equimolar amounts of tailed or untailed mRNAs were compared, a significant enhancement of translation efficiency was evident (Figure 6). HiScribe T7 ARCA mRNA Synthesis Kit (with tailing) (NEB #E2060) includes E. coli Poly(A) Polymerase, and enables a streamlined workflow for the enzymatic tailing of co-transcriptionally capped RNA.  [<https://www.neb.com/en/tools-and-resources/feature-articles/minding-your-caps-and-tails> ] |
| 27th of July 2024 Participants:  * Henriete   GFP sample has 13.3 ug = 1.33 times more than required in protocol, so the volumes of materials for the GFP sample were increased. To make 1X E. coli Poly(A) Polymerase (5X) Reaction Buffer: 4ul E. coli Poly(A) Polymerase + 16 ul water =20 ul 1x E. coli Poly(A) Polymerase Reaction Buffer  Add the following components in the order specified:   1. RNA + MilliQ to a total volume of 15 ul for F8 (19.95 ul for GFP) 2. 5X E. coli Poly(A) Polymerase Reaction Buffer: 2 µl (1X) for F8 (2.66 ul for GFP) 3. ATP (10mM): 2 µl for F8 (2.66 ul for GFP) 4. E. coli Poly(A) Polymerase: 1 µl for F8 (1.33 ul for GFP) 5. Total volume 26.6 ul for GFP and 20 ul for F8 6. Incubate reaction at 37°C for 30 minutes. 7. Stop the reaction by adding EDTA to a final concentration of 10 mM or directly proceeding to the cleanup step.  Participants:  * Henriete  1. 80 ul (73.4 ul for GFP sample) MilliQ and 100 ul of chloroform was added and shaken vigorously 2. samples were shaken vigorously in the fume hood for 15 seconds. 3. centrifuged at 12000 G at 4C for 2 min 4. the aqueous phase was transferred to another epp 5. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol 6. samples were shaken vigorously in the fume hood for 15 seconds. 7. centrifuged at 25000G at 4C for 10 min 8. 50 ul of 70% ethanol was added 9. samples were shaken vigorously in the fume hood for 15 seconds. 10. centrifuged at 25000G at 4C for 5 min 11. Pellet was dried and 10 ul MilliQ was added.   Note: in step 5 only 70% ethanol was added instead of 100%. The 30% water might have dissolved the rna. The samples were centrifuged at 30000G for 5 min again. 100 ul 100% ethanol was added and samples were centrifuged for 5 min at 25000G. Participants:  * Henriete  1. 100 ml TBE 1x buffer : 90 ml Milliq + 10 ml TBE (10X) 2. 50 ml buffer + 0.35g agarose + 2.5 ul EtBr. 3. Gel was set to solidify for 50 min  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad130-1.webp) Figure 1. gel electrophoresis of modified mRNA (well 11: modified F8 mRNA 1, 12: modified F8 mRNA 2, 13: modified GFP mRNA, 14: positive control) samples to the left belong to RAD131.  Only the modified GFP mRNA is visible on this gel. Suggesting that capping and tailing of this RNA was successful, but that of the F8 RNA was not. This could be because the F8 RNA was already less stable.  **Conclusion:** The modification was successful on the GFP RNA, but the F8 RNA should be repeated. To get around our problems with purifying the F8 RNA, Frank tried our method. |
| RAD132: Capping and tailing of GFP (sample G) and F8 mRNA **Background:** In this experiment, a second attempt at tailing and capping is done. In the last experiments (RAD129-RAD130), a small amount of GFP RNA was successfully modified. modification of F8 RNA was not yet successful, but we received a large amount of purified F8 RNA from our supervisor Frank which might have a better quality and will hopefully stay intact during the tailing and capping. |
| 4th of September 2024 Participants:  * Lea * Thijmen   Capping:  a new 2 mM SAM dilution was prepared: 2uL (32mM)SAM + 30uL MilliQ. Sample prep:   1. 3.3 uL of sample G was used to get 10ug and diluted with MilliQ to a final volume of 15uL 2. 3.6 ul F8 RNA was used to get 10ug and diluted with MilliQ to a final volume of 15uL 3. Heated at 65C for 5 min 4. placed on ice for 5 min 5. the following components were added in the order specified:    * 10X Capping buffer: 2.0 µl    * GTP (10 mM):1.0 µl    * SAM (2 mM, dilute 32 mM stock to 2 mM): 1.0 µL    * Vaccinia Capping Enzyme: 1.0 µl. 6. Incubate at 37°C for 30 minutes. (left the sample on ice for a bit while waiting for the heating block to cool down   purification: (same protocol as in RAD129)   1. 80 ul MilliQ and 100 ul of chloroform was added to the sample and shaken vigorously in the fumehood for 15 seconds 2. centrifuged at 12000 G at 4C for 2 min 3. the aqueous phase was transferred to another epp 4. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol. samples were shaken vigorously in the fume hood for 15 seconds. 5. centrifuged at 25000G at 4C for 10 min 6. supernatant was removed and discarded 7. 50 ul of 70% ethanol was added 8. samples were shaken vigorously in the fume hood for 15 seconds. 9. centrifuged at 25000G at 4C for 5 min 10. supernatant was removed and the pellet was dried to the air 11. pellet was resuspended in 15ul milliQ |
| 5th of September 2024 Participants:  * Lea * Thijmen   tailing: (same protocol as RAD130)   1. reaction mixtures for both samples were prepared by adding the following components:    * RNA: purified sample    * 4ul 5X E. coli Poly(A) Polymerase Reaction Buffer    * 2ul ATP (10mM)    * 1ul E. coli Poly(A) Polymerase 2. Incubate at 37°C for 30 minutes 3. purification:(same protocol as above) 4. NOTE: a different centrifuge was used with a max RPM of 20000 instead of 25000 |
| 6th of September 2024 Participants:  * Maks * Thijmen   analysis:  The two samples were analyzed on a 0.7% 50 min 110V Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad132-1.webp) Figure 1. After capping and tailing the gel showed no stain  Even though the capping went well (pellet was seen), the tailing went bad (no pellet, even though the pellet should become even bigger). This might be due to absence of Mg2+ in the tailing enzyme.  **Conclusion:** The capping procedure from the experiment RAD 132 worked out. However, after the tailing procedure no product was seen. The gel showed no stain. The Mg2+ will be added to the tailing enzyme. |
| RAD133: Capping and tailing of GFP (sample G) and F8 mRNA **Background:** The capping procedure from the experiment RAD 132 worked out. However, after the tailing procedure no product was seen. The gel showed no stain. No MnCl2 was added in the last experiment, this would have inhibited the poly(A) formation. However, it does not explain the lack of yield. In this experiment, the procedure is repeated with minor adjustments and with |
| 11th of September 2024 Participants:  * Kate * Lea * Thijmen  Experimental:  * 2 mM SAM dilution was prepared: 2uL (32mM)SAM + 30uL MilliQ * 8 samples were prepared (4GFP & 4 F8): * F8: 3.6 ul F8 RNA was used to get 10ug and diluted with MilliQ to a final volume of 15uL (4x) * GFP(1): 3.3 uL of sample G was used to get 10ug and diluted with MilliQ to a final volume of 15uL * GFP(4): 6.68 ul GFP from RAD120 1 to get 10ug and diluted with MilliQ to a final volume of 15uL * GFP(2,3): 5.47 ul GFP from RAD120 2 to get 10ug and diluted with MilliQ to a final volume of 15uL * NOTE: G sample is empty.  ![Data table](https://static.igem.wiki/teams/5342/images/notebook/rad-133-0.webp) Samples were incubated at 65C for 5 min  Samples were cooled on ice for 5 min  The following components were added in the order specified:   * 10X Capping buffer: 2.0 µl * GTP (10 mM):1.0 µl * SAM (2 mM, dilute 32 mM stock to 2 mM): 1.0 µL * Vaccinia Capping Enzyme: 1.0 µl.   Incubate at 37°C for 30 minutes  Purification: (same protocol as in RAD129)  For each sample:   1. 80 ul MilliQ and 100 ul of chloroform was added to the sample and shaken vigorously in the fumehood for 15 seconds 2. centrifuged at 12000 G at 4C for 2 min 3. the aqueous phase was transferred to another epp 4. 10ul of 3 mol sodium acetate was added together with 300 ul ethanol. 5. centrifuged at 25000G at 4C for 10 min 6. Supernatant was removed and discarded 7. 50 ul of 70% ethanol was added 8. samples were shaken vigorously in the fume hood for 15 seconds. 9. centrifuged at 25000G at 4C for 5 min 10. supernatant was removed and the pellet was dried to the air 11. Pellet was resuspended in 11 uL milliQ 12. Samples were stored at -20C |
| 13th of September 2024 Participants:  * Lea * Thijmen   Tailing:  For every sample: the following components were added:   * RNA: purified sample in 11 uL milliQ * 4ul 5X E. coli Poly(A) Polymerase Reaction Buffer * 2ul 25mM MnCl2 (final conc. Of 2.5 mM) * 2ul ATP (10mM) * 1ul E. coli Poly(A) Polymerase * Final volume: 20uL   NOTE: potential small pipetting error in F8-1 with adding PAP  Incubated at 37C for 30 min.  Purification:  Analysis:   1. 0.7% agarose gel in 1×PBE buffer 2. We recieved 3 samples (2xF8, 1xEGFP) capped and toilet RNA from Frank to also be analysed -> named FF81, FF82 and FGFP 3. Samples were prepaired with 9ul loading dye, 1ul sample, heated to 70C for 20min 4. Samples were loaded in the following order: F81-4, GFP1-4, FF81-2, FGFP.  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad133-1.webp)  * 0.7% Agarose gel electrophoresis of denatured mRNA samples: * 1st, 2nd, 3rd, 9th row shows a smear (F8), 11th row shows a band (GFP form Frank) * Other lanes are empty * Reason assumed: concentrations of the sample are too low to be visible.   Nanodrop results:   |  |  | | --- | --- | | F8 1 | 417.6 ng/ul | | F8 2 | 284.3 ng/ul | | F8 3 | 464.4 ng/ul | | F8 4 | 586.4 ng/ul | | GFP 1 | 501.5 ng/ul | | GFP 2 | 767.2 ng/ul | | GFP 3 | 426 ng/ul | | GFP 4 | 848.8 ng/ul |   **Conclusion:** The concentration of RNA was too low to be seen on the gel, but due to the time constraints, it was decided to move on with this RNA. |

Ionizable Lipids Preparation

31/05/24

* Adrian
* Kate
* Lea

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| RAD201: Preparation of 246 ionizable lipids **Background:** The ionizable lipids are synthesized by an epoxide ring-opening reaction: ![epoxide ring-opening reaction](https://static.igem.wiki/teams/5342/images/notebook/rad201-1-webp.webp) The lipid nanoparticle comprising an ionizable lipid significantly affects the characteristics of the LNPs. They can increase or decrease the drug encapsulation efficiency; create more or less uniformly-sized particles; and improve the effectiveness of the drug delivery into the hepatocytes or LSEC cells.  Ionizable lipids are crucial for LNP preparation, and they provide effective mRNA encapsulation, as well as safe LNP delivery in the body. ![table](https://static.igem.wiki/teams/5342/images/notebook/rad201-2-webp.webp) <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9691360/>  **Equipment:**   * Flash column * Magnetic stirrer 300 rpm * Three neck round bottom flask (25 ml) * Reflux cooler. * Drying neck on top * Hot plate * Oil bath * Temperature probe * TLC plates, can * Micropipette, 1000 ul   **Chemicals/Solutions**   * 1,4-Bis(3-aminopropyl) piperazine were purchased from Sigma-Aldrich * 1,2-epoxydodecane; Sigma-Aldrich   **Safety aspects and precautions:**   * Epoxide: Irritative, Category 10 flammable. * 1,4-Bis(3-aminopropyl) piperazine: Corrosive (Causes severe skin burns and eye damage), 8A – Combustible. Bis (aminopropyl) piperazine appears as a colorless liquid with a faint fishlike odor. Corrosive to tissue. Burns, although requiring some effort to ignite. Produces toxic oxides of nitrogen during combustion. |
| 9th of July 2024 Participants:  * Adrian * Kate * Lea  Experimental:  1. The 5 ml of 1,2-epoxydodecane were added to 25 ml roundbottom flask. 2. The 0.981 ml 1,4-Bis(3-aminopropyl) piperazine were added to the vessel as well. Upon addition, the mixture became whitish-cloudy and then cloudiness has dissappeared. 3. The reaction was heated up to 90C. 4. TLC was conducted on the 1,2-epoxydodecane & 1,4-Bis(3-aminopropyl) piperazine:    1. 10%MeOH/DCM: 0.5ml/4.5ml + triethyl amine: 0.2ml; Rf(1,2-epoxydodecane) = 0; Rf(1,4-Bis(3-aminopropyl) piperazine) = 1.5/6.3 = 0.24  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-3-webp.webp)    2. 15%MeOH/DCM: 1.5ml/8.5ml + triethyl amine: 3 drops; Rf(1,2-epoxydodecane) = 0; Rf(1,4-Bis(3-aminopropyl) piperazine) 1/6.8= 0.15  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-4-webp.webp) 5. TLC was conducted on the product & 1,4-Bis(3-aminopropyl) piperazine:    1. 15%MeOH/DCM: 2ml/11ml + triethyl amine: 5 drops (0.4 ml); Almost no starting material was seen. However, the Rf value was too big. Rf(product) = 4.7/5.1 = 0.92; Rf(1,4-Bis(3-aminopropyl) piperazine) = 0.5/4.4 = 0.113  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-5-webp.webp)    2. 5%MeOH/DCM: 0.5ml/9.5ml + triethyl amine: 0.3 ml; Two spots were accidentally left for the product next to each other; Rf prod = 1.2 / 3.4 = 0.35; Rf epoxide = >0.95; Rf amine = 0.07;  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-6-webp.webp)    3. Reaction was left to run for 1 more day. TLC: 5%MeOH/DCM: 0.5ml/9.5ml + triethyl amine: 0.3 ml; TLC showed presence of product and epoxide and no signs of NH2, indicating that the reaction has gone to completion. RF product = 0.38; RF epoxide = 9.25; RF NH2 = 0.09  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-7-webp.webp) 6. The product was left in the freezer at -25C, as yellow crude oil.  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-8-webp.webp) 7. After 2 days, the product was taken out of the freezeer, it became yellowish solidified liquid. The weight of the unpurified compound was measured: 4.224 g.  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-9-webp.webp) 8. The compound of the amount of: 1.018 g was run on the flash column. 9. The following column was prepared: 50 g silica, 3 cm diameter column, 15 cm height silica. 10. Eluent was prepared: 50 ml MeOH/ 950 ml DCM (5%MeOH/DCM), but triethylamine was not added. 11. Fractions 4, 5, 14-26 were stained with annis stain. And fractions 4, 5, 6, 14 - 26 were stained with permanganate stain.  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-10-webp.webp) 12. TLC (5%MeOH/DCM + triethylamine 3 drops) has been done on the fractions: 3, 4, 5, 14, 18, 22, 26.  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-11-webp.webp) 13. New fractions 1\*-13\* were made. 14. Fractions 14-3\* showed the presence of compound, and were combined.  ![](https://static.igem.wiki/teams/5342/images/notebook/rad201-12-webp.webp) 15. Fractions 14-3\* were rotovapped 900-500 mbar, 55C, ~15min. 16. Samples were submitted for the 1H, 13C, COSY - NMR in CDCl3 and LC-MS.  Results and Discussion: Yield: crude yellow oil, 1.018 g (pure), 4.224 g (total - not pure), 0.00451 mol, 94.5%; 1H-NMR (300; CDCl3, RT), ppm: 0.9 (A, CH3, m), 1.3 (B, CH2, m), 2.15 (acetone), 2.5 (C, NC, m), 3.5 (D, COH, m), 5.25 (DCM), 7.3 (CDCl3); 13C-NMR (300; CDCl3, RT), ppm: 15 (A, CH3, s), 25-35 (B, CH2, many s), 30 (acetone), 50-60 (C, CN, many s), 60-70 (D, COH, many s), 78 (CDCl3); COSY-NMR (300; CDCl3, RT), ppm: 1 to 1.5, 2.5 to 3.5, 1.5 to 3.5; HSQC-NMR (300; CDCl3, RT), ppm: 3.75 (H) - 70 (C); 2.5 (H) - 60 (C); 1.5 (H) - 25~35 (C); 1(H) - 10 (C).  The results of the analysis show that the 1H-NMR, 13C-NMR, COSY-NMR, and HSQC-NMR have the needed compound. However, COSY-NMR did not show the specific bond found between the B and D region. This was considered a possible mistake in the machine, and the needed structure was confirmed by the HSQC-NMR and LC-MS. ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad201-13.webp) Figure 1. 1H-NMR of ionizable lipids in CDCl3 at RT. ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad201-14-webp-1.webp) Figure 2. Reference NMR spectra from the [1]. ![Figure 3](https://static.igem.wiki/teams/5342/images/notebook/rad201-15-webp.webp) Figure 3. 13C-NMR of ionizable lipids in CDCl3 at RT. ![Figure 4](https://static.igem.wiki/teams/5342/images/notebook/rad128-16.webp) ![Figure 4](https://static.igem.wiki/teams/5342/images/notebook/rad201-17-webp.webp) Figure 4. COSY- NMR of ionizable lipids in CDCl3 at RT.  **Conclusion:** The compound was synthesized correctly, with high purity and high enough yield for the next experiments. **References:**  * Kim, M., et al. “Engineered Ionizable Lipid Nanoparticles for Targeted Delivery of RNA Therapeutics into Different Types of Cells in the Liver.” Science Advances, vol. 7, no. 9, 26 Feb. 2021, https://doi.org/10.1126/sciadv.abf4398. * HYUKJIN, LEE , et al. “Ionizable Lipid Compounds, Lipid Nanoparticles Comprising Same and Therapeutic Uses Thereof.” Worldwide.espacenet.com, 27 Feb. 2024, worldwide.espacenet.com/patent/search/family/074140901/publication/US11911485B2?q=pn%3DUS11911485B2. Accessed 30 June 2024. |

Trial Yeast mRNA LNPs

26/06/24 - 16/09/24

* Adrian
* Kate
* Maks

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| RAD112: IVT of the GFP and Factor VIII RNA **Background:**To understand how the formulation of the LNPs go, an experiment without using LNPs at first must be conducted. LNP preparation followed by the “[LNP procedure](https://static.igem.wiki/teams/5342/documents/protocols/lnp-procedure.pdf)”. Ionizable lipids were formulated in RAD201. Cholesterol, PBS buffer, and compounds to make citrate buffer 10mM pH4 are present. The compounds DOPC & DOPE - 50 ul of 50 mg/ml; DSPE PEG - 2000 - 50 ul of 50 mg/ml have been received from the Zainab Javed. mRNA should be stable in citrate at 4 pH [1]. At high pH, mRNA is not comfortable. Yeast mRNA used: torula yeast RNA.  **Methods:**  Ethanol injection - provides low mRNA encapsulation efficiency, but is cheap and fast. [2]  DLS - size and Z-potential  **Calculations:**  1 attempt: 250 uL liposomes, 10 ul DOPE, 1 - 2 ul charged lipids, 1uL chol - 7 day (half of these for LNPs) |
| 26th of August 2024 Participants:  * Adrian * Kate  Experimental:  * The samples DSPE PEG - 2000, DOPC, & DOPE were dissolved in chloroform, which we have evaporated. * In 26 minutes, DOPC evaporated under N2. * In 31 minutes, DSPE PEG - 2000 (PEG) evaporated under N2. * In around 30 minutes, DOPC evaporated under N2. * These samples were vacuumed in a desiccator chamber for 15 minutes, and then left overnight to dry. |
| 27th of August 2024 Participants:  * Adrian * Kate   The next day they were resuspended in 50 ul of ethanol, concentration - 50 mg/ml for DOPC and DOPE, and 10 mg/ml of PEG. |
| 28th of August 2024 Participants:  * Adrian * Kate  * The samples were nitrogen blown for 15 seconds to remove oxygen traces. * Then the samples were sonicated for 5 minutes. * Citrate 10mM was prepared by the procedure listed in the “[LNP procedure.](https://static.igem.wiki/teams/5342/documents/protocols/lnp-procedure.pdf)" |
| 29th of August 2024 Participants:  * Kate   I weighed 1.5 mg and added it to 70 ml of 10 mM, 4 pH citrate solution. ![table](https://static.igem.wiki/teams/5342/images/notebook/rad202-0table.webp) Lipids were sonicated for 5 min at RT.   |  |  |  | | --- | --- | --- | |  | Added ul | Theoretical ul | | Ionizable lipids | 3.88 | 3.881271282 | | DOPE | 2.325 | 2.32520745 | | Cholesterol | 5.24 | 5.236352597 | | PEG-lipid | 3.259 | 3.258548536 | | Ethanol | 985 | 985.2986201 |   I added the mixture of lipids 1 ml to 3 ml mRNA in a citrate solution with a syringe. I stirred them vigorously for 10 min. I stored LNP samples, citrate, and cholesterol in RT, the rest of components at -20C.   * The samples DSPE PEG - 2000, DOPC, & DOPE were dissolved in chloroform, which we have evaporated. * In 26 minutes, DOPC evaporated under N2. * In 31 minutes, DSPE PEG - 2000 (PEG) evaporated under N2. * In around 30 minutes, DOPC evaporated under N2. * These samples were vacuumed in a desiccator chamber for 15 minutes, and then left overnight to dry. |
| 30th of August 2024 Participants:  * Kate  Experimental:  * I added the LNP sample to fill the whole volume of the membrane chamber, and let it centrifuge for 15 minutes, 5000 g, RT. (I through out salts) * I added 10xPBS in the space which got freed up by the removed salts, and then let it sit in the centrifuge for 30 minutes, 5000 g, RT. (I collected salts) * Again, I added 10xPBS in the space which got freed up by the removed salts, and then let it sit in the centrifuge for 30 minutes, 5000 g, RT. (I collected salts) * I did not add PBS and just let the left sample run in the centrifuge for 30 minutes, 5000 g, RT. (No salts were present.) * LNPs1 were collected. * I analyzed the samples with a DLS machine |
| 3rd of September 2024 Participants:  * Kate * Maks  * Solution of purified LNPs1 turned out purple today. * In two 1.5 ml membranes, in each, around 1 ml of sample was inserted, and then filled up with a 10x PBS buffer. * Then, the samples were weighed, and inserted into a centrifuge for an hour at 5000 g, RT. * Retained mixture was dissolved up to 1500 ul & the elution was also collected. |
| 5th of September 2024 Participants:  * Adrian  * Solution of unpurified LNP (2x1.5ml) pipetted into membrane opening of 2 centrifugal ultrafiltration tubes. * Tubes are filled with a 10x PBS buffer until the volume reaches the level of the bead of the tube. * Tubes weighed to be within 0.6g of each other and centrifuged for 1 hr at RT at maximum speed (3750 RPM). * LNPs2 were collected. * On the next day, solution LNPs2 also turned purple.  ![](https://static.igem.wiki/teams/5342/images/notebook/rad202-1-webp.webp)Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad202-2-webp.webp) Figure 1. DLS. Size measurement of LNPs1 in the Liposome material mode; Dispersant PBS: Average; 3 single measurements. ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad202-3-webp.webp) Figure 2. DLS. Z-potential measurement of LNPs1 in the Liposome material mode; Dispersant PBS: Average; 3 single measurements.  Cholesterol affects the size of the lipids, maybe this is why bigger than 100. ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad202-4-webp.webp) Figure 3. DLS. Size measurement of LNPs1 in the PEG2000 material mode; Dispersant PBS: Average; 3 single measurements. ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad202-5-webp.webp) Figure 4. DLS. Z-potential measurement of LNPs1 in the PEG2000 material mode; Dispersant PBS  The results above show that our LNP size is around the desirable 100 nm. However, it is a bit more, it is assumed that it is due to the ethanol injection methods uncertainty, so this could be improved with microfluidic mixing.  The Z-potential showed weird results. This is assumed to be related to the high acidity of the sample due to its improper purification - always add the sample only to the highest mark in the membrane chamber, and the rest is buffer. This is probably the reason why some ions and salts did not go away.  Solution of purified LNPs1 turned out purple after several days. This is assumed due to a highly acidic environment, which also proves the above point.The LNPs were not sonicated, and this is why they showed bigger size. The purification method was changed, so it is strange that the LNPs still showed high pH. It potentially might be due to not sufficient dialysis steps (only one hour).  **Conclusion:** LNPs have turned their color to purple, which suggests that either some reaction with LNPs occurred during their storage, or some contamination happened. LNPs were stored in RT, now they will be stored at 4C. LNPs were taken up by syringe during the dialysis analysis. Now, an aliquot will be taken for the analysis, since there is a suspicion that metal from the syringe might have colored the sample.  Always add the sample only to the highest mark in the membrane chamber, and the rest is a buffer. Also, samples should be purified for more than 1 hour. (Potentially, the PBS buffer should be not 10x, but 1x). The Z-potential was most probably affected by purification.  Finally, sonication should always be performed before the DLS measurement.  **Advice:** always sonicate LNPs before analysis/usage, since they agglomerate  **References:**   1. Hamatani, Kiyohiro & Eguchi, Hidetaka & Takahashi, Keiko & Koyama, Kazuaki & Mukai, Mayumi & Ito, Reiko & Taga, Masataka & Yasui, Wataru & Nakachi, Kei. (2006). Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 54. 773-80. 10.1369/jhc.5A6859.2006. 2. Jung, Han Na, et al. “Lipid Nanoparticles for Delivery of RNA Therapeutics: Current Status and the Role of in Vivo Imaging.” Theranostics, vol. 12, no. 17, 2022, pp. 7509–7531, https://doi.org/10.7150/thno.77259. 3. “Cell Press: STAR Protocols.” Cell.com, 2024, star-protocols.cell.com/protocols/3495#step-by-step-method-details. |
| RAD203: Formulation of LNPs with Torula Yeast mRNA inside, second attempt **Background:** The LNPs from the RAD202 experiment have turned out the needed size, but have changed color, and showed weird Z-potential. The goal of RAD203 is to figure out why this might be happening, as well as trying to introduce a new analysis method - encapsulation analysis using fluorescence spectroscopy of LNPs. The protocol: [LNP encapsulation efficiency](https://static.igem.wiki/teams/5342/documents/protocols/lnp-encapsulation-efficiency.pdf). Yeast mRNA used: torula yeast RNA.  **Methods:**   * Ethanol injection - provides low mRNA encapsulation efficiency, but is cheap and fast. [2] * Encapsulation efficiency - The amount of encapsulated mRNA was calculated by subtracting the amount of free mRNA from the total mRNA. Encapsulation efficiency was determined by dividing the amount of encapsulated mRNA by the amount of total mRNA. [3] * DLS - size and Z-potential   **Safety: SYBR® Gold - Mutagenic; dissolved in buffer - keep in fridge, -4C (months); regular - in freezer, -20C.** |
| 3rd of September 2024 Participants:  * Maks  Experimental: Compounds were mixed together in the same way as RAD202. NOTE: Yeast mRNA in citrate solution was left out of the fridge overnight. |
| 9th of September 2024 Participants:  * Kate  ![figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad203-1-webp.webp) Figure1. NOTE: Upon storage at -4C, and not interacting with DLS metallic syringe, the LNPs have not turned purplish!  Encapsulation efficiency analysis  Add 0.24 ul of LNPs (stock - 147.5 g/ml) to 10 ml of solvent (PBS 1x) -> 0.0036 g/ml LNPs  Yeast mRNA (was stored at -20C) was warmed to liquid.  The following delusion scheme was prepared: 6 samples dissolve each 2-fold:   |  |  | | --- | --- | |  | Dilusions: mg/ml | | 1 | 0.01455476731 | | 2 | 0.007277383655 | | 3 | 0.003638691828 | | 4 | 0.001819345914 | | 5 | 0.0009096729569 | | 6 | 0.0004548364784 | |
| 11th of July 2024 Participants:  * Kate   DLS analysis   * The LNPs were taken out of the freezer, and then transferred to another vial with glass pipette. NOTE: LNPs were not purple after withdrawal from the fridge. * LNPs were NOT sonicated before the measurement. * LNPs were then sonicated, and measured twice in size, and only one Z-potential measurement (B, C measurements). * LNPs have turned purple after a while after the first measurement  ![figure 0](https://static.igem.wiki/teams/5342/images/notebook/rad203-2-webp.webp) Figure 0. Purple color change observed after DLS. Reason Unknown. Potentially - metal on the needle/ processes that happen within a DLS machine.  Another aliquot of LNPs was taken, sonicated and analyzed on DLS (D). Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad203-3-1-webp.webp) ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad203-3-webp.webp) Figure 1. Figure 1. DLS before sonification. Size measurement of LNPs3 in the PEG2000 material mode; Dispersant PBS ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad203-4-webp.webp) Figure 2. DLS before sonification. Z-potential measurement of LNPs2 in the PEG2000 material mode; Dispersant PBS ![Figure 3](https://static.igem.wiki/teams/5342/images/notebook/rad203-4-2.webp) Figure 3. DLS after sonication. Size measurement of LNPs2 in the PEG2000 material mode; Dispersant PBS; DLS after sonication (top 3). Z-potential measurement of LNPs2 in the PEG2000 material mode; Dispersant PBS (middle one); DLS after sonication. Size measurement of LNPs2 in the Liposomes material mode; Dispersant PBS (bottom 3)  After DSL LNPs turn purple for unknown reasons. In the 1st measurement RAD203 I did sonicate LNPs (helps agglomerate them), so I measured them again after sonication, and received too big of sizes of LNPs which proved that something changes them after DLS and purple color is the consequence. ![Figure 4](https://static.igem.wiki/teams/5342/images/notebook/rad203-5-webp.webp) Figure 4. Setup of the DLS  The LNPs in the first DLS measurement were not sonicated, which gave results with a bigger size. They were then sonicated after the measurement, and taken for a second DLS measurement.  After the first DLS measurement, the LNPs have turned purplish, and gave weird results in the second DLS measurement.  Encapsulation efficiency  Analysis was not successful, potentially due to the procedure performed in a rush. The background noise value was for some reason showing values bigger than the measured values, which of course can not be the case.  **Conclusion:** The LNPs hich are measured for the second time after DLS show weird change to purple color, and potentially form conglomerates, since the size values of LNPs in the second DLS increased significantly.  Z potential still behaves weirdly. It is assumed that the following behavior might be due to the low pH in the LNP solution which makes ionizable lipids gain positive charge, so in the next experiment, 1xPBS with higher pH (~7) will be used to check if Z will change. Encapsulation efficiency analysis will be performed more carefully in the following experiments, more time will be provided for the procedure to prevent potential mistakes. |
| RAD204: Formulation of LNPs with Yeast mRNA inside, second attempt **Background:** The purplish color behavior was related to the DLS - procedure. However, the Z-potential from the experiment RAD 202-203 can still not be changed. This is why, in this experiment PBS1x will be used instead of PBS10x to see if this affects the Z-potential.  Yeast mRNA used: torula yeast RNA. |
| 13th of September 2024 Participants:  * Adrian * Kate  Experimental:  * The LNP beaker was cleaned with ethanol & then acetone. * Lipids were sonicated for 5 min at RT, and formulated in LNPs with following proportion:    |  |  |  |   | --- | --- | --- |   |  | Added ul | Theory ul |   | ionizable lipids | 3.88 | 3.881271282 |   | DOPE | 2.325 | 2.325207452 |   | cholesterol | 5.24 | 5.236352597 |   | PEG-lipid | 3.259 | 3.258548536 |   | Ethanol | 985 | 985.2986201 | * Dialysis tubes were cleaned after experiment RAD203, LNP, cleaning of dialysis tubes. * I added the mixture of lipids 1 ml to 3 ml mRNA in a citrate solution with a pasteur pipette dropwise. * Lipids were mixed together for 10 (+-2) min. * I have dialysed the LNPs at 3270 g for 1 h in PBS1x, then resuspended them in PBS1x, and set them for dialysis for another hour. * The LNPs were suspended up to 1.5 ml PBS. Total volume - 3 ml. * LNP4 were stored at -4C for 3 days. |
| 16th of September 2024 Participants:  * Kate   DLS analysis. Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad204-1-webp.webp) Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment. ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad204-2-webp.webp) Figure 2. DLS Z-potential measurement of LNPs204 in the Liposome material mode; Dispersant PBS  In this experiment, LNPs were sonicated before the measurement, so the size of theirs is as expected - around 100 nm [1]. However, the Z-potential was still showing weird numbers - high positive which means that particles are very dispersed in the mixture, but for some reason positively charged. It is assumed that these particles are behaving this way because the LNP composition is different from [1]. PEG-2000 is used, and no mannose PEG is present. It is assumed that the mannose must provide LNPs with some sort of shielding effect, negating their positive charge.  **Conclusion:** The Z potential seems to be high due to high dispersion of the LNP particles, and high which is a difference from the reference [1] possibly due to different LNP composition. This means that PBSx1 or PBSx10 do not affect the Z, but the properties of particles.  **References:**   1. https://www.science.org/doi/full/10.1126/sciadv.abf4398?rfr\_dat=cr\_pub++0pubmed&url\_ver=Z39.88-2003𝔯\_id=ori%3Arid%3Acrossref.org |

F8 and GFP mRNA LNPs

16/09/24 - 27/09/24

* Kate
* Maks
* Thijmen

|  |
| --- |
| Formulation of LNPs with GFP mRNA or F8 mRNA inside, first attempt **Background:** The following samples from RAD133 will be inserted in the LNPs: 5xFV GFP (or F GFP in 133RAD), GFP 4 (4 samples in LNPs and not-inserted); F8 3, 5xFV F81 (4 samples in LNPs and not-inserted).   * Concentrations: * 5xFV GFP = 201.9 ng/ul * GFP 4 = 848.8 ng/ul; 7.5 ul * 5xFV F81 = 38.6 ng/ul; * F8 3 = 464.4 ng/ul; 7.5 ul   Also control GFP samples will be borrowed from Martin Emmaneel, CODE: 3C1B - T10e mRNA AN\_MEI\_06d, and encapsulated.  **Methods:**   * Ethanol injection - provides low mRNA encapsulation efficiency, but is cheap and fast. [2] * DLS - size and Z-potential * TEM microscopy |
| 16th of July 2024 Participants:  * Kate  Experimental: Lipids were sonicated for 5 min at RT, and formulated in LNPs with following proportion:   |  |  |  | | --- | --- | --- | |  | Added ul | Theoretical ul | | Ionizable lipids | 3.88 | 3.881271282 | | DOPE | 2.325 | 2.32520745 | | Cholesterol | 5.24 | 5.236352597 | | PEG-lipid | 3.259 | 3.258548536 | | Ethanol | 985 | 985.2986201 |   For 5xFV GFP, the stock was diluted by 96.11865483 |
| 17th of July 2024 Participants:  * Kate   The following set-up was used: ![set-up](https://static.igem.wiki/teams/5342/images/notebook/rad205-1-webp.webp) Hot plate for f8 got heated to 33C for 5 min, then switched off. LNPs were added not while mixing, but mixing started right away. 10 minutes. Then, 7th sample - no-mRNA LNPs were prepared separately, and lipids were added correctly - during the spinning. |
| 18th of July 2024 Participants:  * Kate * Maks   LNPs were purified with a dialysis machine. 1h, 3290xg, RT 2x times in PBS10x buffer. |
| 18th of July 2024 Participants:  * Kate   TEM   * Sample 3 fell * Sample 4 carbon film might be worse than for the other grids * Sample 1: no mRNA - 15 LNPs/view frame: 12000x magnification |
| 19th of July 2024 Participants:  * Kate   DLS was conducted. Results and Discussion: It is important to note that the samples with mRNA inside were prepared wrong - lipids were added BEFORE the mixing, and fast, and not during the mixing. This might affect the results of the mRNA LNP complexes. ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad205-2-webp.webp) Figure 1. DLS size measurement of LNPs205 LNPs with no mRNA in the Liposome material mode; Dispersant PBS ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad205-3-webp.webp) Figure 2. DLS Z-potential measurement of LNPs205 LNPs with no mRNA in the Liposome material mode; Dispersant PBS ![Figure 3](https://static.igem.wiki/teams/5342/images/notebook/rad205-4-webp.webp) Figure 3. DLS size measurement of LNPs205 LNPs with GFP control mRNA in the Liposome material mode; Dispersant PBS ![Figure 3](https://static.igem.wiki/teams/5342/images/notebook/rad205-5-webp.webp) Figure 4. DLS Z-potential measurement of LNPs205 LNPs with GFP control mRNA in the Liposome material mode; Dispersant PBS  In the figures above, you can see that the size of the LNPs is off. Both, for LNPs with no mRNA and with mRNA inside. Even though, when the mixing was done correctly for the LNPs with no mRNA, the size improved, but is still bigger, since no sonification was performed (the concentration of lipids for the no-mRNA was high).  This could indicate that upon injection of ethanol, the ethanol and aqueous phase mix, and do not provide for the right mixing between mRNA and lipids, leading to lipid clusters suspended in the liquid. This is observed only now, and not in previous experiments, since the lipid concentration now is very low, and this affects the method results a lot. It is recommended to use the pulsification method or microfluidics device to prevent this in the future.  TEM ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad205-6-webp.webp) Figure 1. LNPs with no mRNA under TEM ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad205-7-webp.webp) Figure 2. LNPs with mRNAs under TEM - none present  **Conclusion:** It is crucial to add the lipids DURING the mixing step, and not before, since it prevents from forming LNP-mRNA complex. LNPs almost do not form with the ethanol injection method at very low concentration. It is recommended to use higher concentration of lipids, and implement a pulsification method or microfluidics device to prevent this in the future. The TEM does not show any LNPs with mRNA, but shows the LNPs without. It also shows that encapsulation is complex, and might lead to no results if some aspect is not done correctly (in this case - mixing, and low concentration).   * different version of T7 poly. * Add RNAse inhibitor. * Reduce temperature to prevent autocatalytic breakdown. |

HEK Cells

09/07/24 - 30/08/24

* Lea
* Vasilis

|  |
| --- |
| RAD301: Transfection of mRNA in HEK cells **Background:** eGFP mRNA made by us and control eGFP mRNA was transfected into HEK cells to check the functionality of our synthesized mRNA. In this transfection, RNA is diluted with buffer and formed into liposomes using a transfection reagent, this is mixed with HEK cells and incubated overnight. It was planned to also transfect LNPs containing eGFP mRNA, but this was postponed due to an experimental error in synthesizing these LNPs. |
| 19th of September 2024 Participants:  * Lea  Experimental:  * transfection solution was prepared: * 50 ul rNA buffer containing 400ng RNA was mixed with 1ul reagent, mixed and left for 15 min:    |  |  |  |  |   | --- | --- | --- | --- |   |  | mRNA iGEM | 3CB mRNA | Mock |   | RNA | 0.8 ul | 4ul | - |   | Buffer | 49.2ul | 46ul | 50ul |   | Reagent | 1ul | 1ul | 1ul | * 3CB is the batch number of the EGFP mRNA given to us from the lab that is known to be functional. This is our positive control. * 25 ul of transfection mix was added to one well containing HEK cells with +- 50% confluency, 2 wells per sample. * Cells were incubated with the transfection mix for +- 19h  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad301-1-webp.webp) ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad301-2-webp.webp) ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad301-3-webp.webp) ![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad301-4-webp.webp) The 3CB control shows visible expression indicating transfection was successful. However, the expression of the control was expected to be higher. Low expression can be explained by the fact that the cells were not very healthy when the transfection was performed. The expression of our synthesized mRNA is a lot lower, but some cells expressing GFP can be found - like the one indicated by the red arrow. The negative control is empty.  **Conclusion:**We produced functional mRNA. However, the expression is not optimal. From this we can conclude that our method for synthesizing mRNA works, but could be improved. Due to the low expression of our mRNA, it might be beneficial to use the 3CB GFP mRNA to test the efficiency of the LNPs. |
| RAD302: Transfection of mRNA in HEK cells, attempt 2 **Background:** It will be attempted to insert mRNA from experiment RAD133 and LNPs from experiment RAD206. The following scheme of transfection will be implemented: ![scheme](https://static.igem.wiki/teams/5342/images/notebook/rad302-1-webp.webp) mRNAs used: GFP 1, GFP2, F8 1, 5x FV F8 2, control GFP; LNPs used: LNPs with no mRNA, GFP1 LNPs, GFP2 LNPs, F8 1 LNPs, 5xFV F8 2 LNPs, GFP cont LNPs; one slot with just cells, one slot with method for mRNA transfection.  LNP transfection was done by using the following procedure: [HEK Cell experiment](https://static.igem.wiki/teams/5342/documents/procedure/hek-cell-experiment-procedure.pdf). No Western blot was done. Control GFP samples will be borrowed from Martin Emmaneel, CODE: 3C1B - T10e mRNA AN\_MEI\_06d. |
| 24th of September 2024 Participants:  * Vasilis  1. A dilution of 25 ul was made using 23ul of mRNA buffer 2. 0.5 ul of transfection reagent was added to every sample 3. The samples were incubated for 15 min 4. The cells were labeled with the same numbers as the samples 5. 1ul of each sample was added to each well with the cells  Results and Discussion:![Figure 1](https://static.igem.wiki/teams/5342/images/notebook/rad302-2-webp.webp) Figure 1. The control GFP mRNA is expressed ![Figure 2](https://static.igem.wiki/teams/5342/images/notebook/rad302-3-webp.webp) Figure 2. Our mRNA samples and LNPs (also the one with control GFP) with mRNA, no GFP detected (same picture for all) ![Figure 3](https://static.igem.wiki/teams/5342/images/notebook/rad302-4-webp.webp) Figure 3. Trans picture of cells with the LNPs inside (same picture for all) ![Figure 4](https://static.igem.wiki/teams/5342/images/notebook/rad302-5-webp.webp) Figure 4. Cells without fluorescence - survived  Only the sample with control GFP was expressed. This implies that the mRNA samples are nonfunctional (GFP 1, which was slightly expressed in RAD301, was not visible here, potentially due to denaturation). Since control was expressed by regular transfection, and not with LNPs, the LNP synthesis procedure in the moment is not efficient.  SDS-PAGE gel was considered to be unnecessary for analysis of F8 protein presence, since if there was no expression of control GFP, than yes, but since there is no expression of the experiment with GFP whose RNA also looked better from the start, I think it is a waste of time and resources. In the final effort, I would put my money on getting the transfection both with JetMessenger ánd LNPs to work before analyzing protein extracts of cells.  **Conclusion:**Combining the results from the RAD206, RAD133, and RAD203, it can be said that the reason for the following transcription result is a combination of the results from all 3 factors: HEK cells are used instead of the cells used in the reference [1] - HeLa or LSEC cells, different lipid composition was used: instead of DSPE-PEG 2000, C16-PEG ceramide (PEG-lipid) and DSPE-PEG-mannose lipids were used, and the mRNA experiments provided very low yield of functionable mRNA.  This resulted in no transfection fluorescence of just GFP mRNA samples (except for the control one), since the mRNA from RAD133 also was not visible on the gel, and in no transfection fluorescence of GFP in LNPs. However, the LNP procedure was also not efficient - very little amount of LNPs produced in combination with different compounds used for production.  It is advised to make more mRNA samples after consultation on the current results, procedure and storage should be revised. Different cell cultures should be used: either HeLa or LSEC cell lines. Also, for LNPs, it is recommended to use higher concentration of lipids, and implement a pulsification method or microfluidics device to prevent low yields of LNPs in the future. It is also recommended to purchase more expensive compounds for LNP formulation, as in the referenced article [1]: instead of DSPE-PEG 2000 - C16-PEG ceramide (PEG-lipid) and DSPE-PEG-mannose lipid which might provide for the better targeting during transfection.  **References:**   1. https://www.science.org/doi/full/10.1126/sciadv.abf4398?rfr\_dat=cr\_pub++0pubmed&url\_ver=Z39.88-2003𝔯\_id=ori%3Arid%3Acrossref.org |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Our success in the Engineering Design Cycle{% endblock %}
{% block page\_content %}

### *Our Engineering Success*

#### *Design*

Our team started brainstorming ideas in November 2023 the topic of hemophilia caught our interest early on, vesicle delivery was also an early concept, but the exact design of our project changed a lot during our literature research as we were learning more and more about hemophilia and the different types of vesicle delivery.
Our project design started to crystallize at the beginning of march when we decided to use ionizable lipid nanoparticles (LNPs) for mRNA delivery.
This method has already been well-researched in the last decades.
Our design is largely based on a paper called “Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver” by Kim et al. 2021
This article gives instructions for formulating LNPs that can deliver siRNA as well as mRNA to specifically the liver sinusoidal endothelial cells, which was our original target, where factor VIII is normally produced.
These LNPs contain Ionizable lipid (synthesized from 1,4-Bis(3-aminopropyl) piperazine and 1,2-epoxydecane) , DOPE, cholesterol, and DSPE PEG C16 ceramide in a molar ratio of 26.5:20:52:1.5.
To specifically target LSECs, the LNPs include conjugated DSPE PEG-manose lipids that interact with the scavenger receptors on LSECs.
Our goal was to recreate these LNPs with Factor VIII mRNA. In addition, we wanted to implement these LNPs for intra nasal delivery.
Because the Factor VIII protein coding sequence was not yet in the registry, we chose to buy a plasmid by Robert Peters containing the full sequence for the human FVIII protein from addgene.

#### *Build*

To achieve our goal, we needed two things: the Factor VIII mRNA and the LNPs. We worked on these in parallel.
The goal was to recreate the LNPs described by Kim et al. However, due to the cost of some components, we used a model LNP made out of cheaper alternatives. DSPE PEG C16 ceramide and DSPE PEG-manose were both substituted with the more affordable DSPE PEG-2000.

Producing the mRNA in a chassis organism was not feasible, so the plan was to synthesize the mRNA for Factor VIII using in vitro transcription and synthetically modifying it with a 5’-cap and 3’ poly(A)-tail.
Amplifying the Factor VIII coding sequence before transcription proved to be difficult because of its length.
After trying PCR with multiple sets of primers, the whole plasmid was amplified in E.coli to then be linearised.
In our multiple tries of in vitro transcription, we managed to finetune our protocol and produce enough RNA to make LNPs.
Before the RNA could be used in LNPs, it had to be purified, and modified with a 5’-cap and a poly-A tail.
Purification was done with phenol-chloroform extraction, which also gave us some difficulties and it took us some time to troubleshoot the protocol.
Working with Factor VIII RNA proved more difficult than expected, because the length made it prone to degradation and none of our team members had previous experience working with RNA.

#### *Test*

The LNPs by Kim et al. specifically targeted the sinusoidal endothelial cells, but several factors, including cost and availability, made it hard for us to test our LNPs on these cells. As an alternative, we used the HEK293T/17 cell line.
To test our LNPs we tried to deliver mRNA encoding for the eGFP protein. We
We tested the properties of our synthesized mRNA without LNPs by transfecting them into HEK cells to compare with the delivery of RNA with our LNPs.
Analysis of the LNPs was done using dynamic light scattering (DLS) and transmission electron microscopy (TEM) to determine the size and zeta potential of the LNPs. The encapsulation efficiency was determined with the use of
The concentration of RNA was also frequently measured using a NanoDrop spectrophotometer.

#### *Learn*

During our building stage, we were already confronted with some problems that made us reconsider our approach and rethink our design.
The length of the Factor VIII RNA forced us to rethink our method of gene amplification.
We also changed the concentration of the PBS buffer after measuring the zeta potential of our first batch of LNPs.
Due to the cost of LSECs and some LNP components, we were forced to construct an affordable model that would still be able to prove our concept.
We decided to use a generic HEK cell line and because there would be no specific targeting possible with these cells, we could also replace the expensive PEG-manose lipid with a more affordable option.

#### *Improve*

During our building stage, we were already confronted with some problems that made us reconsider our approach and rethink our design.
The length of the Factor VIII RNA forced us to rethink our method of gene amplification.
We also changed the concentration of the PBS buffer after measuring the zeta potential of our first batch of LNPs.
Due to the cost of LSECs and some LNP components, we were forced to construct an affordable model that would still be able to prove our concept.
We decided to use a generic HEK cell line and because there would be no specific targeting possible with these cells, we could also replace the expensive PEG-manose lipid with a more affordable option.

#### References:

Kim M, Jeong M, Hur S, Cho Y, Park J, Jung H, et al. Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver. Science Advances. 2021 Feb 26;7(9)

pCDNA4/Full length FVIII from Robert Peters (Addgene plasmid # 41036 ; http://n2t.net/addgene:41036 ; RRID:Addgene\_41036)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describing the research, experiments, and protocols we used in for InfinityF.{% endblock %}
{% block page\_content %}

### Experimental Design

![factor-8-process](https://static.igem.wiki/teams/5342/images/home/factor8-process.webp)

Our project consists of 2 main parts: the LNP synthesis and the mRNA production of factor VIII.
Furthermore, the experiments involved GFP expression as an assessment method for LNP’s in HEK cells.
Lastly, the mRNA expression in HEK cells together with the insertion of the mRNA into the vesicles, along with the detection method and the evaluation of the engineered product towards the expression of factor VIII(Fig.1).
You can refer to our protocols for the detailed experimental procedure.

![Visual representation of design](https://static.igem.wiki/teams/5342/images/experiments/procedure.webp)

Figure 1. Overview of the process to reach the required protein Factor VIII.

The process timeline starts with transfecting pCDNA4 plasmids into E.coli bacteria strain where they are amplified. The linearization is carried out via XhoI restriction enzyme cleavage.
The pcDNA is transcribed into an mRNA sequence which undergoes 5’capping and 3’ poly-A tailing. The modified mRNA is encapsulated in our produced lipid nanoparticles.
These nanoparticles are introduced in HEK cell line where the the full FVIII protein is translated.

### DNA FVIII production

We made the DNA of FVIII by transfecting Top 10 and XL 1 blue E.Coli bacterial suspensions, with pCDNA4 plasmid in a medium containing ampicillin.
The pc DNA from both bacterial colonies was extracted using EcoRI and XhoI and was analyzed for size with gel electrophoresis.
We determined the concentration of the isolated plasmids and the XL1 blue had a higher concentration, thus we used it for mRNA production.
The XL1 blue was linearised using Xhol digestion following the protocol: “Enzyme digestion”.

### GFP Production

We wanted to amplify eGFP DNA, which would serve as control mRNA for the lipid encapsulation.
The eGFP DNA was amplified by PCR using the following protocol:
[“PCR procedure Radboud University team”](https://static.igem.wiki/teams/5342/documents/protocols/pcr-procedure-radboud-university-team.pdf) and
[PUC-18 GFP template.](https://www.addgene.org/220495/)
After a sufficient amount of GFP DNA was received, we continued with the purification of the DNA.
For the PCR purification, we used
[QIAquick PCR Purification Kit](https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/qiaquick-pcr-purification-kit)
and the protocol QIA PCR purification was followed
[(“QIAquick® PCR Purification Kit”)](https://static.igem.wiki/teams/5342/documents/protocols/qiaquick-pcr-purification-kit.pdf).

### mRNA Production

The FVIII and GFP mRNA were produced by T7 RNA polymerase from the cp FVIII DNA and the GFP DNA using the protocol listed as “In vitro synthesis of RNA using T7 RNA polymerase”.
RNAase inhibitor was used to prevent the RNA from degrading.
This step was repeated several times with several adjustments in the protocol for a high yield of mRNA.
The concentration was assessed using the
[nanodrop Uv-vis spectrometer](https://www.fishersci.com/shop/products/nanodrop-one-spectrophotometer/13400518).
After the production of a sufficient amount of RNA, the next step was its purification using the
[Phenol-Chloroform](https://www.carlroth.com/nl/nl/a-to-z/rotiphenol-chloroform-isoamyl-alcohol/p/a156.1)
solution and extraction method using the protocol
[“RNA cleanup by phenol-chloroform”](https://www.protocols.io/view/rna-clean-up-by-phenol-chloroform-yxmvmw46g3pe/v2?step=null)
After the extraction, the mRNA had to be modified to prevent its degradation, thus it was capped and tailed.
The mRNA was capped following the protocol
[Cap-0 synthesis using Vaccinia Capping enzyme](https://www.neb.com/en/protocols/0001/01/01/capping-protocol-m2080)
and the Vaccinia (NEB#2080) enzyme to form the complete Cap-0 structure.
Later the mRNA was tailed following the protocol
[“Poly(A) Tailing of RNA using E.coli Poly(A) polymerase”](https://www.neb.com/en/protocols/2014/08/13/poly-a-tailing-of-rna-using-e-coli-poly-a-polymerase-neb-m0276)
using E. coli Poly(A) Polymerase (NEB #M0276).

### LNP production and evalution

For the LNP production, ionizable lipids 246 were synthesized using the following protocol:
[“Ionizable Lipid synthesis”.](https://static.igem.wiki/teams/5342/documents/protocols/ionizable-lipid-synthesis.pdf)
After the synthesis of our lipids, we continued with the LNP preparation using the protocol:
[“LNP procedure”](https://static.igem.wiki/teams/5342/documents/protocols/lnp-procedure.pdf)
with torula yeast mRNA as a trial attempt, which involved the mixing of the lipids (ionizable lipids 246, DOPE, cholesterol, DSPE PEG-2000, ethanol) and the encapsulation of the yeast mRNA using the ethanol injection method.
The size of the LNPs was assessed with DLS (Dynamic Light Scattering).
Then we continued with making LNPs encapsulating the GFP and FVIII mRNA made previously.
The LNPs were checked again using DLS and the encapsulation was assessed using TEM (Transmission Electron Microscopy) and the protocol:
[“LNP encapsulation efficiency”](https://static.igem.wiki/teams/5342/documents/protocols/lnp-encapsulation-efficiency.pdf).

### mRNA evaluation

The produced mRNA’s of FVIII and GFP had to be evaluated for their translative abilities.
This is why we performed experiments using Hek 293 cell lines.
We encapsulated the mRNA into our LNPs and transfected the HEK cells using the lipotransfection method with the Jet messenger transfection reagent.
For the GFP mRNA transfection, we used the following protocol:
[“Polyplus Short-protocol- jetMESSENGER-mRNA”](https://static.igem.wiki/teams/5342/documents/protocols/polyplus-short-protocol-jetmessenger-mrna.pdf)
and incubated the cells overnight.
After the incubation, the cells were evaluated for any GFP production using the EVOS M5000 fluorescence microscope.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## Results mRNA

### Summary

Our project consisted of three main parts: mRNA production, lipid nanoparticle production and cell line transfection (Figure 1). In result we did not manage to produce a full line of living cells with expression FVIII protein after transfecting them with LNPs containing this mRNA. However, there are multiple points throughout our project that can be considered as a success and basis for possible future research. For example, the cells transfected with our produced nanoparticles were seen to be alive which gave an indication of the LNP safety. The BioBrick of our project was successfully produced in a form of functional cpDNA - **BBa\_K5342000**.

![1](https://static.igem.wiki/teams/5342/images/results/mrna-number-1.webp)

**Figure 1. Overview of the process to reach the required protein Factor VIII.**

The process timeline starts with transfecting pCDNA4 plasmids into E.coli bacteria strain where they are amplified. The linearization is carried out via XhoI restriction enzyme cleavage. The pcDNA is transcribed into an mRNA sequence which undergoes 5’ capping and 3’ poly-A tailing. The modified mRNA is encapsulated in our produced lipid nanoparticles. These nanoparticles are introduced in the HEK cell line where the full FVIII protein is translated.

### Main points of mRNA results

#### PCR of FVIII and GFP cDNA

As the first part of our project, polymerase chain reaction (PCR) was carried out on FVIII, GFP and eGFP cDNA to obtain higher concentration of DNA which was then further transcribed via IVT (In-Vitro transcription). Polymerase chain reaction is a well known nucleic acid chain elongation procedure that can be used for both DNA and RNA sequences. It consists of denaturation, renaturation, elongation steps [3].
For our project we implemented the use of Pfu polymerase. The reaction efficiency was then analysed by agarose gel electrophoresis to detect the amplified DNA and any impurities.
A smearing in the sample rows could be seen frequently, the band size of F8 cDNA was faint or the purity of acquired samples was not optimal (Figures 2-5).
On 03/07 engineered GFP was produced via PCR (Figures 6 and 7). The result was assessed on agarose gel on 04/07. A clear band of GFP cDNA was detected on the gel and a concentration of 60.2 ng/ul was acquired via nanodrop (Figures 8, 9).

![2](https://static.igem.wiki/teams/5342/images/results/mrna-number-2.webp)

**Figure 2. Agarose gel electrophoresis of amplified cDNA via PCR. (24/05/24)**

Indication of rows: 1st DNA ladder, 2nd positive control, 3rd FVIII template, 4th negative control.
Visible bands after amplifying the Factor 8 cDNA via polymerase chain reaction. Correct FVIII DNA band visible at around 7.5 kb. A smearing is detected. A decision is made to extract the DNA from this gel, purify and amplify it further to acquire a larger quantity of the DNA.

![3](https://static.igem.wiki/teams/5342/images/results/mrna-number-3.webp)

**Figure 3. Agarose gel electrophoresis of amplified cDNA with a different set of primers in PCR. (03/06/24)**

Indication of rows: 1st DNA ladder, 2nd negative control, 3rd positive, 4th PCR reaction with a new set of primers, 5th eluted DNA, 6th another DNA ladder.
The purified DNA is visible as a clear band at the expected height (5th row), the vagueness of the band is due to the sample not being amplified. The negative control shows a smear, the positive control is not detectable.

![4](https://static.igem.wiki/teams/5342/images/notebook/rad-103-2.webp)

**Figure 4. Agarose gel electrophoresis of amplified cDNA via PCR. (28/06/24)**

Indication of 4 rows (from left to right): gene ladder, negative control (using 2nd set of primers), positive control, cDNA with primers (using 1st set of primers). The 4th slot contains a very slight band indicating the amount of amplified DNA is very scarce. A slight band at around 7.5 kB can be seen. The amplification still results in big smears.

![5](https://static.igem.wiki/teams/5342/images/notebook/rad-104-1.webp)

**Figure 5. Agarose gel electrophoresis of amplified cDNA via PCR. (01/07/2024)**

Indication of rows:
1st DNA ladder, 2nd – 6th FVIII, 7th GFP, 8th DNA ladder.
Very clear smears visible after PCR of F8 cDNA with another set of new primers and GFP with the standard GFP primers. 5th row is blank due to loss of the sample. In row 7 very vague bands are seen - low in the bottom of the gel and at the top. The size of GFP cDNA is 730 bp.

![6](https://static.igem.wiki/teams/5342/images/notebook/rad-105-1.webp)

**Figure 6. Agarose gel electrophoresis of amplified cDNA via PCR. (03/07/2024)**

Indication of rows:
1st DNA ladder, 2nd -4th eGFP cDNA, 5th DNA ladder.
An eGFP cDNA correctly received at around the 1000bp region (see the ladder). Bands are very dim due to low concentration.

![7](https://static.igem.wiki/teams/5342/images/notebook/rad-108-1.webp)

**Figure 7. Agarose gel electrophoresis of amplified eGFP cDNA via PCR. (04/07/2024)**

Indication of rows: 1st DNA ladder, 2nd to 5th eGFP cDNA.
All samples show a clear band at ~1000kb.

![8](https://static.igem.wiki/teams/5342/images/notebook/rad-109-1.webp)

**Figure 8. Agarose gel of purified eGFP via PCR (done on 03/07/2024)**

Indication of rows: 1st DNA ladder, 2nd purified eGFP cDNA.
A little smear at the top. The band is detected at around 1000 bp, the expected eGFP size.

![9](https://static.igem.wiki/teams/5342/images/notebook/rad-109-2.webp)

**Figure 9. Analysis of eGFP cDNA by nanodrop. (04/07/2024)**

Measured samples of the GFP cDNA show a concentration of 60.2 ng/ul. The ratio of A260/280 is 1.92. This cDNA is further used for GFP mRNA production.

##### Analysis and conclusion:

The smearing during PCR of the F8 and GFP DNA might have resulted from primers not being optimally designed for the amplification, or from cross-contamination during pipetting and other steps of the experiment.
The generally low amplification of F8 cDNA may have been due to the large size of the DNA chain and poor primer design which led to improper primer annealing. Although, in experiments where the primers would have worked well normally, the yield of mRNA was very poor or highly faulty. A deeper insight has to be done to assess the optimal amount of cycles during PCR to obtain a large quantity of cDNA. This can be very time consuming as even the previously calculated conditions did not meet the resultative expectations.

#### Amplification of pcDNA via bacterial plasmids

It was suggested by our instructor to try out pcDNA amplification. From 03/07 till 08/07 pcDNA amplification via bacterial plasmids was executed out with successful results. The bacterial cultures used in this part were Top10 and XL1Blue. The plasmids we used had been retrieved from DH5alpha bacteria strain with ampicillin resistance (Figure 12) [1]. The plasmids we used were a mammalian expression vector with 5021 bp backbone and an insert size of 7065 bp. The 5' cloning site was BsiWI and 3' cloning site NotI. A H. Sapiens FVIII gene had been inserted in the specified cloning site. [1]
These plasmids were transfected into bacteria cultures Top10 and XL1 Blue. The isolation of plasmids showcased better results from XL1 Blue bacteria colonies, thus, these were used further. The plasmids were digested by the means of XhoI and EcoRI restriction enzymes. The XhoI cleavage sequence is C'TCGAG at position 7126 of the plasmid (Figures 11, 12) [1]. This sequence is found once in the plasmid. In result, a 12.1 kbp linearized plasmid was received. The EcoRI cleavage was used to assess the accuracy of plasmid construct parts.
An agarose gel electrophoresis was prepared to assess the purification and digestion of FVIII pcDNA plasmids (Figure 10). The digested plasmids displayed clear bands at the expected length of the vector – 12086 base pairs. [1] This attested the capability of these samples for further run-off IVT.

![10](https://static.igem.wiki/teams/5342/images/notebook/rad111-1.webp)

**Figure 10. Agarose gel analysis of digested F8 pcDNA plasmids. (08/07/2024)**

Indication of rows: 1st DNA ladder, 2nd unpurified digested plasmids, 3rd-4th remnants of sample 7th – 10th empty wells, 11th-12th purified digested plasmids, 13th DNA ladder, 14th-15th purified digested plasmids.
Digestion was done via XhoI restriction enzyme. The plasmids show clear bands at the expected length of 12086 bp.

![11](https://static.igem.wiki/teams/5342/images/results/mrna-number-11.webp)

**Figure 11. pCDNA4/Full length FVIII sequence[1].**

The length of F8 is noted as a yellow arrow. The restriction enzyme used is XhoI which cleaves in sequence C'TCGAG.

![12](https://static.igem.wiki/teams/5342/images/results/mrna-number-12.webp)

**Figure 12. Full sequence map of pCDNA4/Full length FVIII [1].**

The length of F8 is noted as a yellow arrow.

##### Conclusion

pcDNA amplification resulted in much better yield of DNA as can be seen in figure 10. The purified digested plasmids of 12086 base pairs were visualised as bright bands above the 10000 bp mark. This proved the viability of these samples to be used for further run-off IVT.

#### IVT of FVIII pcDNA and eGFP cDNA.

From 09/07 till 30/08 In-Vitro transcription was carried out for F8 pcDNA and eGFP cDNA to obtain functional mRNA. A nanodrop assessment on 28/08 revealed high concentrations of mRNA samples. The average concentration of F8 and GFP mRNA was calculated to be 2480,41 ng/ul and 1845,70 ng/ul, correspondingly. An average A260/280 ratio of F8 and GFP mRNA was calculated to be 2,10 and 2,14, respectively. And, an average A260/230 ratio for GFP mRNA was calculated to be 2,46 (Table 1).
On 29/08 the transcribed samples were analysed via agarose gel electrophoresis. A clear, but vague band was seen, indicating the presence of GFP mRNA. The rest of the samples were not observable, including a positive control sample (Figure 13).
On 30/08 a new agarose gel electrophoresis was carried out for the purpose of analysing the samples from 28/08 again. A bright band of GFP mRNA was noticed. The positive control appeared in the form of a vague band. The GFP sample was further used for capping, tailing and analysis.

![13](https://static.igem.wiki/teams/5342/images/results/mrna-number-13.webp)

**Table 1. Nanodrop analysis of transcribed F8 and GFP mRNA.**

The A260/280 ratio detects protein contamination in samples. The A260/230 ratio detects salt contamination, data for samples H, I, J were lost during notebook keeping. The average concentration (ng/ul) is 2480 and 1847 for F8 and GFP, respectively. The average A260/280 ratio is 2.1 for both mRNA types. The average GFP A260/230 ratio is 2.5.

![14](https://static.igem.wiki/teams/5342/images/notebook/rad131-3.webp)

**Figure 13. Agarose gel analysis of transcribed samples via IVT. (29/08/2024)**

Indication of rows from left to right: A-B-C-D-E-F-G-H-I-J-F8(1) -F8(2)-GFP-Positive sample (see, Table 1). A clear, vague band on row 3 (sample C – GFP) and 13 (modified GFP). The rest of the samples are not visually detectable.
F8(1), F8(2), and GFP indicate capped and tailed (modified) mRNA samples acquired from previous experiments.

![15](https://static.igem.wiki/teams/5342/images/results/mrna-number-15.webp)

**Figure 14. Agarose gel analysis of IVT transcribed samples. (30/08/2024)**

Indication of rows from left to right:
B-D-E-F-G-H-I-J-F8(2)-Positive sample (processed samples) (see Table 1).
On the 5th row a clear band can be seen with a smear. The supposed sample is GFP mRNA. A clear band is visible in the 10th row as the positive sample. F8(2) indicates the capped and tailed mRNA sample acquired from previous experiments.

##### Analysis and conclusion

Throughout the IVT experiments no clear products were obtained. Frequent cases with large smearing were observed or even no viable mRNA could be found when assessing the gel. Multiple reasons were listed as causation of these results. For example, due to the large size of F8 cDNA, a larger volume of T7 polymerase was required. As can be seen in Figure 19, the samples, although with smears, showed higher translational efficiency when using 4 µl T7 polymerase instead of 2 µl as requested in the protocol. Even with the samples of lower efficiency a phenomenon of uneven band strength was observed. A reason might have been contamination and RNA degradation prior to analysis.
Additionally, The FVIII RNA was visible at 7000 bp, but the expected is at 1200 pb. This could also have resulted from RNA degradation, self-splicing, due to the properties of the gel and the non-linearity of the RNA.
As can be seen in Figure 21 the GFP RNA did not show clear bands in both sample rows. Likely reason was the template. The calculations on the amount required might have been incorrect. Further trials were tested including RNAse inhibitors. This showed partially promising results. Some samples were seen with a clear band, others – smears. Furthermore, an empty row was noticed. The cause was concluded to be a blunder – the falling of pipette tip from the pipette (Figure 20). Further discussion can be made to assess whether such blunders of improper pipetting could have caused absent results and contamination not only in this particular experiment but also throughout the project. Lastly, in Figure 13 no positive control sample band was detected. This requires a deeper insight whether different properties are required for optimal agarose gel analysis.
One of the IVT experiments did show promising results when analysing samples via nanodrop. The concentrations of both GFP and F8 were found to be high, the A260/280 and A260/230 ratio gave a good indication of purity. These ratios can estimate whether the sample is contaminated with protein structures and salts, respectively (Table 1). When analysing these samples on gel, no band presence was visualised. Possible cause thought to be mRNA splicing which would not have been detected on the 0.7 % agarose gel. All small RNA samples would have ‘run out’ during the electrophoresis process (Figure 15).

![16](https://static.igem.wiki/teams/5342/images/notebook/rad113-1.webp)

**Figure 20. Agarose gel of eGFP with wild type T7 polymerase and RNAse inhibitor. (10/07/24)**

Indication of rows: 1st – 5th and 7th – 11th eGFP mRNA, 6th DNA ladder.
Row 3 sample is absent (due to blunder error). Samples of rows 4 and 5 are visible with smearing. Samples 2 and 7 have a significant smear.

![17](https://static.igem.wiki/teams/5342/images/notebook/rad112-1.webp)

**Figure 21. Agarose gel electrophoresis of IVT samples. (09/07/24)** (To prevent waste, the same agarose gel is used for multiple experiments. The crossed out lanes are from a previous experiment.)

Indication of rows (excluding crossed out ones): 1st F8 mRNA, 2nd- 4th F8 mRNA, 5th GFP mRNA, 6th DNA ladder, 7th – 8th F8 mRNA, 9th GFP mRNA.
1st, 2nd and 4th F8 mRNA samples with 2 µL T7 polymerase does not show high transcriptional efficiency while the 3rd seems to have been transcribed well. Samples of rows 7 - 9 show a possible band but with significant smearing.

#### Modification of mRNA samples

The modification of the mRNA samples was carried out from 26/08 till 13/09. The samples were capped with 3’ poly-A tail sequence and tailed with 5’ cap, with purification included in-between both steps. The 5’ cap is a modified guanosine which consists of a methyl group on the 7-position. This process is vital for the maturity of the mRNA and translational competence. The poly-A tail ensures protection from enzymatic degradation and ensures the translational process [2].
Multiple sets of samples were assessed on the gel which included normal, unprocessed transcribed samples as can be seen in figures 13 and 14. This section of our research did not bear a highly positive outcome. Our instructor provided several samples of mRNA transcribed via his methods. All present samples were shown on an agarose gel predominantly as smears, vague bands or blank rows (Figures 13,14, 15).
Additionally, low concentration of mRNA was assessed via nanodrop after the modification (Table 2). Due to time constraint, further transfection was carried out with the samples we had acquired.

![18](https://static.igem.wiki/teams/5342/images/results/mrna-number-18.webp)

**Figure 15. 0.7% Agarose gel electrophoresis of denatured processed mRNA samples. (13/09/2024)**

Indication of Rows: 1st-4th F8 mRNA, 5th – 8th GFP mRNA, 10th – 11th F8 mRNA (given by our instructor), 12th GFP mRNA (given by our instructor). A smear in 1st - 3rd and 9th row (F8), a band (GFP received form instructor) in 11th row. Other wells indicate little to no mRNA presence.

![19](https://static.igem.wiki/teams/5342/images/results/mrna-number-19.webp)

**Table 2. Nanodrop analysis of modified F8 and GFP mRNA.**

The samples were processed via addition of 5’cap and poly-A tail. The numbering of 1-4 indicates the sample number.

##### Analysis and conclusion

The acquired samples were further modified with a poly-A tail and 5’ cap to ensure the stability and translational efficiency in-vitro. The protocols used were suggested to be reliable by our instructor. But, as can be seen in Figures 14 and 15, vague band visibility was detected. Large blunders might have occured during the purification and extraction in-between the capping and tailing phases. As was noted in many experiments, Phenol:Chloroform:Iso-amyl alcohol extraction was not very reliable in the sense of acquiring a good yield of pure mRNA. There were instances when a pellet of mRNA was received after purification of 5' capped samples but none after proceeding with poly-A tailing. For future experiments, it would be advised to review the protocols for this exact purification and assess if another purification method could be more optimal. As explained previously, the modification of mRNA is crucial for further translational experiments.

## Results - LNPs

**Ionizable lipid synthesis**
Our team synthesized the ionizable lipids needed for lipid nanoparticle formation. We recreated the ionizable lipids produced by Kim et al [1], who first synthesized this lipid by an epoxide ring-opening reaction with amines. To confirm that the product was correct and pure, the lipids were analyzed with nuclear magnetic resonance (NMR) spectroscopy.

![1.1](https://static.igem.wiki/teams/5342/images/results/ion1.webp)

Figure 1. 1H-NMR of ionizable lipids in CDCl3 at RT.

![2.1](https://static.igem.wiki/teams/5342/images/results/ion2.webp)

Figure 2. Reference H-NMR spectra by Kim et al. [1].

In NMR, each nucleus of a specific type (for example H1 or C13) produces an electromagnetic signal when in the presence of an oscillating magnetic field. These signals can shift based on their chemical environment so that every nucleus in a unique environment produces a unique signal which allows chemists to elucidate a molecule's structure. In this case we are looking at a 1H-NMR which shows all the unique hydrogen nuclei.
This lipid is a very complex molecule which makes it impossible to assign every single peak. However, when comparing with the spectrum from the publication by Kim (figure 2), the same peak groups can be observed. We see the same tall peak around 0.88 ppm, the high peak at 1.27 ppm and the low broad series of peaks at 1.44 and 2.5 ppm as well as the three small peaks at the end.

**Synthesis results:**
Yield: crude yellow oil, 1.018 g (pure), 4.224 g (total - not pure), 0.00451 mol, 94.5%; 1H-NMR (300; CDCl3, RT), ppm: 0.9 (A, CH3, m), 1.3 (B, CH2, m), 2.15 (acetone), 2.5 (C, NC, m), 3.5 (D, COH, m), 5.25 (DCM), 7.3 (CDCl3); 13C-NMR (300; CDCl3, RT), ppm: 15 (A, CH3, s), 25-35 (B, CH2, many s), 30 (acetone), 50-60 (C, CN, many s), 60-70 (D, COH, many s), 78 (CDCl3); COSY-NMR (300; CDCl3, RT), ppm: 1 to 1.5, 2.5 to 3.5, 1.5 to 3.5; HSQC-NMR (300; CDCl3, RT), ppm: 3.75 (H) - 70 (C); 2.5 (H) - 60 (C); 1.5 (H) - 25~35 (C); 1(H) - 10 (C).

**LNPs**
The ionizable lipids were used to form ionizable lipid nanoparticles. LNPs were analyzed for their size, zeta potential and encapsulation efficiency. Size and Z-potential can be acquired through dynamic light scattering (DLS), while the encapsulation efficiency is measured with a fluorescence assay.
The LNPs described by Kim [1] had a size of 60-100nm. Our LNPs had a size of just over 100nm (figure 3a-c), which is a bit larger. This can be attributed to our mixing method, which was less precise than their microfluidics. Batch 2 (figure 3b) shows an increase in LNPs with a diameter of 10,000nm. This is the result of LNP aggregation, which happens if LNPs are not sonicated before analysis. The zeta potential described in the article by Kim was -0.997 mV. We measured a different, highly positive zeta potential. Zeta potential is a measure for the dispersion of the particles in a solution. a high (positive or negative) zeta potential means that the particles are dispersed in solution while a potential close to zero signifies that particles are likely to aggregate. A zeta potential that deviates from literature would mean that our LNPs have different interactions with each other which could also change how they interact with cells.

![1.1](https://static.igem.wiki/teams/5342/images/results/lnp-number-1.webp)

Figure 3. DLS analysis of the first 3 batches of trial LNPs containing torula yeast RNA. Size distribution of batch 1 (a) and batch 2 (b) in PEG2000 material mode; Dispersant PBS: Average; 3 single measurements. (c) Overview of average Z-potential PEG2000 material mode; Dispersant PBS.
To address the unusual zeta-potential, the fourth batch of LNPs was made in 1x PBS buffer instead of 10X PBS. This time the LNPs were sonicated before analysis so there is no aggregation. The average zeta potential was measured at 7.156 mV in Liposome material mode; Dispersant PBS. It was concluded that a change in buffer does not impact the zeta potential. It was concluded that the difference in zeta potential from the source was due to the component substitutions we made.

![2.1](https://static.igem.wiki/teams/5342/images/results/lnp-number-2.webp)

figure 4. DLS analysis of batch 3 (LNP204) of trial LNPs containing torula yeast RNA with 1x PBS buffer. Size measurement in Liposome material mode; Dispersant PBS
To test the transfection properties of our ionizable LNPs in vitro, LNPs were produced containing mRNA coding for GFP. DLS revealed that the size of the LNPs containing GFP mRNA was 1000nm, way higher than it should be (figure 5a). The negative control LNPs without RNA had a slightly larger size, again a result of aggregation since the sample had not been sonicated before analysis (figure 5b). Analysis with transmission electron microscopy (TEM) showed that the lipids formed clusters in the aqueous phase without incorporating any of the mRNA as the result of incorrect mixing (figure 5c). It is important to note that lipids should always be added to the aqueous phase during mixing to prevent this. It is also recommended to use other mixing methods like microfluidics that are better suited for smaller volumes.

![3.1](https://static.igem.wiki/teams/5342/images/results/lnp-number-3.webp)

Figure 5. Analysis of LNPs produced for transfection. (a) DLS size measurement of LNPs205 LNPs with GFP mRNA, Liposome material mode; Dispersant PBS. (b) DLS size measurement of LNPs205 LNPs with no mRNA, Liposome material mode; Dispersant PBS. (c) TEM pictures of LNPs205 LNPs with GFP mRNA show that no encapsulation occurred. (d) TEM pictures of LNPs205 LNPs with no mRNA show LNP formation. (e) DLS Z-potential measurement of LNPs205 LNPs with no mRNA and with GFP mRNA, Liposome material mode; Dispersant PBS.
On 23/09 a second batch of LNPs was prepared for the purpose of transfection.
LNPs were formulated with previously prepared mRNA:
GFP1, GFP2, F8 1, 5xFV F8, GFP control. Control GFP samples will be borrowed from Martin Emmaneel. DLS size analysis showed that the prepared LNPs did not have the desired size, most had a size between 1 and 10 nm or a size between 100 and 1000 nm (figure 6).

![4.1](https://static.igem.wiki/teams/5342/images/results/lnp-number-4.webp)

figure 6. DLS size measurement, Liposome material mode; Dispersant PBS. (a) negative control LNPs, no mRNA. (b) positive control LNPs, control GFP mRNA. (c) GFP2 LNPs. (d) GFP1 LNPs. (e) 5xVF8-2 LNPs. (f) F8-1 LNPs.
With TEM, some LNPs could be seen, although in extremely low abundance - in total 6-10 LNPs were observed in 3 samples.

![5.1](https://static.igem.wiki/teams/5342/images/results/lnp-number-5.webp)

figure 7. TEM pictures of F8 1 mRNA LNP
With this, we conclude that the formulation method is not efficient. LNPs almost do not form with the ethanol injection method when using low lipid concentrations. It is recommended to use higher concentration of lipids and implement a pulsification method or microfluidic mixing.
Encapsulation efficiency of the last batch of LNPs was measured by making a dilution series with known concentrations of the same RNA that is encapsulated in the LNPs. By lysing the LNPs and measuring the fluorescence with SYBR® Gold Nucleic Acid Gel Stain, the concentration of mRNA encapsulated in the LNP could be measured.
The encapsulation efficiency measurement of the last batch was not effective since the LNPs did not form correctly.

![6.1](https://static.igem.wiki/teams/5342/images/results/standard-curve-igem.webp)

Figure 8. Encapsulation efficiency. Standard curve of GFP mRNA
In Figure 8, It can be seen that the intensity of mRNAs in the standard curve is not correlated to the decreasing concentration values of mRNA which proved to us the SYBR Gold dye in the end was not applicable for this analysis. It is important to note that the SYBR Gold dye is also usually used for analysis of DNA and not RNA [2], and was used in this case due to the lack of budget and materials.

M. Kim et al. ,Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver.Sci. Adv.7,eabf4398(2021).DOI:10.1126/sciadv.abf4398

Fisher, Thermo. “SYBRTM Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO).” Thermofisher.com, 2022, www.thermofisher.com/order/catalog/product/S11494.

## HEK293 cell transfection

Cell transfection into HEK293T/17 cell (Human Embryonic Kidney) culture with mRNA and lipid nanoparticles (LNPs) was carried out from 19/09 till 24/09. This is a reliable cell strain which is frequently used in therapeutic research. The results were assessed via fluorescence microscopy. The positive control exhibited fluorescence of GFP mRNA without presence of lipid nanoparticles (Figure 18). The synthesised eGFP mRNA was seen being expressed in the cells in low concentration. This batch of samples did not contain mRNA inserted in LNPs (Figure 19, Table 3). A new batch of HEK cells was transfected with our synthesised F8 and GFP mRNA in LNPs on 25/09. The fluorescence microscopy results indicated no evident fluorescence but the cells were seen to have survived after transfection with LNPs (Figures 16 and 17; Table 3).

![20](https://static.igem.wiki/teams/5342/images/results/mrna-number-20.webp)

**Figure 1. Expression of positive control GFP mRNA via fluorescence microscopy in HEK cells.(24/09/2024)**

Fluorescence detected after transfecting HEK293 cells with positive control GFP mRNA.

![21](https://static.igem.wiki/teams/5342/images/results/mrna-number-21.webp)

**Figure 2. The expression of synthesised eGFP mRNA via fluorescence microscopy in HEK cells. (19/09/2024)**

The cells expressing GFP are indicated by the red arrow. Translational efficiency is low.

![22](https://static.igem.wiki/teams/5342/images/results/mrna-number-22.webp)

**Figure 3. Radboud iGEM teams’ mRNA samples and LNPs with F8 and eGFP mRNA (including control GFP). (24/09/2024)**

No fluorescence detected in all HEK cells.

![23](https://static.igem.wiki/teams/5342/images/results/mrna-number-23.webp)

**Figure 4. HEK cells without fluorescence. (24/09/2024)**

Live cells after transfection of LNPs.

![24](https://static.igem.wiki/teams/5342/images/results/mrna-number-24.webp)

**Overview of results from experiment (done on 24/09/2024)**

No fluorescence observed in cell cultures transfected with F8, GFP mRNA and lipid nanoparticles. Cells with empty transfected LNPs show signs of survival (see Figure 18 and 19).

##### Analysis and conclusion

The transfection of the acquired GFP and F8 mRNA was preceded in HEK cells. The fluorescence of eGFP was observed to not be optimal indicating low viable concentration of the mRNA. Moreover, the cells transfected with F8 and eGFP mRNA with LNP included did not show any visible fluorescence. The latter could have resulted from low concentration of lipid nanoparticles produced prior to transfection. The cells that got transfected with these LNPs did show signs of survival which proved the safety and non-toxicity of the produced nanoparticles. It has to be taken into account that the cell line used was not the originally planned HELA cell strain. This was due to cost restrictions and availability of usable HELA cells.

### Future plans

For now, if we would consider continuing our project, we would implement changes in LNP production to acquire better yield and functionality of the vesicles and change the structure of mRNA production. We observed that methods which were normally used for experiments such as amplification did not work well. We would delve deeper in research about our protein and its characteristics to find the optimal way of its production. Also, in the final step we would like to implement the nasal spray delivery system of our mRNA-LNP product. This would be the groundbreaking stage of our research for a safer and cheaper alternative of Hemophilia A therapy.

### Project achievements

All experiments were listed with 3 letter abbreviation RAD (Radboud University) with the number of the experiment corresponding to the project part and the execution order.

*For example, RAD302 = second experiment done in the third part of our project, specifically, HEK cell transfection.*

**A list of successful results during our project from our  [Radboud University Team Lab Notebook](https://2024.igem.wiki/radboud-university/notebook) :**

* RAD105* RAD108* RAD107* RAD109* RAD110* RAD111* RAD113* RAD114* RAD115* RAD120* RAD124* RAD127* RAD128* RAD129* RAD130* RAD201* RAD204* RAD205* RAD206* RAD301* RAD302

**A list of unsuccessful results during our project from our  [Radboud University Team Lab Notebook](https://2024.igem.wiki/radboud-university/notebook) :**

* RAD101* RAD102* RAD103* RAD104* RAD106* RAD112* RAD116* RAD117* RAD118* RAD119* RAD121* RAD122* RAD123* RAD125* RAD126* RAD131* RAD132* RAD133* RAD202* RAD203* RAD302

### Project achievements

1. Biochemical and functional characterization of a recombinant monomeric Factor VIII-Fc fusion protein. Peters RT, Toby G, Lu Q, Liu T, Kulman JD, Low SC, Bitonti AJ, Pierce GF. J Thromb Haemost. 2012 Dec 4. doi: 10.1111/jth.12076. 10.1111/jth.12076 PubMed 23205847- Passmore, L. A., & Coller, J. (2021). Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression. Nature Reviews. Molecular Cell Biology, 23(2), 93–106. https://doi.org/10.1038/s41580-021-00417-y- Passmore, L. A., & Coller, J. (2021). Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression. Nature Reviews. Molecular Cell Biology, 23(2), 93–106. https://doi.org/10.1038/s41580-021-00417-y

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