iGEM 2024 — Team sheffield

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_title %}

# Notebook

{% endblock %}
{% block page\_index %}

Week 1

* 17th June
* 18th June
* 19th June
* 20th June
* 21st June

Week 2

* 24th June
* 25th June
* 26th June
* 27th June

Week 3

* 1st & 2nd July
* 4th July

Week 4

* 8th July
* 9th July
* 10th July
* 11th July
* 12th July

Week 5

* 15th July
* 16th July
* 17th July
* 18th July
* 19th July

Week 6

* 22nd July
* 23rd July
* 24th July
* 25th July
* 26th July

Week 7

* 29th July
* 30th July
* 31st July
* 1st August
* 2nd August

Week 8

* 5th August
* 6th August
* 7th August
* 8th August
* 9th August

Week 9

* 13th August
* 14th August
* 15th August

Week 10

* 20th August
* 21st August
* 22nd August
* 23rd August

Week 11

* 26th August
* 27th August
* 28th August
* 29th August

Week 12

* 2nd September
* 3rd September
* 4th September
* 5th September
* 6th September

Week 13

* 9th September
* 10th September
* 11th September
* 13th September

{% endblock %}
{% block page\_content %}

## Week by Week write up

---

### WEEKS

![week timetable](https://static.igem.wiki/teams/5172/timetable.jpg)

## W/C 17th June Lab Notebook

---

### 17th June 2024

### Experiments:

* PafA-Pet21a was transformed into BL21 E.Coli using transformation protocol.~13 ng of DNA.
* Our advisor generated five fragments via PCR that can then be golden gate clones together to develop a pet21a\_BsaI plasmid backbone and a pet21a\_sfGFP backbone.

  | Desired Fragment Length (bp) | Primers used (see table below) | What is this fragment? |
  | --- | --- | --- |
  | 3918 | 1, 4 | Backup pET21a with the BsaI site in AmpR removed. BsaI site added at MCS. Fragment 1 of 2. |
  | 1526 | 3, 2 | Backup pET21a with the BsaI site in AmpR removed. BsaI site added at MCS. Fragment 2/2 |
  | 3908 | 1, 6 | pet21a(+)-sfGFP |
  | 1504 | 5, 2 | pET21a(+)-sfGFP |
  | 883 | 7, 8 | pJUMP21-1A |

  | Primer Label | Primer Name | Sequence |
  | --- | --- | --- |
  | 1 | pET21a\_BsaI\_MCS\_GFP\_REV | GCCGGTGAGCGTGGcTCTCGCGGTATCATTGCAGCAC |
  | 2 | pET21a\_BsaI\_AmpR\_REV | AATGATACCGCGAGAGCCACGCTCACCGGCTCC |
  | 3 | pET21a\_BsaI\_MCS\_FWD | AGAAGGAGATATACAAATGCGAGACCGGTCTCTGCTTTACACCACCACCACCACCACTG |
  | 4 | pET21a\_BsaI\_MCS\_REV | GACCGGTCTCgCATtTGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGG |
  | 5 | pET21a\_BsaI\_MCS\_GFP\_FWD | CGCTGGTCTCTGCTTTACACCACCACCACCACCACTG |
  | 6 | pET21a\_BsaI\_MCS\_GFP\_REV | ACATTTGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGG |
  | 7 | pET21a\_BsaI\_GFP\_FWD | TTTAACTTTAAGAAGGAGATATACAAATGTGAGACCGGAGTTGACGGCTAG |
  | 8 | pET21a\_BsaI\_GFP\_REV | GGTGGTGGTGGTGTAAAGCAGAGACCAGCGTGCAACTCAGT |

  Fragments were mixed together to form pet21\_sfGFP and the fragments were mixed to form pet21\_BsaI. This was assembled using golden gate assembly. Following NEBs protocol, but instead of using the mixture of BsaI and ligase they provide, we instead add 1µl of BsaI and 1µl of Ligase.

### Results:

* Transformed BL21 were plated on an LB Agar plate containing ampicillin.
  The gel of the five fragments is seen below and all bands are around the right size, confirming a successful PCR.
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/06-17-24.png)

### Extra Notes:

* All members of the team became aware of where all necessary equipment and materials for the project were located.
* The appropriate safety disclaimers were signed by each member of the team and demonstrations were given for how to safely use all equipment that would be needed throughout the project.
* All members of the team completed digital health and safety and waste management training.
* Team leaders completed autoclave training.

## W/C 17th June Lab Notebook

---

### 18th June 2024

### Experiments:

* If the transformation was successful, add a colony of the transformed BL21 to a mixture of LB broth and ampicillin and allow it to grow overnight.

### Results:

* The transformation from the 17th of June was successful. Therefore a colony of BL21 from the petri dish was added to a mixture of 5ml of LB broth and 5μl of ampicillin. The sample was then left to grow overnight.
* It was also confirmed that a control plate showing a positive pJUMP28 transformation had been successful. This plate was then discarded.

### Extra Notes:

* Transformation training was carried out for all members of the lab team.
* Restriction digest training was also completed by all lab members.

## W/C 17th June Lab Notebook

---

### 19th June 2024

### Experiments:

* Produce a 1ml glycerol stock of PafA-BL21.
* Inoculate 2x duran bottles containing 500ml LB and ampicillin using overnight samples of PafA from 18th June. Then allow this to grow to an optical density of 0.4-0.6. Then induce by adding 500μM of IPTG and leave to express PafA overnight by incubating at 18°C.
* Our advisors (Brooks Rady and Isaac White) helped us to develop a backbone to enable easier screening of colonies= pet21-sfGFP with BsaI sites surrounding the GFP. This plasmid was transformed into DH5α.

### Results:

* Glycerol stock was produced
* PafA was induced and left to incubate overnight
* Transformation of an advisor’s backbone structures

### Extra Notes:

* Agarose gel training was carried out
* PCR training was completed

## W/C 17th June Lab Notebook

---

### 20th June 2024

### Experiments:

* Overnights made using Brook’s Pet21a-sfGFP
* Buffers for protein purification were made

### Results:

* PCR products (amplified PhoA) were run on a gel. First three lanes were the correct size. The corresponding PCR product was purified and pooled together on 25/6/24. The Nanodrop gave a result of 210ng/μL, 38μL.
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/06-20-24.png)

### Extra Notes:

* MiniPrep Training Completed
* PCR Cleanup Training Completed
* Team leaders trained on how to use sonicator and high speed centrifuge

## W/C 17th June Lab Notebook

---

### 21st June 2024

### Experiments:

* Miniprep of Brooks’ Pet21a-sfGFP ligation completed.

### Results:

* No Results, nanodrop of miniprep pending.

### Extra Notes:

* Thomas provided training for all other members of the lab team on how to code on the Wiki and how to add, commit and push changes onto it.

## W/C 24th June Lab Notebook

---

### 24th June 2024

### Experiments:

* Nanodropping pet21a-sfGFP minipreps (from 21st June)
* SDS-PAGE of purified PafA and wash steps to identify how effective our purification was using premade SDS gels (Novex 16% Tris-Gycine Plus Wedge Well Gel - ref code = XP00160BOX) which are stored in the fridge. This gel was then stained with QuickBlue Protein Stain for half an hour
* Digesting pet21a-sfGFP with BsaI-HFV2

### Results:

* Pet21a-sfGFP DNA concentrations from nanodropping 5 colonies from Pet21(+)BSaI-sfGFP (ng/µl)
* | Colony No. | Concentration (ng/µl) |
  | --- | --- |
  | 1 | 28 |
  | 2 | 39 |
  | 3 | 40 |
  | 4 | 40 |
  | 5 | 38 |
* SDS-PAGE
* ![gel](https://static.igem.wiki/teams/5172/notebook/06-24-24-gel.png)
* FT = Flow through, BB = Binding Buffer, WB = Wash Buffer, E = Elution, \*The first lane contains 4µl of Geneflow BlueStar Plus prestained protein marker.

### Extra Notes:

Verified with our supervisors that this gel looks good - whilst there's extra banding suggesting some impurity, this looks around average for their gels of this enzyme. In the future we should consider eluting past 5 elutions until no more protein is left to elute.

## W/C 24th June Lab Notebook

---

### 25th June 2024

### Experiments:

* Gel electrophoresis of pet21a-sfGFP w/ BsaI (from 24/06/2024)
* Gel electrophoresis of PhoA w/ BsaI
* Purification of PafA
* SDS-PAGE of purified PafA
* Determine PhoA DNA conc. & digest w/ BsaI-HFV2
* Gel electrophoresis of PhoA w/ BsaI
* Transformation of pet21a-sfGFP into DH5⍺
* Overnight incubation of pet21a-sfGFP DH5⍺

### Results:

* pet21a-sfGFP w/ BsaI gel electrophoresis -
  ![gel](https://static.igem.wiki/teams/5172/notebook/25-06-1.jpg)
  \*Lane 1 was ladder and then the proceeding was colonies 1-5
* PhoA w/ BsaI gel electrophoresis -
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/25-06-2.jpg)
  1. 100bp DNA Ladder - Invitrogen
  2. BsaI digest on PhoA →- Right size!
  3. pET21a-sfGFP-1
  4. pET21a-sfGFP-2
  5. pET21a-sfGFP-3
  6. pET21a-sfGFP-4
  7. pET21a-sfGFP-5
  8. 100bp DNA Ladder - Invitrogen
* SDS-PAGE w/ PafA -
  ![gel](https://static.igem.wiki/teams/5172/notebook/25-06-3.jpg)
  + FT = Flow through
  + BB = Binding buffer
  + WB = Wash buffer
  + E = Elution
* PhoA w/ BsaI digest was run on a gel and showed a band of the correct size so PhoA is ready for ligation

## W/C 24th June Lab Notebook

---

### 26th June 2024

### Experiments:

* SDS-PAGE with product of PafA purification (in addition to other liquids from the nickel column to ensure no PafA had been lost)
* pet21a-sfGFP ligation
* Transformation of pet21a-sfGFP digest into DH5⍺
* Minipreps of pet21a-sfGFP and pet21a backbones then nanodropping to determine concentration
* 20ml of o/n pet21a-sfGFP Colony 1 transformation produced
* 10ml of o/n pet21a(+) produced

### Results:

* PafA SDS-PAGE:
  ![gel](https://static.igem.wiki/teams/5172/notebook/26-06.jpg)
  Wells:
  1. BLUeye Pre-Stained Protein Ladder (10-245kDa) - GeneFlow - 4µl
  2. FT
  3. BB
  4. WB
  5. FT of PD10
  6. Sample Concentrator (blue lid, screwy centrifuge tube)
  7. FT in sample concentrator (EB1 & EB2)
  8. FT in sample concentrator (EB3, EB4, & EB5)
  9. Purified PafA
  10. BLUeye Pre-Stained Protein Ladder (10-245kDa) - GeneFlow - 4µl
* Transformation of pet21a-sfGFP digest (Both glowing and non-glowing colonies were observed):

  | Miniprep No. | Pet21a-sfGFP Miniprep DNA conc (µg/ml) | Pet21a(+) Miniprep conc (µg/ml) |
  | --- | --- | --- |
  | 1 | - | 74 |
  | 2 | - | 58 |
  | 3 | - | 50 |
  | 4 | 111 | 51 |
  | 5 | 38 | 47 |

### Extra Notes:

Minipreps:
All solutions were resuspended in 30µl of mP6 in the final step APART from no. 4 Pet21a Backbone (w/o vsGFP)\* which was resuspended in 50ng/µl.

## W/C 24th June Lab Notebook

---

### 27th June 2024

### Experiments:

* Miniprep O/Ns of pet21a-sfGFP (Colony 1) and pet21a(+)
* More PhoA amplified from g-block
* Golden Gate assembly of pet21a-sfGFP (Colony 1), PhoA, BsaI-HFv2 using T4 DNA ligase buffer
* Gel electrophoresis of Golden Gate results

### Results:

pet21a-sfGFP Miniprep conc (µg/ml):

* 54
* 70
* 79
* 25

Pet21a(+) Miniprep conc (µg/ml):

* 60
* 66

Golden Gate assembly gel electrophoresis:

![gel scan](https://static.igem.wiki/teams/5172/notebook/27-06.jpg)

Wells:

1. 1kb DNA Ladder
2. Digested pET21a-sfGFP (Colony 1) - same that was sent for sequencing after transforming, amplifying and mini prepping again
   * We can see two clear bands next to each other. We think the upper one is undigested plasmid and the second one is the backbone fragment.
   * If we change the light exposition we think we can faintly see a band ~850bp, the size of sfGFP.
3. Digested pET21a(+)\*

### Extra Notes:

\*Mistake made - it was meant to be pET21a-BsaI (the backup backbone).

## W/C 1st July Lab Notebook

---

### 1st & 2nd July 2024

### Experiments:

* Colony PCR was performed on six transformant colonies containing PhoA in the pet21a vector using pet forward and reverse primers with a 1 minute 45 second extension time. We used 35 µl of PCRBIO Taq 2x Polymerase red mix, 2.8 µL of pet forward+reverse primer (10 µM), 32.2 µL of sterile water to make up a total volume of 70 µL.
* We added 10 µL of this solution to small Eppendorfs and added the transformant colonies containing PhoA in the pet21a vector. The colonies were then streaked on scratch plates and the pipette tip was dropped into an LB overnight made of 5ml agar and 5l ampicillin.
* A gel electrophoresis was done on the PhoA samples using 1 kb Plus DNA ladder.

### Results:

* ![gel scan](https://static.igem.wiki/teams/5172/notebook/07-01-24.png)
* ![gel scan](https://static.igem.wiki/teams/5172/notebook/07-01-24-2.png)

Electrophoresis of PhoA samples. A 1 kb Plus DNA was used for both these gels.

### Extra Notes:

* The growth colony electrophoresis results implied that it should have worked because only a few bands glowed and the rest were not glowing. However we are not sure how the pet21a could have turned back into a plasmid without having PhoA as the backbone. This could have been due to some ligation error as the labelling was not clear and we weren’t sure if the PhoA had BsaI sites. So we will do ligation again with newly amplified PhoA.
* The colony PCR protocol was revised with new concentrations to account for inaccuracies.

## W/C 1st July Lab Notebook

---

### 4th July 2024

### Experiments:

* Ligation of PhoA and Pet21a was done using BsaI Golden Gate assembly protocol.
* 3 Ligations of Pet21a and PhoA were made:
* + 2.5 µl of 70ng/µl pet21-sfGFP + 13.0 µl of distilled water
  + 4.0 µl of 54ng/µl pet21-sfGFP + 11.5 µl of distilled water
  + 2.5 µl of 79ng/µl pet21-sfGFP + 13.0 µl of distilled water
* All 3 ligations also had 2µl of T4 DNA ligase buffer, 1µl of BsaI-HFv2 and 1µl of ligase added to them.

## W/C 8th July Lab Notebook

---

### 8th July 2024

### Experiments:

* Attempted a colony PCR of DH5α with our ligated construct of PhoA-pet21a. Used a lesser volume of primers than in future experiments.

### Results:

* No band at ~1500bp, possibly due to having a low concentration of primers.

### Extra Notes:

* Another colony PCR will need to be done tomorrow and a Miniprep in order to run a diagnostic digest.

## W/C 8th July Lab Notebook

---

### 9th July 2024

### Experiments:

* Completed a colony PCR using the PhoA BsaI primers.
* Miniprepped colony PCR from 8th July in order to perform a diagnostic digest with BsaI using colonies 10 and 11.
* Colony 10 used to run a digest.
* Made a 1:1 dilution of BsaI-HFv2 due to increased accuracy when pipetting higher volume.

### Results:

![gel-scan](https://static.igem.wiki/teams/5172/notebook/07-09-24.png)

* Results from colony PCR of Colonies 12,13,14,15,16 - Only 13 seems to have worked ~1300bp

![gel-scan](https://static.igem.wiki/teams/5172/notebook/07-09-24-2.png)

* Digest results unclear due to low concentration of DNA again

### Extra Notes:

* Now need to transform colony 10 into DH5α to triple check using colony PCR.

## W/C 8th July Lab Notebook

---

### 10th July 2024

### Experiments:

* Miniprepped colony 13
* Transformed colony 13 into BL21
* Colony 13 Miniprep was taken for sequencing. Volume and concentration taken beforehand.
* Colony PCR of 5 colonies from colony 10 that was digested on 9th July and produced unclear results. We also created scratch plates and LB overnights for each.
* A glycerol stock of PafA in BL21 was produced
* A bradford assay of PafA was made

### Results:

* Miniprep of colony 13 gave a concentration of 40.00 µg/ml.
* Approximately 6ml of 80% glycerol was produced. This was then used to create a glycerol stock of PafA in BL21, which was ~1ml in total and consisted of the cells from a 5ml Overnight colony (supernatant) with 0.4ml of 80% glycerol, 0.6ml of LB agar broth, and 1µl of 100mg/ml Ampicillin.
* A spectrophotometer reading of the bradford assay was performed after 10 minutes. The spec readings for the standard max’d in at 0.6 (OD595), whereas the PafA was 2.6 ish. Therefore we are repeating with a 1 in 5 and a 1 in 10 dilution (10µl PafA +90µl, 20µl PafA +80µl).

### Extra Notes:

* To make the spec a bit easier, we are using 0.2, 0.4, 0.6, 0.8 and 1mg/ml BSA concs rather than the full range- most protocols describe this anyway we were just being extra precautious.

## W/C 8th July Lab Notebook

---

### 11th July 2024

### Experiments:

* Made 3 Overnight Colonies of Colony 13, each with 5ml of LB and 5µl of 100µg/ml Ampicillin.
* Produce an agarose gel and then run colony PCR of colony 10
* Stepped up overnight PafA using 500ml of LB broth and 500µl of ampicillin, when this reached an OD of 0.419, induced with 250µl of IPTG.
* Conical flask for this had not been autoclaved so plated this onto an agar plate with ampicillin to ensure there was no contamination.
* Also produced an overnight colony of PafA using 5ml LB broth and 5 µl of 100µg/ml Ampicillin in case the conical flask used for the step up was contaminated and needed to be repeated.

### Results:

* ![gel scan](https://static.igem.wiki/teams/5172/notebook/07-11-24.png)
* ![gel scan](https://static.igem.wiki/teams/5172/notebook/07-11-24-2.png)

Results from the gel

## W/C 8th July Lab Notebook

---

### 12th July 2024

### Experiments:

* Checked for contamination of agar plates.
* Transferred PafA into 10x50ml test tubes.
* Centrifuged to produce a pellet.
* Remove the supernatant.
* Store test tubes in cool room to begin characterisation work on 15th July.

### Results:

* No contamination as nothing grew on the agar plates, therefore went ahead and centrifuged the induced PafA and removed the pellet. Then stored at 4°C to begin characterisation work on 15th July.

## W/C 15th July Lab Notebook

---

### 15th July 2024

### Experiments:

* Made Overnight Colonies of PhoA and PafA, each with 5ml of LB and 5µl of 100µg/ml Ampicillin.

## W/C 15th July Lab Notebook

---

### 16th July 2024

### Experiments:

* PafA and PhoA O/Ns both stepped up in 500ml LB broth + 500µl Ampicillin. When both reached an OD ~0.4 they were induced with 250µl of IPTG and left overnight at 18°C.

## W/C 15th July Lab Notebook

---

### 17th July 2024

### Experiments:

* Protein purification of PafA.

### Extra Notes:

* Double the usual volume of PafA protein went through the nickel column.
* Not all the FT was collected for PafA.

## W/C 15th July Lab Notebook

---

### 18th July 2024

### Experiments:

* Nickel Column was stripped.
* SDS-PAGE carried out for PhoA, PafA, and elutions.
* O/N of colonies 10 & 13 done for tomorrow

### Results:

* ![gel](https://static.igem.wiki/teams/5172/notebook/07-15-24.png)
* ![gel](https://static.igem.wiki/teams/5172/notebook/07-15-24-2.png)

### Extra Notes:

* Gel for PhoA did not work - perhaps due to a problem with PhoA itself or due to a problem regarding the bacteria’s metabolism.

## W/C 15th July Lab Notebook

---

### 19th July 2024

### Experiments:

* Colony PCR of PhoA colonies 10 & 13.
* Gel electrophoresis on the Colony PCRs using 1kbp DNA ladder.
* Desaltification using P10 desalting column. Column equibrilated with 25ml storage buffer. Once flowed through, 2.5ml of purified PafA protein elution from previous day was added. Then 3.5ml storage buffer added (after complete flow-through of PafA). Then protein and storage buffer were alternated until no more protein was left.
* Miniprep of O/N PhoA Colonies 10 &13.

### Results:

* PhoA Colony 10 DNA conc. - 192.835ng/µl
* PhoA Colony 13 DNA conc. - 467.111ng/µl

### Extra Notes:

* Each of the 3.5ml elutions of protein collected from the desalting column beads by the storage buffer were labelled in individual tubes, numbered 1 to 5.
* Both colonies 10 & 13 showed peaks at wavelength ~260 on nanodrop (good sign of the DNA being present).

## W/C 22nd July Lab Notebook

---

### 22nd July 2024

### Experiments:

* Digest PhoA colonies 10 & 13
* Digest pet21a-sfGFP
* PCR of pet21a-sfGFP and PhoA colonies 10 & 13

### Results:

* PCR on gel:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/22-07-24.png)

### Extra Notes:

* Gel: Pet21sfGFP digest showed that the BsaI enzyme is working in the ligations. However, given the amount added in (1µl of 550ng/µl), sfGFP band is ~850bp, out of a total of 6500bp. Therefore roughly 7.5x less bright on a gel than the concentration…should be around a 70ng/µl band brightness. This most definitely isn't so the digest or enzyme wasn't working very efficiently- tho bands in ladder’s brightness barely seems to change. Did it for 1hr at 37°C.

## W/C 22nd July Lab Notebook

---

### 23rd July 2024

### Experiments:

* 4 O/Ns of Colony 10 PhoA (with Ampicillin) prepared.
* Needed individual colonies for 10 so a streak plate of Colony 10 was prepared and incubated in 37°C.
* Gel electrophoresis done of PhoA 10 + Nco, PhoA 13 + Nco, and PhoA 10 (miniprep DNA control) using a 1kbp DNA ladder.

### Results:

* Gel electrophoresis:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/23-07-24.png)
  + Lane 1 = colony 10 digested w/ NcoI
  + Lane 2 = colony 13 digested w/ NcoI
  + Lane 3 = miniprepped colony 10 (control)

### Extra Notes:

* Gel: Band in lane 1= ~30ng/µl linearised PhoA containing plasmid as NcoI only cuts PhoA. Assuming linearised, not as streaky as bright as the miniprep but seeing something vs Col 13 not. Col 10 may contain PhoA - will be checked via sequencing tomorrow.

## W/C 22nd July Lab Notebook

---

### 24th July 2024

### Experiments:

* 4 minipreps of PhoA colony 10 were carried out, two done by students (with expired kit), two by an advisor (with newer kit).
* 4 O/Ns of Colony 10 PhoA prepared (using yesterday’s streak plate), each at 7ml.

### Results:

* PhoA 10 streak plate from the previous day was successful.
* Miniprep concentrations: 22.6ng/ml, 59.6ng/ml, 28.2ng/ml, 31.6ng/ml.

## W/C 22nd July Lab Notebook

---

### 25th July 2024

### Experiments:

* Mini preps of previous day’s 4 PhoA Colony 10 O/Ns.

### Results:

* Miniprep DNA Concentrations - 44ng/µl, 19ng/µl, 33ng/µl, 61ng/µl.

## W/C 22nd July Lab Notebook

---

### 26th July 2024

### Experiments:

* Making enzyme concentrations at 200nM, 20nM, and 2nM in the following manner:
  + 6ml of FINAL conc 10nM enzyme = making a 200nM enzyme = 80µl in 720µl (1:10) then 800µl in 5.2ml (1:7.5)
  + 1ml of FINAL conc 1nM = 1:10 dilution of the one above (200nM ← 20nM)
  + 1ml of 100pM PafA = 1:10 dilution of above (20nM ← 2nM)
* Buffers put in water bath at 28°C for 30 minutes.
* PNPP solutions made according to protocols then concentrations were tested in the spectrophotometer (at 405nm) in the following manner:
  + 1 = 100µl enzyme at final conc of 10nM + 900µl 22.5mM PNPP - BLANK 1 = Blanked against 100µl reaction buffer + 900µl PNPP 20mM
  + 2 = 100ul enzyme at final conc of 1nM - BLANK 2 = Blanked against 100µl enzyme at final conc of 1nM + 900µl SB
  + 3 = 100ul enzyme at final conc of 100pM - BLANK 3 = Blanked against 100µl enzyme at final conc of 100pM + 900µl SB
* To each at time 0 seconds, 900µl of 20mM PNPP was added. This was put in the spec and recorded every minute

### Results:

* Spectrophotometer results for PNPP:

  | Time(s) | 40 | 100 | 160 | 220 | 280 | 340 | 400 | 460 | 520 | 580 |
  | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
  | 100nM 0.010 | 0.019 | 0.027 | 0.036 | 0.045 | 0.055 | 0.064 | 0.074 | 0.084 | 0.094 | |
  | 1nM | 0.000 | 0.001 | 0.001 | 0.002 | 0.002 | 0.003 | 0.003 | 0.004 | 0.005 | 0.005 |
  | 100pM | -0.050 | -0.001 | -0.002 | -0.002 | -0.002 | -0.002 | -0.003 | -0.003 | -0.003 | -0.003 |

  | Time(s) | 640 | 700 | 760 | 820 | 880 | 940 | 1000 | 1060 | 1120 | 1180 |
  | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
  | 100nM | 0.104 | 0.113 | 0.123 | 0.134 | 0.143 | 0.153 | 0.163 | 0.173 | 0.182 | 0.192 |
  | 1nM | 0.006 | 0.006 | 0.007 | 0.008 | 0.008 | 0.009 | 0.009 | 0.009 | 0.010 | 0.011 |
  | 100pM | -0.003 | -0.003 | -0.003 | -0.003 | -0.003 | -0.003 | -0.003 | -0.003 | -0.003 | -0.003 |

  | Time(s) | 1240 | 1300 | 1360 | 1420 | 1480 | 1540 | 1600 | 1660 | 1720 | 1780 |
  | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
  | 100nM | 0.202 | 0.212 | 0.222 | 0.232 | 0.243 | 0.252 | 0.262 | 0.271 | 0.282 | 0.294 |
  | 1nM | 0.011 | 0.012 | 0.012 | 0.013 | 0.013 | 0.014 | 0.015 | 0.015 | 0.016 | 0.016 |
  | 100pM | -0.003 | -0.003 | -0.003 | -0.003 | -0.002 | -0.002 | -0.002 | -0.002 | -0.002 | -0.001 |

  | Time(s) | 1840 | 1900 | 1960 | 2020 | 2080 | 2140 | 2200 | 2260 | 2320 | 2380 |
  | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
  | 100nM | 0.305 | 0.315 | 0.328 | 0.337 | 0.345 | 0.354 | 0.364 | 0.373 | 0.383 | 0.393 |
  | 1nM | 0.017 | 0.017 | 0.018 | 0.018 | 0.019 | 0.019 | 0.020 | 0.020 | 0.021 | 0.021 |
  | 100pM | -0.001 | -0.001 | -0.001 | -0.001 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |

  | Time(s) | 2440 | 2500 | 2560 | 2620 | 2680 | 2740 |
  | --- | --- | --- | --- | --- | --- | --- |
  | 100nM | 0.404 | 0.412 | 0.422 | 0.433 | 0.442 | 0.451 |
  | 1nM | 0.022 | 0.023 | 0.023 | 0.024 | 0.024 | 0.025 |
  | 100pM | 0.003 | 0.003 | 0.003 | 0.003 | 0.003 | 0.003 |

  ![graph](https://static.igem.wiki/teams/5172/notebook/26-07-24.png)

### Extra Notes:

* Autoclaved substrate buffer and reaction buffer.
* Leftovers from the enzyme dilution trial will be stored in the freezer in the reaction buffer as the trisHCL should stabilise the enzyme for short term storage.

## W/C 29th July Lab Notebook

---

### 29th July 2024

### Experiments:

* PNPP assay with differing concentrations of 200nM, 2nM, and 200pM.

### Results:

* PNPP assay: very low absorbance on plate readings, reactions occur too slowly. Maybe due to too much freeze-thawing of the enzyme which deactivated it.

## W/C 29th July Lab Notebook

---

### 30th July 2024

### Experiments:

* Step up of 2x PhoA O/ns (10ml) from the previous day and 2x PafA O/ns (10ml).
* SDS of elutions from the PD10 column (from 19/07/24)

### Extra Notes:

* PafA and PhoA were induced when they reached an OD of 0.4-0.6.
* Produced more 100mg/ml Ampicillin stocks
* Produced more PD10 columns - may not have filled with the correct volume of beads though

## W/C 29th July Lab Notebook

---

### 31st July 2024

### Experiments:

* Span all the 500ml cultures (2xPhoA, 2xPafA) down in the centrifuge and discarded the liquid, then proceeded to sonicate.
* A 5ml storage buffer was run through a PD10 column containing 5cm of Sephardic beads. The action was repeated 5 times making up for 25ml storage run through column.
* A 2.5 ml of elation was added to the column and run in a centrifuge. The elution was collected in the tube labelled FT PD10 E1.
* A 3.5ml of 20% glycerol, hepes and NaCl buffer. It was centrifuged and collected in the tube named PD10 1. Then it was transferred to blue eppendorf tubes and stored in the small freezer at -80°C.
* Both elation and glycerol adding was repeated more times with according labels on the tubes (E1,E2,E3) and all PD10 collected were stored in the freezer in eppendorf tubes.

## W/C 29th July Lab Notebook

---

### 1st August 2024

### Experiments:

* SDS gel of protein purification
* Reaction buffer put in 30℃ water bath for later assay
* Measured PafA concentration two samples labelled 1 and 2 kept in fridge using extinction coefficient: 82170.00 M-1cm-1
  1= 3.53mg/ml= 59.98µM and 2=0.868mg/ml= 14.5µM

### Results:

* SDS gel:

  | Lane | Contents of Lane |
  | --- | --- |
  | 1 | Protein ladder 4µl |
  | 2 | FT 10µl +10µl Dye |
  | 3 | BB 10µl +10µl dye |
  | 4 | WB 10µl +10µl dye |
  | 5 | 1st elution 10µl + 10µl dye |
  | 6 | 2nd elution 10µl + 10µl dye |
  | 7 | FT PD10 E1 10µl + 10µl dye |
  | 8 | FT PD10 E2 10µl + 10µl dye |
  | 9 | E1 blue eppendorf (pd10) 10µl + 10µl dye |
  | 10 | E2 blue ependorf pd10 10µl + 10µl dye |

  ![gel](https://static.igem.wiki/teams/5172/notebook/01-08-24.jpg)

### Extra Notes:

* It is possible that PafA samples 1 and 2 were not mixed well.

## W/C 29th July Lab Notebook

---

### 2nd August 2024

### Experiments:

* PNPP assay carried out
* PCR of PhoA:

  | Component | Volume (µl) |
  | --- | --- |
  | PhoA Gblock ( should be at 10ng/µl according to IDT’s respension advice) | 1.00 |
  | PhoA BsaI Fwd iGEM09 (10µM) | 1.25 |
  | PhoA BsaI New Rev iGEM13 (10µM) | 1.25 |
  | Q5 2X Master Mix | 12.5 |
  | Sterile water | 9.00 |
* Did another PCR of PhoA exactly the same mixture, but altered the PCR cycle by making the annealing temp start at 71 and each of the 30 cycles go down by 0.4 so that it covers a range of 71-59. This is as benchling and NEB predict greatly different Tms for the same primers. (~71 for NEB and ~60 for benchling).
* Pet21sfGFP nano dropped by an advisor to check for any GFP contamination - a common problem from past minipreps.
* To double check the concentration the plasmid was linearised in different amounts to confirm on a gel that the concentration of pet21sfGFP is correct.
* Digest of pet21sfGFP:
  Digesting 6µl of the pet21sfGFP in 50µl total volume:
  + 5µl rCutSmart
  + 6µl of Pet21sfGFP A
  + 1.5µl KpnI based on the fact that we might have 3000ng and it’s 5-10 units per 1000n
  + 38µl Sterile distilled water
* Gel electrophoresis of PhoA and pet21sfGFP

### Results:

* Our nanodrop gives us an A260/280 of ~1.9. Online research tells us that below 1.8 indicates protein contamination so it seems unlikely that it is contaminated.
* Gel (45mins at 100V):
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/02-08-24.jpg)

  | Lane | Samples |
  | --- | --- |
  | 1 | 1kb GeneRuler ladder Invitrogen |
  | 2 | PhoA 71 1:10 |
  | 3 | PhoA 71-59 1:10 |
  | 4 | PhoA 71 1:100 |
  | 5 | PhoA 71-59 1:100 |
  | 6 | 1kb GeneRuler ladder Invitrogen |
  | 7 | Undiluted linearised pet21sfGFP |
  | 8 | 1:2 linearised pet21sfGFP |
  | 9 | 1:10 linearised pet21sfGFP |
  | 10 |  |
  | 11 | Undiluted linearised pet21sfGFP 2 |
* Golden gate assembly (using miniprepped pet21sfGFP A)

  | Reagent | Calculations using NEBioCalculator | Amount (µl) |
  | --- | --- | --- |
  | Pet21a-sfGPF A from mini prep which Issac did with a conc of 500ng/µl | Mass we want 168.6 ng (0.05pmol). Did a 1:3 dilution | 1 of 1:3 Dilution |
  | PhoA from a conc of 200ng/µl | Mass we want 83.71ng. Did a 1:2.5 dilution | 1 of 1:2.5 dilution |
  | T4 DNA ligase buffer (10x) | - | 2µl |
  | BsaI-HFv2 | - | 1 |
  | Ligase | - | 1 |
  | Sterile distilled water | To 20µl | 14 |

  This was then put in the thermocycler for 30 cycles of 37°C for 2 minutes and then 16oC for 2 minutes, followed with 60°C for 5 minutes and then held at 4°C overnight

### Extra Notes:

* Sequencing results came back with a large gap/deletion in the PhoA gene- there’s also 4 or 5 A bases on either side of the deletion. We think it's likely to be an assembly problem rather than a sequencing problem, so we're going to reclone with the new PhoARev primer.

## W/C 5th August Lab Notebook

---

### 5th August 2024

### Experiments:

* Colony PCRs of the PhoA transformation (colonies 11-20) from the previous Friday
* Streak plate (with Ampicillin) made using colony PCRs
* O/N produced from the streak plate

### Results:

* Plate from the PhoA transformation done on Sunday by an advisor:
  ![plate](https://static.igem.wiki/teams/5172/notebook/05-08-24.jpg)
* Colony PCR result on gel:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/01-08-24-1.png)

### Extra Notes:

* Ran colonies 11-20 in PCR machine on following:
  1. 5 minutes at 95°C
  2. 35 cycles of 94°C for 30
  3. 62°C going down by 0.1°C every cycle for 45 seconds
  4. 72°C for 40 cycles
  5. Following cycles final extension at 72°C for 5 minutes

## W/C 5th August Lab Notebook

---

### 6th August 2024

### Experiments:

* Miniprep of O/Ns 11, 12, & 20
* PhoA colonies 11-20 used to make Ampicillin streak plates of colonies 11, 13, 16, & 17
* Freeze-thaw cycles 1-10 carried out on PafA, 10 minute intervals of alternately freezing at -80°C and thawing on ice.
* Transformation of PhoA colony 11 into BL21
* Protein digest on minipreps of colonies 12 & 20 (and a repeat of colony 11) using following mixture for each:
  + 11.5µl of miniprep
  + 12.5µl of rCutSmart buffer
  + 0.5µl of BsaI-HFv2
  + 0.5µl of NdeI

### Results:

* Minipreps' nanodrop results:
  + Colony 11 - 43µg/ml:
    ![nandropper result](https://static.igem.wiki/teams/5172/notebook/06-08-24.jpg)
  + Colony 12 - 55µg/ml:
    ![nanodropper result](https://static.igem.wiki/teams/5172/notebook/06-08-24-2.jpg)
  + Colony 20 - 52µg/ml:
    ![nanodropper result](https://static.igem.wiki/teams/5172/notebook/06-08-24-3.jpg)
* Protein digest results on gel:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/06-08-24-4.jpg)

  Lanes:

  1. 1kb Ladder
  2. Colony 12 miniprep digested with NdeI and BsaI
  3. Colony 20 miniprep digested with NdeI and BsaI
  4. Empty
  5. Colony 11 PCR run again (used on 05/08 with no dye)

### Extra Notes:

* PhoA colony 11 sent off for sequencing

## W/C 5th August Lab Notebook

---

### 7th August 2024

### Experiments:

* Did O/Ns of transformed PhoA colonies 10 & 11.
* Protein Conc Via Biodrop with PafA E1 (g/100ml) at 13.73 (blanked against 20% glycerol storage buffer)

### Results:

* Streak plates of PhoA colonies 11, 13, 16, & 17 from the previous day:
  ![petri dish](https://static.igem.wiki/teams/5172/notebook/07-08-24.jpg)
  ![petri dish](https://static.igem.wiki/teams/5172/notebook/07-08-24-2.jpg)
  ![petri dish](https://static.igem.wiki/teams/5172/notebook/07-08-24-3.jpg)
  ![petri dish](https://static.igem.wiki/teams/5172/notebook/07-08-24-4.jpg)
* Transformation plate of PhoA Colony 11 into BL21:
  ![petri dish](https://static.igem.wiki/teams/5172/notebook/07-08-24-5.jpg)
* Biodrop of PafA (from freeze-thaw cycles 1-10):
  + 1 = 2.268mg/ml = 37.89µM
  + 2 = 2.739mg/ml = 45.76µM
  + 3 = 2.875 mg/ml = 48.03µM
  + 4 = 3.520mg/ml = 58.81µM
  + 5 = 2.751mg/ml = 45.96µM
  + 6 = 3.785mg/ml = 63.24µM
  + 7 = 2.996mg/ml = 50.06µM
  + 8 = 3.682mg/ml = 61.52µM
  + 9 = 0.723mg/ml = 12.08µM
  + 10 = 1.761mg/ml = 29.42µM
  + Stored in freezer = 2.432mg/ml = 40.63µM
  + \*Stored in fridge = 10nM
* PNPP assay of freeze-thaw cycles - each well had 90µl PNPP added but concentrations of wells varied between 11.1mM, 1.1mM, and 0.11mM. Each reaction was started by adding 10µl of 1nM PafA (of varying freeze-thaw cycle numbers)\*\*

### Extra Notes:

* \*Biodrop of fridge-stored solution of PafA was stored and diluted in reaction buffer prior to this so may not be the best comparison.
* \*\*We hadn’t made up enough PNPP solutions so did more dilutions. The 11.1mM original PNPP went into B1-B11. 11.1mM was remade and went into B12- C12.

## W/C 5th August Lab Notebook

---

### 8th August 2024

### Experiments:

* Did PCR colonies for the streak plate made of Colony 11\*

### Extra Notes:

* \*Was the only colony with signs of PhoA growing

## W/C 5th August Lab Notebook

---

### 9th August 2024

### Experiments:

* PCR of an advisor’s gBlock amplification (using Platinum SuperFi II DNA Polymerase from thermofisher) then cleanup with Omega Monarch Kit. Was eluted in 11µl then incubated for 5 minutes before spinning.
* Carried out a Colony PCR with Pet21a+ transformation plate and with the spread plates from Colonies 13, 16 and 17. Four samples from each spread plate were taken and 8 were taken from the Pet21a+ transformation plate. This was also used to make overnights and scratch plates to isolate a band at 1000bp.
* Gel electrophoresis of both this day’s Colony PCR and of the PCR (pre and post cleanup) carried out by an advisor.
* Colonies 20 & 21 used to make O/Ns.

### Results:

* Concentration of PCR cleanup was 277ng/µl
* Gel electrophoresis:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/09-08-24.jpg)

  | Lane | Samples |
  | --- | --- |
  | 1 | 1kb ladder invitrogen generuler |
  | 2 | Brook’s PCR of Gblock PhoA (0.5µl + 10µl water + 2µl dye) |
  | 3 | Clean up of the PCR of the gblock PhoA (1µl + 9µl water + 2µl dye= 20-25ng/µl) |
  | 4 |  |
  | 5 | 13a - colony PCR of restreaks from 13 which had two bands on a previous gel. |
  | 6 | 13b |
  | 7 | 13c |
  | 8 | 13d |
  | 9 |  |
  | 10 | 16a - colony PCR of restreaks from 16 which had two bands on a previous gel. |
  | 11 | 16b |
  | 12 | 16c |
  | 13 | 16d |
  | 14 |  |
  | 15 | 17a - colony PCR of restreaks from 17 which had two bands on a previous gel. |
  | 16 | 17b |
  | 17 | 17c |
  | 18 | 17d |
  | 19 | 1kb invitrogen generule |
* Gel electrophoresis:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/09-08-24-2.jpg)

  | Lane | Colony PCRs from Brook’s restreak of transformation with new PhoA ligation |
  | --- | --- |
  | 1 | 1kb invitrogen generule |
  | 2 | 20 |
  | 3 | 21 |
  | 4 | 22 |
  | 5 | 23 |
  | 6 | 24 |
  | 7 | 25 |
  | 8 | 26 |
  | 9 | 27 |
  | 10 | Skipped due to bubbles |
  | 10 | 1kb invitrogen generule |
  | 11 | 28 |

### Extra Notes:

* After PCR cleanup, to ensure the correct volume for future elutions, in a separate tube, a further 9µl was eluted and left for 5 minutes prior to spinning and got a concentration of 22ng/µl therefore we should only elute in 11µl or it will lower our total DNA concentration.
* Scratch plates (of pet21a+ and Colonies 13, 16, and 17) were mistakenly left over the weekend at room temperature.

## W/C 12th August Lab Notebook

---

### 13th August 2024

### Experiments:

* O/N of PafA (10ml, containing 10µl Ampicillin) was mixed with 500ml LB broth (and 500µl Ampicillin) and placed in the shaker at 100rpm and 37°C. Was induced at 236 minutes with an absorbance of 0.518 (wavelength used on spectrophotometer was 600nm).
* 30.1, 30.2, 50.1, 50.2, and 70.1 gBlocks underwent PCR\* and subsequent PCR cleanup.
* A portion of each gBlock (10ng) diluted to 1:100 (100pg) and 1:1000 (10pg).
* Golden gate assembly for each gBlock (apart from 70.2) was then carried out, each gBlock at each different *insert:vector* ratio of 5:1, 3:1, 2:1, and 0:1 (only vector).

### Results:

* Gel electrophoresis of gBlocks (10ng) and their 1:100 dilutions:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/13-08-24.png)
  + Lane 1 - 1kb GeneRuler
  + Lane 2 - 30.1
  + Lane 3 - 30.1 (1:100)
  + Lane 4 - 30.2
  + Lane 5 - 30.2 (1:100)
  + Lane 6 - 50.1
  + Lane 7 - 50.1 (1:100)
  + Lane 8 - 50.2
  + Lane 9 - 50.2 (1:100)
  + Lane 10 - 70.1
  + Lane 11 - 70.1 (1:100)
* Gel electrophoresis of gBlock 1:100 and 1:1000 dilutions\*\*:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/13-08-24-2.png)
* Concentrations of original 10ng gBlocks post-cleanup:

  | 30.1 | 30.2 | 50.1 | 50.2 | 70.1 |
  | --- | --- | --- | --- | --- |
  | 170ng/µl | 26ng/µl | 42ng/µl | 140ng/µl | 158ng/µl |
* Concentrations of 1:100 and 1:1000 dilutions post-cleanup:

  | Initial gBlock concentration | 30.1 | 30.2 | 50.1 | 50.2 | 70.1 |
  | --- | --- | --- | --- | --- | --- |
  | 100pg (1:100 dilution) | 4 | 9 | 2 | 3 | 6 |
  | 10pg (1:1000 dilution) | 3 | 2 | 3 | 7 | 3 |

### Extra Notes:

* GBlocks of 30.1, 30.2, 50.1, 50.2, and 70.1 arrived (70.2 was still in transit and arrived the following day). Each had an initial concentration of 10ng/µl.
* \*Each gBlock used the following primers for this and all future PCRs:
  + 30.1 - iGEM\_16 and iGEM\_17
  + 30.2 - iGEM\_18 and iGEM\_19
  + 50.1 - iGEM\_20 and iGEM\_21
  + 50.2 - iGEM\_22 and iGEM\_23
  + 70.1 - iGEM\_24 and iGEM\_25
* \*\*No visible bands shown; 1:100 and 1:1000 dilutions were excessive dilutions.

## W/C 12th August Lab Notebook

---

### 14th August 2024

---

### Experiments:

* The induced PafA culture was centrifuged at 4500rpm for 15 minutes, supernatants were discarded and pellets were frozen (according to Protein Purification Protocol).
* The 1:1000 dilutions of gBlocks 30.2, 50.1, and 70.2 underwent PCR amplification, using their accompanying primer pairs - each primer was diluted by 10x. PCR cleanup was performed then each sample was nanodropped and stored at -20°C.
* Pet21a-PhoA colonies 20a-h and 21a-f underwent colony PCR.
* Each golden gate assembly (5:1, 3:1, 2:1, and 0:1) of gBlocks 30.1, 30.2, 50.1, 50.2, and 70.1 underwent transformation. Each was plated with both the normal concentration and a 10x dilution.
* pH PNPP assay carried out.
* Kinetics PNPP assay repeated.
* Two solutions of PafA (A and B) nanodropped against a blank solution of glycerol, HEPES, and sodium chloride.

### Results:

* Colony PCR for Pet21a-PhoA colonies 20a-h & 21a-f along with 1:1000 dilutions of 30.1, 50.1, and 70.1\*:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/14-08-24.png)
  ![table for gel](https://static.igem.wiki/teams/5172/notebook/14-08-24-2.jpg)
* Concentrations of 1:1000 dilutions of 30.2, 50.1 and 70.2 post-cleanup:
  + 30.2 - 76µg/ml
  + 50.1 - 37µg/ml
  + 70.2 - 132µg/ml
* PafA solutions A and B concentrations:
  + A - 2.18µg/ml
    ![molar calculation](https://static.igem.wiki/teams/5172/notebook/14-08-24-3.jpg)
  + B - 2.55µg/ml
    ![molar calculation](https://static.igem.wiki/teams/5172/notebook/14-08-24-4.jpg)

### Extra Notes:

* Fresh LB agar + Ampicillin plates were poured
* \*Colony PCR was mistakenly carried out with 100bp ladder (instead of usual 1kb ladder), but results are still distinguishable

## W/C 12th August Lab Notebook

---

### 15th August 2024

---

### Experiments:

* Colony PCR of the 3:1 golden gate assembly of 70.1 (as its transformation from the previous day was successful). Results run on gel.
* Pet21sfGFP was digested using NdeI (in rCutSmart buffer), then run on gel with normal concentration, 1:2 dilution, and 1:10 dilution.
* 70.2 golden gate assemblies 5:1, 3:1, 2:1, and 0:1 underwent transformation and were plated with both normal concentrations and 10x dilutions.
* Overnights for 70.1 were made from (after successful gel results) using colonies 13, 14, 16, 17, and 18.
* Overnights made for PhoA colonies 21a-f.

### Results:

* Colony PCR of 70.1 (3:1) results on gel:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/15-08-24.jpg)
  ![table for gel](https://static.igem.wiki/teams/5172/notebook/15-08-24-2.jpg)

## W/C 19th August Lab Notebook

---

### 20th August 2024

### Experiments:

* Golden gate assembly was carried out for 30.1, 30.2, 50.1, and 50.2 then subsequently transformed into DH5α.\*
* Colony PCRs 70.2 colonies 1-18 carried out (using Pet 98 & 99 primers). Colonies 8, 13, 14, 16, 17, and 18 were made into O/Ns.
* Miniprepped PhoA (from 16/08/2024) was transformed into BL21.

### Results:

* Colony PCRs for 70.2 colonies 1-18:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/20-08-24.png)

### Extra Notes:

* \*All golden gate assemblies were carried out in insert:vector ratio of 3:1, using BsaI-HFv2. Each assembly had roughly 50ng of gBlock added to the solution.

## W/C 19th August Lab Notebook

---

### 21st August 2024

### Experiments:

* 70.1 (colonies 8, 13, 14, 16, 17, and 18) and 70.2 (colonies 1, 2, 3, 4, 5, 7, 11, and 14) were miniprepped.
* Colony PCRs, scratch plates, and overnights were made from the transformations of 30.1 (10 colonies), 30.2 (10 colonies), 50.1 (3 colonies), and 50.2 (10 colonies).

### Results:

* 70.1 miniprep concentrations (ng/µl):
  + 8 - 7
  + 13 - 160
  + 14 - 47
  + 16 - 39
  + 17 - 44
  + 18 - 42
* 70.2 miniprep concentrations (ng/µl):
  + 1 - 44
  + 2 - 42
  + 3 - 94
  + 4 - 84
  + 5 - 35
  + 7 - 40
  + 11 - 28
  + 14 - 40
* Colony PCR results for 30.1, 30.2, 50.1 and 50.2:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/21-08-24.png)
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/21-08-24-2.png)

### Extra Notes:

* Due to poor gel electrophoresis results of colony PCRs for 30.1, 30.2, 50.1, and 50.2 they will be redone (including scratch plates and O/Ns). 50.1 will be redone using the scratch plate rather than the transformation plate (due to low number of viable colonies on the transformation plate).

## W/C 19th August Lab Notebook

---

### 22nd August 2024

### Experiments:

* Colony PCRs of 30.1, 30.2, 50.1, and 50.2 from the previous day were redone.
* Colony PCR was also redone using the O/Ns of 30.1, 30.2, 50.1, and 50.2 (from 21/08/2024).\*
* PhoA was stepped up into 500ml of LB with 500ul of Ampicillin. It was induced using 250µl of 1M IPTG at an OD of 0.416 and left shaking at 180rpm at 18°C overnight.
* 30.1 (colonies 3-6) and 50.2 (colonies 4-7) were miniprepped.

### Results:

* Colony PCR of 30.1 and 30.2 on gel:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/22-08-24.png)
  ![table contents of gel](https://static.igem.wiki/teams/5172/notebook/22-08-24-2.png)
* Colony PCR of 30.2, 50.1, and 50.2 on gel:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/22-08-24-3.png)
  ![table contents of gel](https://static.igem.wiki/teams/5172/notebook/22-08-24-4.png)
* 30.1 and 50.2 miniprep concentrations\*\*:

  | Colony | Concentration (ng/µl) |
  | --- | --- |
  | 30.1 Col 3 | 57 |
  | 30.1 Col 4 | 32 |
  | 30.1 Col 5 | 32 |
  | 30.1 Col 6 | 48 |
  | 50.2 Col 4 | 56 |
  | 50.2 Col 5 | 51 |
  | 50.2 Col 6 | 32 |
  | 50.2 Col 7 | 45 |

### Extra Notes:

* \*Same Master Mix as for usual PCR was used but 2µl of O/N was added (instead of a pipette tip dipped into a colony).
* \*\*Colonies 3, 4, and 6 from 30.1 and colonies 4, 5, and 7 from 50.2 were sent off for sequencing due to high concentrations and clear peaks at 260nm (the wavelength of DNA).

## W/C 19th August Lab Notebook

---

### 23rd August 2024

### Experiments:

* PNPP assay carried out.

## W/C 26th August Lab Notebook

---

### 26th August 2024

### Experiments:

* PafA WT, PhoA, 30.1, 50.2, 70.1, and 70.2 were stepped up and induced with IPTG.\* Cultures were spun down, supernatant discarded, and pellets frozen.

### Extra Notes:

* \*Using O/Ns made by an advisor on the previous day (Sunday).

## W/C 26th August Lab Notebook

---

### 27th August 2024

### Experiments:

* PCR carried out on 30.2 and 50.1.
* PafA and PhoA proteins were purified.

### Results:

* 30.2 and 50.1 PCR results on gel:
  ![gel scan](https://static.igem.wiki/teams/5172/notebook/27-08-24.png)

  Lanes:

  1. 1kb DNA ladder
  2. 30.2
  3. 50.1

## W/C 26th August Lab Notebook

---

### 28th August 2024

### Experiments:

* 30.1 and 50.2 protiens were purified.

## W/C 26th August Lab Notebook

---

### 29th August 2024

### Experiments:

* PNPP assay trialled for WT PafA and PhoA.
* 70.1 and 70.2 proteins were purified.

## W/C 2nd September Lab Notebook

---

### 2nd September 2024

### Experiments:

* First half of practice run of glass beads in phosphate-recovery device with Dylan Lewis carried out.

## W/C 2nd September Lab Notebook

---

### 3rd September 2024

### Experiments:

* Second half of practice run of glass beads in phosphate-recovery device with Dylan Lewis carried out.
* Size exclusion carried out on proteins 30.1, 50.2, and 70.2.

## W/C 2nd September Lab Notebook

---

### 4th September 2024

### Experiments:

* Circular dichroism carried out with Xander Arsecott-Barber for PhoA, 70.1, 70.2, and 50.2.
* Size exclusion carried out on proteins PafA WT, PhoA, and 70.1.

## W/C 2nd September Lab Notebook

---

### 5th September 2024

### Experiments:

* PNPP kinetics assay carried out with WT PafA and 50.2. \*

### Results:

* Mg/ml of pafA was converted to moles which was 0.00531.

### Extra Notes:

* \*PafA was diluted to 1.1nM.

## W/C 2nd September Lab Notebook

---

### 6th September 2024

### Experiments:

* Phosphate-recovery device tested using WT PafA.

## W/C 9th September Lab Notebook

---

### 9th September 2024

### Experiments:

* Kinetics assays for PafA WT and 70.2 were trialled.
* Temperature assays were trialled using PafA WT.

## W/C 9th September Lab Notebook

---

### 10th September 2024

### Experiments:

* Kinetics assays carried out for PafA WT, PhoA, 30.1, 50.2 and 70.2.
* Temperature assays carried out for PafA WT, 30.1, and 70.2.

## W/C 9th September Lab Notebook

---

### 11th September 2024

### Experiments:

* pH assays carried out for PafA WT, PhoA, 30.1, 50.2, and 70.2.
* Temperature assay was carried out for 50.2.

## W/C 9th September Lab Notebook

---

### 13th September 2024

### Experiments:

* Product inhibition assays carried out for PafA WT, PhoA, 30.1, 50.2, and 70.2.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_title %}

# Engineering

{% endblock %}
{% block page\_index %}

[Introduction](#introduction)
[Example 1](#example1)
[Example 2](#example2)

{% endblock %}
{% block page\_content %}

## Introduction

---

|  |  |
| --- | --- |
| ![engineering cycle](https://static.igem.wiki/teams/5172/engineering/engineering-cycle.jpg) | Synthetic biology is embodied by its iterative engineering approach. The Design, Build, Test and Learn cycle, a cornerstone of this methodology, enables rapid prototyping and optimisation of biological systems. In our project, we rigorously adhered to this framework, ensuring our decision-making processes were always informed. |

## Example 1- Nickel Column Protein Purification

---

![1st nickel cycle](https://static.igem.wiki/teams/5172/engineering/nick1.jpg)

---

|  |  |
| --- | --- |
| ![design](https://static.igem.wiki/teams/5172/engineering/design.jpg) | A critical objective of our project was to purify the phosphatase enzymes intended for testing. We initially designed a purification protocol incorporating sonication, ultracentrifugation and affinity chromatography using a nickel column to purify polyHis-tagged enzymes. |

![how nickel column process works](https://static.igem.wiki/teams/5172/engineering/logoless/eng1.png)

|  |  |
| --- | --- |
| ![build](https://static.igem.wiki/teams/5172/engineering/build.jpg) | To implement our purification strategy, we constructed a nickel column using a PD10 column containing Chelating Sepharose, which was clamped to utilise gravity for pulling the sample through the matrix. |
| ![test](https://static.igem.wiki/teams/5172/engineering/test.jpg) | We applied this gravity-based system to purify our first sample of PafA, using the designed protocol ([link](https://2024.igem.wiki/sheffield/protocols)). The system worked, allowing us to obtain purified enzyme samples for further experiments. |
| ![learn](https://static.igem.wiki/teams/5172/engineering/learn.jpg) | Through this initial test, we identified a key bottleneck; the gravity-driven process was not time-efficient, especially considering the multiple enzyme variants that needed to be purified. This realisation prompted us to reconsider our approach to increase throughput in future purification steps. |

![2nd nickel cycle](https://static.igem.wiki/teams/5172/engineering/nick2.jpg)

---

|  |  |
| --- | --- |
| ![design](https://static.igem.wiki/teams/5172/engineering/design.jpg) | To address the time inefficiencies, we researched alternative methods to accelerate the nickel-column process. One promising approach was to modify the nickel column so it could be spun in a centrifuge, significantly speeding up the flow-through process without compromising sample quality. |
| ![build](https://static.igem.wiki/teams/5172/engineering/build.jpg) | We implemented this idea by drilling a hole in the cap of a Falcon tube to accommodate the P10 column, enabling us to spin the nickel column in a centrifuge. |

![new design for a column](https://static.igem.wiki/teams/5172/engineering/logoless/unnamed.png)

|  |  |
| --- | --- |
| ![test](https://static.igem.wiki/teams/5172/engineering/test.jpg) | Before applying this new method to enzyme samples, we conducted a test spin using a binding buffer to optimise centrifugation parameters. This experiment aimed to find the optimum speed and duration for the centrifugation to ensure efficient flow through the bead matrix without disturbing the column bed. |
| ![learn](https://static.igem.wiki/teams/5172/engineering/learn.jpg) | Centrifugation significantly improved efficiency over the gravity-driven method, allowing us to process samples faster. However, we also learned that excessive speed or prolonged centrifugation could disrupt the Sepharose bead matrix, underlining the importance of carefully balancing these variables in future protein purification steps. |

## Example 2 - The Column Device

---

![1st column cycle](https://static.igem.wiki/teams/5172/engineering/col1.jpg)

---

|  |  |
| --- | --- |
| ![design](https://static.igem.wiki/teams/5172/engineering/design.jpg) | As an early proposed implementation, we sought to develop a column device containing immobilised PafA enzymes capable of hydrolyzing organophosphates. We designed experiments to test this system using p-nitrophenyl phosphate (PNPP) as a substrate, which would pass through a nickel column with His-tagged PafA enzymes immobilised on the matrix. |
| ![build](https://static.igem.wiki/teams/5172/engineering/build.jpg) | We constructed a nickel affinity column containing purified PafA enzymes. This setup allowed us to immobilise the PafA enzymes on the column, creating a stable system for testing their ability to hydrolyse PNPP while immobilised. |
| ![test](https://static.igem.wiki/teams/5172/engineering/test.jpg) | To assess the functionality of the immobilised enzymes, we passed a 40 mM PNPP solution through the column. Our goal was to determine whether the immobilised PafA enzymes could hydrolyze PNPP into p-nitrophenol (pNP) and inorganic phosphate (P). |
| ![learn](https://static.igem.wiki/teams/5172/engineering/learn.jpg) | Our test confirmed that the immobilised PafA enzymes were still functional, successfully catalysing the hydrolysis of PNPP. However, we encountered a challenge: separating the inorganic phosphate from the rest of the flow-through was difficult. This realisation prompted us to consider additional strategies for phosphate capture. |

![2nd col cycle](https://static.igem.wiki/teams/5172/engineering/col2.jpg)

---

|  |  |
| --- | --- |
| ![design](https://static.igem.wiki/teams/5172/engineering/design.jpg) | To address the issue of phosphate capture, we researched various phosphate-binding proteins (PBPs) that could be immobilised in conjunction with PafA on the same nickel column. These PBPs would selectively bind inorganic phosphate, allowing for its separation from the flow-through. |
| ![build](https://static.igem.wiki/teams/5172/engineering/build.jpg) | We synthesised a gBLOCK containing the selected PBP gene and inserted it into our pET21a backbone. Following our established purification workflow, we purified the PBP and immobilised it on the same nickel column as the PafA enzyme, utilising its polyHis-tag for binding. |
| ![test](https://static.igem.wiki/teams/5172/engineering/test.jpg) | We repeated the experiment with the new setup, passing the 40 mM PNPP solution through the column to test whether the immobilised PBP could successfully capture inorganic phosphate. This was confirmed by the lower absorbance at 405 nm in the flow-through compared to the column containing only PafA, indicating that the PBP was effectively binding inorganic phosphate. ([link to column section](https://2024.igem.wiki/sheffield/column)) |
| ![learn](https://static.igem.wiki/teams/5172/engineering/learn.jpg) | Our test demonstrated that the addition of PBP successfully enhanced phosphate capture, confirming the functionality of our dual-enzyme immobilisation system. This breakthrough marked a significant step toward optimising our phosphate recovery strategy, but further optimisation of stability and scalability will be required for practical applications. |

![3rd col cycle](https://static.igem.wiki/teams/5172/engineering/col3.jpg)

---

|  |  |
| --- | --- |
| ![design](https://static.igem.wiki/teams/5172/engineering/design.jpg) | As we progressed, concerns arose about the stability of PafA under the harsher conditions typical of wastewater treatment, which differed from the controlled lab environment with PNPP solution. To address this, we leveraged AI tools like LigandMPNN and AlphaFold 3 to redesign PafA, aiming to enhance its thermostability and pH tolerance for real-world applications. |
| ![build](https://static.igem.wiki/teams/5172/engineering/build.jpg) | We used these AI tools to generate six redesigned versions of PafA. We then ordered the redesigned genes and cloned them into the pET21a backbone. Following our standard recombinant protein production workflow, we purified these new PafA variants for testing. |
| ![test](https://static.igem.wiki/teams/5172/engineering/test.jpg) | To evaluate the improved stability of the redesigned enzymes, we performed circular dichroism analysis. This test confirmed that the redesigned enzymes were significantly more thermostable than the wild-type PafA. While the wild-type enzyme aggregated at 70°C, the redesigned variants remained stable and did not aggregate or unfold even at 95°C, demonstrating their enhanced robustness. |
| ![learn](https://static.igem.wiki/teams/5172/engineering/learn.jpg) | This iteration successfully improved the thermostability of PafA, generating more durable enzyme variants. However, due to time constraints, we were unable to test the immobilisation of these new variants onto the column. Future work will focus on integrating these variants into the column setup, which is crucial for practical implementation. |

![4th col cycle](https://static.igem.wiki/teams/5172/engineering/col4.jpg)

---

|  |  |
| --- | --- |
| ![design](https://static.igem.wiki/teams/5172/engineering/design.jpg) | We sought external feedback on our column design and enzyme immobilisation strategy by consulting with Dr. Eleni Routoula from the University of Sheffield. She provided invaluable advice, recommending that we replace Sepharose beads with glass beads for enzyme immobilisation. Glass beads are more durable, especially in wastewater conditions, and reduce the risk of enzyme unfolding, which can occur with Sepharose. |
| ![build](https://static.igem.wiki/teams/5172/engineering/build.jpg) | To explore this new approach, we collaborated with Dylan Lewis, a PhD student, who allowed us to observe the process of enzyme immobilisation onto glass beads. This process involved the use of Piranha solution, which we could not handle ourselves due to safety restrictions. Unfortunately, we were unable to implement this method within our project timeframe, but it presents a promising avenue for future development. |

{% endblock %}