iGEM 2019 — Team Nottingham

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# Team:Nottingham/Notebook

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    People
    + [Meet the Team](https://2019.igem.org/Team%3ANottingham/Team)
    + [Collaborations](https://2019.igem.org/Team%3ANottingham/Collaborations)
    + [Attributions](https://2019.igem.org/Team%3ANottingham/Attributions)
    + [Sponsors](https://2019.igem.org/Team%3ANottingham/Sponsorship)
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    + [Description](https://2019.igem.org/Team%3ANottingham/Description)
    + [Design](https://2019.igem.org/Team%3ANottingham/Design)
    + [Experiments](https://2019.igem.org/Team%3ANottingham/Experiments)
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    + [Results](https://2019.igem.org/Team%3ANottingham/Results)
    + [Demonstrate](https://2019.igem.org/Team%3ANottingham/Demonstrate)
    + [Hardware](https://2019.igem.org/Team%3ANottingham/Hardware)
    + [Model](https://2019.igem.org/Team%3ANottingham/Model)
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    Parts
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    + [Contribution](https://2019.igem.org/Team%3ANottingham/Contribution)
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      Human Practices
      + [Human Practices](https://2019.igem.org/Team%3ANottingham/Human_Practices)
      + [Outreach](https://2019.igem.org/Team%3ANottingham/Public_Engagement)
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      Awards](https://2019.igem.org/Team%3ANottingham/Medals)
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      Judging Form](https://igem.org/2019_Judging_Form?team=Nottingham)

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# People

---

[Meet the Team](https://2019.igem.org/Team%3ANottingham/Team)
[Collaborations](https://2019.igem.org/Team%3ANottingham/Collaborations)
[Attributions](https://2019.igem.org/Team%3ANottingham/Attributions)
[Sponsors](https://2019.igem.org/Team%3ANottingham/Sponsorship)

![](https://static.igem.org/mediawiki/2019/2/2b/T--Nottingham--project.png)

# Project

---

[Description](https://2019.igem.org/Team%3ANottingham/Description)
[Design](https://2019.igem.org/Team%3ANottingham/Design)
[Experiments](https://2019.igem.org/Team%3ANottingham/Experiments)
[Notebook](https://2019.igem.org/Team%3ANottingham/Notebook)
[Results](https://2019.igem.org/Team%3ANottingham/Results)
[Demonstrate](https://2019.igem.org/Team%3ANottingham/Demonstrate)
[Hardware](https://2019.igem.org/Team%3ANottingham/Hardware)
[Model](https://2019.igem.org/Team%3ANottingham/Model)

![](https://static.igem.org/mediawiki/2019/b/be/T--Nottingham--parts.png)

# Parts

---

[Parts Overview](https://2019.igem.org/Team%3ANottingham/Parts)
[Basic Part Prize](https://2019.igem.org/Team%3ANottingham/Basic_Part)
[Contribution](https://2019.igem.org/Team%3ANottingham/Contribution)

![](https://static.igem.org/mediawiki/2019/1/17/T--Nottingham--human.png)

# Human Practices

---

[Human Practices](https://2019.igem.org/Team%3ANottingham/Human_Practices)
[Outreach](https://2019.igem.org/Team%3ANottingham/Public_Engagement)

# Project

## Notebook

### Take a look at what we've been up to.

### **WARNING:** We've been busy!

![](https://static.igem.org/mediawiki/2019/a/a5/T--Nottingham--notebook.png)

# Subgroups

**Subgroup 1** consisted of Alice, Jacob, Millie, Saniya and Yaseen. Their work was primarily wet-lab based.
Millie and Saniya were responsible for integrating *botR* expressing modules in to the *C. sporogenes* genome. Jacob, Alice and Yaseen were responisble for assembling the reporter expressing plasmids and conjugating these plasmids in to *C. sporogenes*.

**You can view Alice, Jacob and Yaseen's full lab book [here](https://static.igem.org/mediawiki/2019/7/73/T--Nottingham--Acetone_Vector_Results.pdf)**.

**You can view Millie and Saniya's full lab book [here](https://static.igem.org/mediawiki/2019/f/f8/T--Nottingham--botR-Lab-Book.pdf)**.

**Subgroup 2** consisted of Fiona, Marta and Sami. Between them, they had a mix of wet-lab and dry-lab work. Outside the lab, they built an **electronic nose** and gained **sponsorship** for the team. Inside the lab, they performed many growth curves to assist subgroup 1 and the modellers in subgroup 3.

**You can view Fiona and Sami's full lab book [here](https://static.igem.org/mediawiki/2019/a/aa/T--Nottingham--Acetone-Detection-Lab-Folder.pdf)**.

**Subgroup 3** consisted of Dan and James. They worked in the dry lab, **modelling** *C. sporogenes* and building the **wiki**. Predictions based on their models helped to direct the research of subgroups 1 and 2.

Subgroup 1

Subgroup 2

Subgroup 3

[![](https://static.igem.org/mediawiki/2019/7/79/T--Nottingham--pdf.png)
**Click to view the full lab book.**](https://static.igem.org/mediawiki/2019/7/73/T--Nottingham--Acetone_Vector_Results.pdf)

# Week **1**

## 10th - 14th June

**Alice, Jacob and Yaseen**

10/06/2019

* Designed primers using SnapGene. These will be used for the HiFi assembly of APOs (Aceto and Bot), GusA and control constructs.

13/06/2019

* Made chemically-competent DH5-alpha (*E. coli*), which are needed to to clone our HiFi-assembled constructs.

14/06/2019

* Transformed competent DH5-alpha with PUC19 control DNA, as a positive control to test the competencies of cells.

**Millie and Saniya**

* Design of primers
* Amplified the fragments needed to create our 3 constructs: RBS (RBS-botR-Tfdx), P1 (P1-botR-Tfdx) & pLac (pLac:-botR-Tfdx). The terminator and guide RNA were amplified using primer dimers.
* Extract plasmid vector pRECas1-MCS-p15a (for our construct insert to be ligated into.)

# Week **2**

## 17th - 21th June

**Alice, Jacob and Yaseen**

18/06/2019

* Inoculated the plasmid PMTL8215x, into which we will assemble the acetone production operon and GusA constructs.

19/06/2019

* PMTL8215x plasmid extraction (to be digested).
* Overnight digestion of pMTL8215x by restriction enzymes. This will allow for HiFi assembly of our constructs via the cohesive ends.

20/06/2019

* Dephosphorylation of the digestion reaction, thus preventing unwanted religation of the plasmid backbone.
* Gradient PCR – to to amplify our gene fragments.
* Primer PCR and Colony PCR – The primer PCR is to amplify the separate parts.

21/06/2019

* To see whether the PCR worked, we perfomed agarose gel electrophoresis. Correct bands were purified from this gel.
* HiFi assembly of the three final constructs (Aceto, Bot and GusA).

**Millie and Saniya**

* Linear ligation of fragments to create the 3 constructs.
* PCR used to amplify the constructs.
* Agarose gel electrophoresis indicated RBS and P1 were the correct sizes. pLac construct didn’t have any bands (no sample amplified).
* pLac troubleshooting:
  + Check if the problem is due to the PCR failing. By changing the polymerase we were able to identify bands. However, the bands were the wrong size indicating ligation was not successful.
  + We amplified and ligated fragments LHA-pLac and botR-Tfdx-RHA together to then ligate straight into the plasmid vector.

# Week **3**

## 24th - 28th June

**Alice, Jacob and Yaseen**

24/06/2019

* Transformation of constructs into chemcially competent DH5alpha (cloning strain)
* Transformations plated on LB+Cm plates

25/06/2019 & 26/06/2019

* Colony PCR of transformed DH5alpha – to see if the cells have correct constructs.
* Digestion of pMTL82151 – to assemble positive and negative controls for each construct.

27/06/2019

* Dephosphorylation of pMTL82151 digestion.
* Gradient PCR - amplify gene fragments for control constructs.

28/06/2019

* Gel electrophoresis of Gradient PCR and pMTL82151 digestion - purify successful bands.
* HiFi assembly of control constricts for Aceto, Bot and GusA.
* Transformation of control constructs into DH5alpha, plated on LB+Cm plates

**Millie and Saniya**

* Digestion of ligated inserts and pRECas1-MCS-p15a plasmid vector.
* Ligation of inserts (RBS,P1 and pLac) into separate plasmid vectors.
* Transform our plasmid constructs into E.coli TOP10 cloning strain.
* Plate cells and then screen single colonies.
* Colonies with correctly sized plasmids sent for sequencing.

# Week **4**

## 1st - 5th July

**Alice, Jacob and Yaseen**

03/07/2019

* Colony PCR of Transformed DH5alpha.
* Inoculation of correct colonies overnight in Lb+Cm media.

04/07/2019

* Miniprep (plasmid extraction) and culture collection of overnight inoculations.
* Minipreps of previous inoculations of:
  + ViG02
  + ViG03
  + ViG06
  + ViG07

**Millie and Saniya**

* Review of sequencing data:
  + Culture collection of correct RBS and pLac sequences.
  + P1 incorrect – terminator not included in any of the sequencing data.
    - Change of primer to one which binded to the terminator, allowing us to identify from the gel when the terminator had been looped out.
    - Ligated the terminator straight into the plasmid vector - bypassing the PCR step which previously resulted in the terminator looping out.
    - Re-sequence - good!

# Week **5**

## 8th - 12th July

**Alice, Jacob and Yaseen**

09/07/2019

* Minipreps of previous inoculations of:
  + ViG02
  + ViG03
  + ViG06
  + ViG07
* Colony PCR of the successful C. bot control plate (Bot-).
* Inoculations of next lot of vectors:
  + ViG05 – GusA positive control construct
  + ViG08 – GusA negative control construct
  + ViG09 – aceto negative control construct
  + ViG011 – Cbot experimental construct
* Nanodrop of second lot of miniprep.

10/07/2019

* Performed a gel with yesterday's colony PCR.
* Minipreps of second lot of inoculations:
  + ViG05
  + ViG08
  + ViG09
  + ViG011

11/07/2019

* Inoculations of C.bot (-) from the colony aliquot used in colony PCR and GusA+ (VIG04, VIG05) in LB+Cm overnight.

12/07/2019

* Send these assembled vectors for sequencing:
  + ViG02 - Aceto (+)
  + ViG03 - GusA experimental
  + ViG05 - GusA (+)
  + ViG08 - GusA (-)
  + ViG09 - Aceto (-)
  + ViG011 - Cbot experimental

**Millie and Saniya**

* Extract plasmids (with our construct inserts) from TOP10 (cloning strain).
* Transform correct constructs into CA434 (E.coli donor strain).
* Conjugations between CA434 and wild type C. sporogenes.

# Week **6**

## 15th - 19th July

**Alice, Jacob and Yaseen**

15/07/2019

* PCR of primers 25 and 26 – we ran out of this specific part, so had to re-do the PCR so we had enough to remake the aceto and Bot constructs.
* Gel purification
* Inoculation of ViG01 (high and low copy plasmids), bot (+), GusA (+).
* Digestion of the plasmid pMTL8215 – so we have enough for HiFi assembly.

16/07/2019

* Dephosphorylation of the digestion.
* Miniprep of yesterday’s inoculations and make glycerol stocks from these – so we have a stock of these vectors to use in the future and a purified frozen stock to send to sequencing and transform with.
* Gels from the repeated primer PCR of 15/07/2019.
* Primer PCR again – failed so re-done with different template DNA and enzyme (allTaq this time around), Gels came back and showed failure, except for faint line in digested vector, so this was cut out and will be purified tomorrow.
* Primer PCR for a third time, used ViG09 as a template as the sequencing results showed that this sequence has no mutations and should work.
* Inoculation of Aceto (+), GusA (normal) and ViG01 (pMTL82151).

17/07/2019

* Gel from third primer PCR.
* Purification of successful gel from third primer and of vector from digestion.
* HiFi assembly of Aceto and Bot (+) constructs.
* Digestion of low-copy plasmid – to assemble GusA in to this.
* Miniprep of Aceto (+), GusA (normal) and ViG01 (pMTL82151) inoculations.

18/07/2019

* Dephosphorylation low copy plasmid.
* Gel and purification of digestion.
* HiFi of GusA using the low copy digested plasmid.
* Inoculation of Cbot (-) colonies (col 5 and col 6).

19/07/2019

* Miniprep and glycerol stock of yesterday inoculations of Cbot (-), will become ViG17 (68ng/ul) - Colony 5 didn’t grow, colony 6 did and was miniprepped and had a glycerol stock made of it.
* Transfrom HiFi vectors:
  + Aceto
  + C. bot (+)
  + GusA - low copy plasmid
  + GusA (+) - low copy plasmid
  + GusA (-) - low copy plasmid
* Send vectors to sequencing:
  + ViG07
  + ViG13
  + ViG16
  + ViG17

**Millie and Saniya**

* Pick colonies and re-streak onto plates with theophylline the CRISPR cas9 inducer.
* Colony screening with PCR, to check for mutants.
* No mutants :(
* Transformed plasmid into a different donor strain:
  + TOPSEX and S17.1 (transformed by electroporation as before)
  + Plate onto LB/Cm (chloramphenicol plates)
  + TOPSEX showed greater transformation efficiency, S17.1 transformation efficiency much lower so conjugation was carried forward with TOPSEX.

# Week **7**

## 22nd - 26th July

**Alice, Jacob and Yaseen**

22/07/2019

* Colony PCR of transformations.
* Diagnostic gel of transformations.
* Gradient PCR of primers to amplify parts for acetone constructs – this wasn’t analysed as the polymerase had been left out too long.
* PCR of FAST primers – to amplify the FAST gene using overlap extension with 4 primers.
* Gradient PCR – amplify parts for C. bot vectors in two (amplify thl, ctfab, adc and the vector backbone in one and just assemble the promoters in HiFi) this makes less parts from the HiFi so less that can go wrong.

23/07/2019

* PCR using Fast primers to incorporate an overlap in the FAST gene construct.
* Diagnostic gel of FAST PCR.
* Gradient PCR of promoterless vectors (OiG5121) and OiG1920 primer part– so that we can just assemble the promoters via HiFi.
* Digestion of ViG01.

24/07/2019

* Dephosphorylation of ViG01.
* Gel purification of P3 (OiG1920).
* HiFi assembly of FAST vectors with no promoter and with the P3 promoter.
* Gradient FAST PCR of FASTfdx – different annealing temperatures.
* Transformation of HiFi assembled vectors – FASTnop, FASTP3, Aceto, Bot (+) and Bot (+) back up.

25/07/2019

* Colony PCR of transformations.
* Gel purification of FASTfdx.
* HiFi of FAST fdx.
* Inoculation of all colony PCR reactions (only successful ones will be glycerol-stocked, miniprepped and sequenced).

26/07/2019

* Gel of colony PCR.
* Miniprep of successful colonies.
* Send stuff to sequencing:
  + ViG18 – col4 - FASTnoP
  + ViG18 – col6- FASTnoP
  + ViG19 – Bot (+)
  + ViG20 – Aceto (normal)
* Transformation of FASTfdx, FASTp3, Aceto (normal), Bot (+).

**Millie and Saniya**

* Conjugations repeated with TOPSEX strain and C.sporogenes.

# Week **8**

## 29th July - 2nd August

**Alice, Jacob and Yaseen**

29/07/2019

* Conducted a HiFi of FAST-P3 and FAST-Pfdx at 3 different temperatures. Then a gel of the reactions, cutting out the brightest bands - for the highest concentrations of DNA.
* Redid the colony PCR of Bot (+) to check which colonies were correct for overnight innoculation.
* Overnight innoculation of the low copy versions of GusA, GusA + and GusA -.

30/07/2019

* Dephosphorylated the digested VIG01, then heat shocked.
* Purified the FAST-P3 and FAST-Pfdx constructs from yesterday's gel.
* Miniprep of the overnight inoculations (GusA, Gus-, Gus+ and Bot+), then sent the results for sequencing.
* HiFi of the two FAST constructs into the newly digested vector, then transform them into *E.coli*.

31/07/2019

* Checked the FAST transformation plates - no growth.
* Did overnight inoculations of ViG17 and ViG14.
* Started the PCR amplification of APO (thl-Ctfa-Ctfb-adc) operon and OiG35 and OiG36 together.

01/08/2019

* Ran the gels of the amplified APO and Oi3536 – The PCR had failed.
* Prepared to repeat the APO and Oi3536 PCR - but at different temperatures.
* MiniPrep of ViG17 and ViG14.
* Digested ViG14 (low copy vector).

02/08/2019

* Dephosphorylation of ViG14 then heat shock.
* Ran a gel of ViG07, ViG11 and ViG14, then purified ViG07 and ViG11 - no ViG14 band.
* ligation of ViG07 and ViG11.
* Transformation of FAST-P3 and FAST -Pfdx.

**Millie and Saniya**

* Restreaked colonies onto theophylline plate then screened the colonies that grew.
* Screened many colonies to locate correct constructs for RBS,P1 and pLac.

# Week **9**

## 5th - 9th August

**Alice, Jacob and Yaseen**

05/08/2019

* Checked FAST-P3 and FAST-Pfdx transformations - no growth.
* Heat killed ViG07 and ViG11 ligations (10 mins at 65°C).
* Transformed the ligation of ViG07 and ViG11 - to make Bot+.

06/08/2019

* FAST overlap PCR using FAST-noP as a template.
* Check transformations of Bot (+).
* innoculate ViG14.

07/08/2019

* MiniPrep ViG14.
* Colony PCR of BotR (+).
* Diagnostic gel overlap - FAST overlap, colony PCR.
* Digest ViG14.

08/08/2019

* Dephosphorylation of ViG14.
* Purification gel - FAST overlap PCR and ViG14.
* Re-do Colony PCR of Bot (+).
* Innoculate Colony 2 from Bot (+) colony aliquiots.

09/08/2019

* MiniPrep & glycerol stock of Bot (+).
* Purification of Fast-Pfdx and Fast-P3 overlap PCRs and purification of cut vector.
* Linearization PCR of Vector backbones for FAST constructs - used GusA + and GusA as sequencing validated they had the necessary promoter regions.
* Transformation of assembled vectors into TOPSEX (donor strain) - Electroporation.

**Millie and Saniya**

* RSingle colonies selected and restreaked to encourage plasmid loss as the bacteria replicates on antibiotic deficient agar (plasmid contains antibiotic resistance genes).
* Sequencing of mutant RBS and P1 construct showed the insert had been successfully introduced into the C.sporogenes genome.
  + These colonies were given to the acetone vector sub group to insert the reporter plasmid vector.
* Plasmid loss not successful for pLac as the wt C. Sporogenes had taken over.

# Week **10**

## 12th - 16th August

**Alice, Jacob and Yaseen**

12/08/2019

* Culture collection of TOPSEX transformations (yellow caps):
  + ViG09 – Aceto (-)
  + ViG20 – Aceto (normal)
  + ViG02 – Aceto (+)
  + ViG17 – Cbot (-)
  + ViG11 – Cbot (normal)
  + ViG23 – GusA LC (-)
  + ViG21 – GusA LC (normal)
  + ViG22 – GusA LC (+)
  + ViG18 – FASTnoP
* PCR clean-up of LINViG21+22 and DPN1 digestion of LINViG21+22.
* Gel of LINViG21 and LINViG22.
* Overnight inoculations of transformed TOPSEX.

13/08/2019

* Conjugations of Overnight inoculations:
  + ViG09 – Aceto (-)
  + ViG20 – Aceto (normal)
  + ViG02 – Aceto (+)
  + ViG23 – GusA LC (-)
  + ViG21 – GusA LC (normal)
  + ViG22 – GusA LC (+)
* Repeat linearization PCR ViG21+22.
* PCR clean-up of ViG21 and ViG22 and DpnI digestion.

14/08/2019

* Gel purification of LINViG21 and 22 - repeated due to very low DNA concentrations.
* Repeat of Linearisation PCR of LINViG21 and 22.
* HiFi assembly of FAST-fdx and FAST-P3:
  + FAST-fdx-overhang, Pfdx promoter part, digested low-copy vector.
  + FAST-P3-overhang, P3 promoter part, digested low-copy vector.

15/08/2019

* Transformed FAST-fdx and FAST-P3 - 3-part HiFi contains FAST gene with overhangs, promoter part and cut vector).
* Repeated Linearisation PCR of ViG21 and ViG22, PCR purified and Digested with DpnI.
* Gel purified ViG21 - after the DpnI dugestion.
* Re-streaked conjugations.

16/08/2019

* Linearization PCR of ViG22.
* Colony PCR of Re-streaks.
* HiFi FAST-P3 - 2-part HiFi containing the linearised ViG21 vector and the FAST-P3-overhang.

**Millie and Saniya**

* Redid pLac conjugations (of TOPSEX into C. sporogenes) in the hopes of finding a correct construct.

# Week **11**

## 19th - 23rd August

19/08/2019

* PCR clean-up of Transconjugant colony PCR screen, this is so we can send the purified DNA for sequencing to see if our APO vectors have the correct sequence still.
* Send vectors in transconjugants to sequenceing.
* PCR clean-up and digestion of LIN22.
* Colony PCR screen of PCR FAST-fdx and FAST-P3 Transformation - gel to analyse.

20/08/2019

* Purification of LIN22 to be used in 2-part HiFi of FAST-fdx
* HiFi FAST-fdx

21/08/2019

* Transformation of FAST-P3 and FAST-fdx (Two-part HiFi).
* Re-streaking transconjugants using results from colony PCR.

22/08/2019

* Colony PCR of: Aceto(+), GusA(+), GusA(P3) and GusA(-) - from DH5alpha host strains
* colony PCR of: P1Aceto (P3), WT\_GusA(+), RBS\_GusA(P3) and RBS\_GusA(-) from *C. sporogenes* transconjugants - to check the size and sequence of the inserts and see where any mutations arose.
* Subsequent Gel of colony PCR.

23/08/2019

* Screening of FAST-fdx & FAST-P3 colonies.
* Culture collections of transconjugants from re-streaks.

# Week **12**

## 26th - 30th August

28/08/2019

* MiniPrep of FAST-fdx and FAST-P3.
* Sequencing of FAST constructs.
* TOPSEX transformations FAST-fdx and FAST-P3 - both colony 1.
* Cbot (-) and Cbot conjugationed into RBS host strain.
* P1 and WT. Aceto (P3) conjugation in to P1 Host strain.

29/08/2019

* Make culture collection of FAST\_fdx and FAST\_P3 in TOPSEX from transformation plates.
* Streak FAST\_fdx and FAST\_P3 onto LB+Cm plate - For a DH5alpha culture collection.
* Repeat PCR of DH5alpha, TOPSEX and **C. sporogenes**.
* ViG18 digestion - to purify the FAST\_NoP fragment and insert it into a low copy vector for consistency.
* Ligation of FAST\_NoP insert and Low-copy vector.
* Digestion of FAST\_fdx low copy vector - using SalI and NdeI to cut out the fdx promoter.
* Plate conjugations onto selective plates.

30/08/2019

* Conjugation of P3 constructs (Bot\_P3, Ace\_P3 and GusA\_P3) in to PLac.
* conjugation of Aceto\_fdx and GusA\_fdx into WT **C. sporogenes**.
* Sequencing TOPSEX and **C. sporogenes** GusA constructs.
* Transform FAST\_NoP ligations.

# Week **13**

## 2nd - 6th September

02/09/2019

* Screening of the Bot P3 (in RBS, P1, WT), Bot(-) (in WT) and Aceto P3 (in P1).
* Re-streak PLac (Bot\_P3, Ace\_P3 and GusA\_P3) and WT *sporogenes* (Ace\_fdx and GusA\_fdx).
* Screening of the FAST\_NoP ligations.
* Sequencing results from GusA constructs - all correct.

03/09/2019

* Screening PLac + WT conjugations.
* MiniPrep FAST\_fdx - plasmid extraction.
* Sequencing of FAST\_NoP.
* Transform TOPSEX with Aceto (+) (ViG07) - previous transformation of Aceto(+) with ViG02 had a mixture of bands, so when it was conjugated in to *C. sporogenes*, the organism was preferentially choosing the smaller mutated band.
* Re-streak Plac + WT conugations.
* Digestions of FAST\_fdx - using SalI and NdeI to cut the fdx promoter out of the fdx low copy vector.

04/09/2019

* Assays using the Bot APO constructs.
* FAST\_NoP and FAST\_fdx digestion (FAST\_NoP didn’t have the low-copy replicon) - to cut out the low copy replicon from FAST\_Fdx and insert it in to FAST\_NoP.

05/09/2019

* 24hr timepoint for bot assays.
* Sequencing of FAST and PLac constructs.

06/09/2019

* Conjugations of WT\_Ace\_fdx and PLac\_GusA\_P3.
* 48 hr timepoint for Bot Assays.

# Week **14**

## 9th - 13th September

09/09/2019

* Screen fdx characterisation parts and FAST\_NoP ligation (low-copy) – inoculation of successful colonies
* Re-streaks from Plac\_gusA\_P3 and WT\_aceto\_fdx conjugations.

10/09/2019

* Miniprep of FAST\_NoP (low-copy) ligation and fdx characterisation parts.
* Sequencing of FAST\_NoP and fdx.
* Overnight innoculations of Aceto assays.

11/09/2019

* 0hr, 4hr and 8hr timepoint for aceto assays.
* Transform fdx characterisation parts in E. coli (CA434).

12/09/2019

* Culture collections of CA434 transformations.
* 24hr timepoint for aceto assays.

13/09/2019

* 48hr timepoint for aceto assays.
* Conjugations of fdx characterisation vectors into *C. sporogenes.*

# Week **15**

## 16th - 20th September

16/09/2019

* PCR of all assembled constructs in *C. sporogenes* and run a gel
* Set up overnights for OD measurements of fdx characterisation vectors in *E. coli.*

17/09/2019

* Screen conjugations of fdx characterisation vectors.
* Set up overnights for OD measurements of fdx characterisation vectors in *C. sporogenes*.

18/09/2019

* Measurement of OD of fdx characterisation in *C. sporogenes*.

19/09/2019

* FAST assays
* PCR (of fdx characterisation vectors), PCR purification and sent to sequencing to identfy and differentiate the constructs.

# Week **1**

## 10th - 14th June

**Electronic Nose**

* Began to create a basic concept of an electronic nose and realised that we require assistance from an engineer. Then sent a request for help to the University of Nottingham Electronics and Electrical Engineering (UoN EEE) department. Dr Christian Klumpner, from the UoN EEE, agreed to assist us with building the device.

# Week **2**

## 17th - 21th June

**Electronic Nose**

* After contacting Rapid Electronics, they gave us a list of basic components we may need to build the nose: Arduino Uno board, Figaro TGS 822 Organic Solvent Vapors sensor, Orangepip USB cable, 30V Power supply.
* Rapid then connected us with the UoN EEE department technician: Edward Kujawinski, who agreed to help us with putting the electronic components together.

# Week **3**

## 24th - 28th June

**Electronic Nose**

* Contacted Pro Pak foods who are willing to give us non-monetary donations. Gave us basic information on the precautionary measures they take against *C. botulinum*.
* Recieved the components from Rapid, to begin making the electronic nose.
* We met with Dr Klumpner to further discuss the design of the electronic nose. We also learnt how to use an Arduino and electronic circuit design: multimeters, software programming and using a breadboard.

**Wet Lab**

* Ran a Gas Chromatography (GC) to validate the production of acetate by *Clostridium sporogenes*. If **C. sporogenes** cannot produce acetate, then the mutant won't produce acetone. Acetate is the precursor to acetone
* Measured the OD of *C. sporogenes* at different time points. The data was used to produce a growth curve. We can use this in future to compare the growth of the modified **C. sporogenes** to the wild type.
* Counted the number of colonies of *C. sporogenes* to determine the total number of viable cells (The colonies proved to be uncountable because the samples were not diluted enough).
* Met with Dr Klumpner - learnt basic programming on Arduino and how to read a multi-meter and the basics of electronic circuit design.

# Week **4**

## 1st - 5th July

**Electronic Nose**

* Pro-Pak Foods has agreed to donate food samples to the team, so we can test our GM **C. sporogenes** on them.
* Furthermore, they are also inviting us to tour their manufacturing facility in Malton, Yorkshire.
  + This will enable us to understand how food manufacturing processes work and determine how our project can benefit the food industry.
  + It also provides us with an opportunity to interview someone with experience in food manufactured
* Worked alongside our mathematician, Daniel, to create an equation which turns the sensor’s voltage reading into PPM of acetone, essential for the Arduino code for the electronic nose. The equation was produced using the datasheet provided by Rapid Electronics:

  *outputValue = ((double)(142.51)/sensorValue)*

# Week **5**

## 8th - 12th July

**Electronic Nose**

* Met with Dr Klumpner and the modelling team to implement the Voltage-PPM equation into the Arduino code.
* Learnt the basics of electronic hardware design:
  + Reading resistance on resistors
  + distinguish between the positive and the negative end of the LED and which way to connect it in the circuit.
* Found out more about the chosen gas sensor and considered adding a humidity and temperature sensor to improve the accuracy of the device, as both have a great impact on the acetone detection sensitivity.
* Found out more about the chosen gas sensor and decided to add a humidity-temperature sensor to increase the precision of the device.

# Week **6**

# 15th - 19th July

**Electronic Nose**

* Visited Pro Pak foods in Malton. Went on the factory tour and interviewed David Raine, the technical director.
  + Told us about the industry standards for preventing **C. botulinum**
  + Showed us retort machines - makes use of steam and pressure to kill **C. botulinum** bacteria and spores
  + Mentioned that the meat industry might be worth contacting as the are under government pressure to reduce nitrites in their products - nitrites have anti-botulinum effects.
* Learnt how to code more complicated programmes and circuits using the Arduino board through literature and educational videos. Implemented the knowledge gained through coding and building a real life circuit consisting of an LED turned on and off at the press of a button.

# Week **7**

## 22nd - 26th July

**Electronic Nose**

* Left the acetone sensor to heat up for 7 days before testing for its detection ability (a required step in order to calibrate the sensor - one time only requirement)
* Built and wrote code for a prototype circuit for the electronic nose, consisting of a sensor (Figaro TGS 822) and an LED that lights up if the concentration of acetone detected exceeds 50ppm.
  The prototype circuit was created to test the obtained knowledge of coding and circuit design gained in the previous week

**Wet Lab**

* Prof. Peck, a consultant microbiologist from the Quadram Institute visited the University of Nottingham and gave a seminar titled “*Clostridium botulinum* and foodborne botulism”. We saw this as the perfect opportunity to get expert-specific feedback.
* He agreed to speak to us after the seminar, so we presented our project and invited him to a Q&A session.
  + He advised us that our device may be better with a syringe
  + Prof. Peck also told us about a botulinum carrot juice outbreak in North America. Suggested that we use carrot juice as a real food media application to test our GM surrogate in.
* Repeated the growth curve. We also obtained HPLC data to validate the GC data from our first curve. HPLC was also to confirm whether acetone present in the samples was background or not.
  + Also measured the change in pH during the growth curve to determine whether it matches the data gathered from previous pH experiments.
  + The results we got from this were somewhat unexpected - we anticipated the pH to decrease due to the production of acetate, but on the contrary it increased.

![basic circuit design](https://static.igem.org/mediawiki/2019/d/df/T--Nottingham--basic_circuit_design.png)

# Week **8**

## 29th July - 2nd August

**Electronic Nose**

* Performed an acetone detection test on a nail polish remover, to ensure that the sensor stabilized and works correctly after the required 7 days heat up. The sensor could detect the acetone, but not determine the concentration - thus the arduino code was edited.
* Held a Skype call with the Oxford iGEM team to discuss a potential electronic engineering collaboration.
* Planned on using an additional ethanol-detecting sensor, due to the risk of ethanol concentrations in the headspace of food samples potentially affecting the readings of the Figaro TGS 822 for acetone.
* Planned the design for the exterior design of the electronic nose - decided to include a syringe to create a closed space around the gas sensor to obtain more accurate readings from samples being tested.
* Investigated ways of incorporating the LCD screen from both the software and hardware side.

**Wet Lab**

* As per request of the modellers, the growth curve of WT *C. sporogenes* was repeated. The optical density and pH was measured every hour. The plan was to do do gas chromatography, but the machine was too busy, so a high performance liquid chromatography (HPLC) was performed in proxy.
* Prof. Liz Sockett FRS invited us to have coffee with her, She provided us with expertise and things we could do to improve our project. Like Conducting an acetone kill curve - to determine the lethal concentration of acetone for **C. sporogenes*.*

# Week **9**

## 5th - 9th August

**Electronic Nose**

* Modified the prototype code by changing the equation, which turned voltage into PPM, into an equation which also takes resistance into account, resulting in more precise readings. A piece of code was written for the LCD, so that it shows the detected concentration of acetone. Finally, coded and connected the circuit to on/off buttons.
* Researched methods of powering the device so that it is portable and safe. Contacted Dr Klumpner for further opinion and guidance due to the realisation that the battery we have ordered might be too powerful for the Arduino board and, as a result, may destroy it.
* Performed acetone concentration detection tests on the Figaro TGS 822 acetone sensor. The acetone concentrations used ranged from 0 PPM to 150 PPM. The consequent readings fluctuated around the corresponding concentrations that were being tested. However, this was an unscientific test as factors including partial dispersal of acetone into the surrounding air, and varying distances between the acetone samples and the sensor affected the results.
* Decided not to use the additional ethanol sensor, based on the results obtained by the modellers indicating that the ethanol present in the headspace of food samples will not be in concentrations high enough to alter the acetone concentration readings.

**Wet Lab**

* Made Carrot Juice Dilutions (100%, 50%, 30% & 10%),in preparation for a carrot curve with WT **C. sporogenes**
* Ran the HPLC for the repeated growth curve, conducted last week.

# Week **9**

## 12th - 16th August

**Electronic Nose**

* Ordered an LCD and a new battery from Rapid Electronics. The battery change was decided after a consultation session with Dr Klumpner, who has advised on using a lower voltage battery.
* Contacted Sheffield iGEM team for potential help with the auto-CAD design of the electronic nose.
* Completed the first draft of the final nose design.
* Investigated methods of powering the Arduino board with the new battery (connections and needed components).
* Researched the basics of Auto-CAD.

# Week **10**

## 19th - 23rd August

**Wet Lab**

* Conducted a growth curve in varying medias, measuring OD, pH and HPLC. We used TYG adjusted to a pH of 5.5, then TYG with 30, 60 and 100mM of Glucose. We did this because for the adc enzyme to convert acetate to acetone, it needs an environment of pH 5.5. In our previous growth curve, the pH increased instead of decreaseing - against predictions. It was predicted that if extra glucose was added to the growth media, the pH would decrease to pH 5.5.

# Week **11**

## 26th - 30th August

**Wet Lab**

* Conducted a Kill curve to find out what concentration of acetone WT **C. sporogenes** is able to survive and grow in. *Sporogenes* was grown in TYG media with varying concentrations of acetone - 0mM, 25mM, 50mM, 75mM and 100mM. The genetically similar *Clostridium acetobutilicum* is able to grow in these concentrations of acetone. To be a success, our mutant would need to be able to survive and grow in conditions with increasing acetone levels - as it will be producing acetone. Although no significant results were found, it could be concluded that WT *sporogenes* could survive 100mM of acetone.

# Week **12**

## 2nd - 6th September

**Wet Lab**

* Repeated the Kill curve at higher concentrations of acetone - 0mM, 100mM, 200mM, 300mM and 400mM. This was to continue to find out if WT *sporogenes* could survive high levels of acetone.
* Conducted a growth curve in different dilutions of carrot juice - 10%, 30%, 50% and 100%, measuring CFU and HPLC. CFU was used rather than OD because the carrot juice makes the solution too thick to get a proper reading, HPLC was used to detect whether *sporogenes* still produced acetate. The aim of growing *sporogenes* in carrot juice was to determine if it grew as we wanted in "normal" food.

# Week **13**

## 9th - 13th September

* Sent designs for the electic nose casing off for 3D printing.
* Sent the plan for the automated syringe to the Sheffield team (collaboration).

# Week **14**

## 16th - 20th September

* Preparation of Carrot juice, Aceto and botR construct samples to enter Gas Chromatography.
* Modifications to the component placement within the electric nose and subsequent modifications to the interior casing design.

# Week **1**

## 10th - 14th June

**Structural Model**

* Practised using MATLAB
* Learnt about the basics of the COBRA toolbox

**Wiki**

* Worked the landing page

# Week **2**

## 17th - 21th June

**Structural Model**

* BLASTed genes in *C. acetobutylicum* against *C. sporogenes* and *C. botulinum*
* Parsed and analysed BLASTing results

**Wiki**

* Continued to work the landing page animation
* Began development of the team page

# Week **3**

## 24th - 28th June

**Structural Model**

* Compiled reactions and metabolites of *C. sporogenes* into a model
* Added acetone production genes from *C. acetobutylicum* to model* Ran Flux Balance Analysis on the model to confirm that steady-state solution existed for production of acetone

**Wiki**

* Developed project description and inspiration page

# Week **4**

## 1st - 5th July

**Structural Model**

* Ran Elementary Modes Analysis on the model to investigate possible flux distributions across the metabolic network
* Parsed and normalised results of EMA, and examined useful modes

**Wiki**

* Comleted development of landing page

# Week **5**

## 8th - 12th July

**Structural Model**

* Verified model by checking carbon and hydrogen balancing
* Added bifurcating *bcd* gene to model, and examined changes in product spectrum.
* Found a paper with larger model of *C. sporogenes*
* Will use this model going forwards
* Applied similar processes as with small model: EMA, FBA, bifurcating version

# Week **6**

# 15th - 19th July

**Structural Model**

* Added correct reactions/names to new model

**Wiki**

* Created project Notebook page - added content from each subgroups

**Outreach**

* Designed eco-friendly/lab-safety poster and leaflet

# Week **7**

## 22nd - 26th July

**Structural Model**

* Added acetone genes to new model

**Wiki**

* Added dictionary functionality to project pages
* Worked on notebook page

**Outreach**

* Edited press release

**Collaboration**

* Participated in Skype call with Oxford engineer

# Week **8**

## 29th July - 2nd August

**Structural Model**

* Performed Acetate scan on model
* Calculated ATP yields of elementary modes.

**Wiki**

* Added alternative navigation for smaller devices
* Worked on notebook page.

**Outreach**

* Digitised prototype board game

**Collaboration**

* Participated in Skype call with Carroll High School engineer
* Wrote jQuery guide for Carroll High School wiki guide collaboration

# Week **9**

## 5th - 9th August

**Structural Model**

* Performed biomass scan on model

**Wiki**

* Added biggest sponsors to home page
* Started working on experiments and outreach page

# Week **10**

## 12th - 16th August

**Wiki**

* Planned and started development on outreach page

# Week **11**

## 19th - 23rd August

**Structural Model**

* Wrote first draft of structural modelling report
* Balanced lumped reactions in model
* Associated reactions with genes in model

**Wiki**

* Planned human practises page

# Week **12**

## 26th - 30th August

**Structural Model**

* Wrote second draft of structural modelling report

**Wiki**

* Worked on human practices page

# Week **13**

## 2nd - 6th September

**Structural Model**

* Worked on structural section of modelling writeup

**Wiki**

* Worked on human practises page and experiments page
* Stanardised wiki colors with Millie, to match presentation and poster

# Week **14**

## 9th - 13th September

**Structural Model**

* Worked on structural section of modelling writeup
* Made further refinements to model
* Ran further analysis: robustness analysis and production envelope

**Wiki**

* Created modelling diagrams for wiki

# Week **15**

## 16th - 20th September

**Wiki**

* Finished the developing the human-practices page

# Week **16**

## 23rd - 27th September

**Wiki**

* Finished the developing the outreach page

# Week **17**

## 30th September - 4th October

**Modelling**

* Edited LaTeX modelling report

**Wiki**

* Added content several pages:
  + Safety page
  + Hardware page
  + Modelling page
  + Medals page
  + Collaboration page

**Outreach**

* Edited the press report for 'The Biologist'

# Week **18**

## 7th October - 11th October

**Modelling**

* Added diagrams to LaTeX modelling report

**Wiki**

* Worked on design page on wiki
* Worked on modelling page on wiki

**Presentation**

* Designed modelling slides in presentation
* Wrote modelling script for presentation

# Week **19**

## 13th October - 18th October

**Wiki**

* Finalised pages for wiki freeze

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iGEM Nottingham *2019*


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# Team:Nottingham/Experiments

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    People
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    Parts
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      Human Practices
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    * [![](https://static.igem.org/mediawiki/2019/7/7f/T--Nottingham--awards.png)

      Awards](https://2019.igem.org/Team%3ANottingham/Medals)
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      Judging Form](https://igem.org/2019_Judging_Form?team=Nottingham)

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# People

---

[Meet the Team](https://2019.igem.org/Team%3ANottingham/Team)
[Collaborations](https://2019.igem.org/Team%3ANottingham/Collaborations)
[Attributions](https://2019.igem.org/Team%3ANottingham/Attributions)
[Sponsors](https://2019.igem.org/Team%3ANottingham/Sponsorship)

![](https://static.igem.org/mediawiki/2019/2/2b/T--Nottingham--project.png)

# Project

---

[Description](https://2019.igem.org/Team%3ANottingham/Description)
[Design](https://2019.igem.org/Team%3ANottingham/Design)
[Experiments](https://2019.igem.org/Team%3ANottingham/Experiments)
[Notebook](https://2019.igem.org/Team%3ANottingham/Notebook)
[Results](https://2019.igem.org/Team%3ANottingham/Results)
[Demonstrate](https://2019.igem.org/Team%3ANottingham/Demonstrate)
[Hardware](https://2019.igem.org/Team%3ANottingham/Hardware)
[Model](https://2019.igem.org/Team%3ANottingham/Model)

![](https://static.igem.org/mediawiki/2019/b/be/T--Nottingham--parts.png)

# Parts

---

[Parts Overview](https://2019.igem.org/Team%3ANottingham/Parts)
[Basic Part Prize](https://2019.igem.org/Team%3ANottingham/Basic_Part)
[Contribution](https://2019.igem.org/Team%3ANottingham/Contribution)

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# Human Practices

---

[Human Practices](https://2019.igem.org/Team%3ANottingham/Human_Practices)
[Outreach](https://2019.igem.org/Team%3ANottingham/Public_Engagement)

# Experiments

## & Protocols

### We have made our experiments reproducible, standardised and accessible to align with iGEM's core ethos.

![](https://static.igem.org/mediawiki/2019/f/fc/T--Nottingham--experiment.png)

*Double-Click* on a card for further details.

Selecting an experiment will cause the corresponding protocols to be shown.

View all Protocols

# Experiments

# Protocols

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iGEM Nottingham *2019*

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# Team:Nottingham/Results

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    People
    + [Meet the Team](https://2019.igem.org/Team%3ANottingham/Team)
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    + [Sponsors](https://2019.igem.org/Team%3ANottingham/Sponsorship)
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    Project
    + [Description](https://2019.igem.org/Team%3ANottingham/Description)
    + [Design](https://2019.igem.org/Team%3ANottingham/Design)
    + [Experiments](https://2019.igem.org/Team%3ANottingham/Experiments)
    + [Notebook](https://2019.igem.org/Team%3ANottingham/Notebook)
    + [Results](https://2019.igem.org/Team%3ANottingham/Results)
    + [Demonstrate](https://2019.igem.org/Team%3ANottingham/Demonstrate)
    + [Hardware](https://2019.igem.org/Team%3ANottingham/Hardware)
    + [Model](https://2019.igem.org/Team%3ANottingham/Model)
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    Parts
    + [Parts Overview](https://2019.igem.org/Team%3ANottingham/Parts)
    + [Basic Part Prize](https://2019.igem.org/Team%3ANottingham/Basic_Part)
    + [Contribution](https://2019.igem.org/Team%3ANottingham/Contribution)
  * [![](https://static.igem.org/mediawiki/2019/1/1c/T--Nottingham--Safety-Icon.png)

    Safety](https://2019.igem.org/Team%3ANottingham/Safety)* ![](https://static.igem.org/mediawiki/2019/1/17/T--Nottingham--human.png)

      Human Practices
      + [Human Practices](https://2019.igem.org/Team%3ANottingham/Human_Practices)
      + [Outreach](https://2019.igem.org/Team%3ANottingham/Public_Engagement)
    * [![](https://static.igem.org/mediawiki/2019/7/7f/T--Nottingham--awards.png)

      Awards](https://2019.igem.org/Team%3ANottingham/Medals)
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      Judging Form](https://igem.org/2019_Judging_Form?team=Nottingham)

![](https://static.igem.org/mediawiki/2019/9/90/T--Nottingham--back-arrow.png)

[![](https://static.igem.org/mediawiki/2019/6/6f/T--Nottingham--home.png)](https://2019.igem.org/Team%3ANottingham)
![](https://static.igem.org/mediawiki/2019/9/9d/T--Nottingham--team.png)
![](https://static.igem.org/mediawiki/2019/2/2b/T--Nottingham--project.png)
![](https://static.igem.org/mediawiki/2019/b/be/T--Nottingham--parts.png)
[![](https://static.igem.org/mediawiki/2019/1/1c/T--Nottingham--Safety-Icon.png)](https://2019.igem.org/Team%3ANottingham/Safety)
![](https://static.igem.org/mediawiki/2019/1/17/T--Nottingham--human.png)
[![](https://static.igem.org/mediawiki/2019/7/7f/T--Nottingham--awards.png)](https://2019.igem.org/Team%3ANottingham/Medals)
[![](https://static.igem.org/mediawiki/2019/9/9d/T--Nottingham--judging.png)](https://igem.org/2019_Judging_Form?team=Nottingham)

![](https://static.igem.org/mediawiki/2019/a/a4/T--Nottingham--cancel.png)

![](https://static.igem.org/mediawiki/2019/9/9d/T--Nottingham--team.png)

# People

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[Meet the Team](https://2019.igem.org/Team%3ANottingham/Team)
[Collaborations](https://2019.igem.org/Team%3ANottingham/Collaborations)
[Attributions](https://2019.igem.org/Team%3ANottingham/Attributions)
[Sponsors](https://2019.igem.org/Team%3ANottingham/Sponsorship)

![](https://static.igem.org/mediawiki/2019/2/2b/T--Nottingham--project.png)

# Project

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[Description](https://2019.igem.org/Team%3ANottingham/Description)
[Design](https://2019.igem.org/Team%3ANottingham/Design)
[Experiments](https://2019.igem.org/Team%3ANottingham/Experiments)
[Notebook](https://2019.igem.org/Team%3ANottingham/Notebook)
[Results](https://2019.igem.org/Team%3ANottingham/Results)
[Demonstrate](https://2019.igem.org/Team%3ANottingham/Demonstrate)
[Hardware](https://2019.igem.org/Team%3ANottingham/Hardware)
[Model](https://2019.igem.org/Team%3ANottingham/Model)

![](https://static.igem.org/mediawiki/2019/b/be/T--Nottingham--parts.png)

# Parts

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[Parts Overview](https://2019.igem.org/Team%3ANottingham/Parts)
[Basic Part Prize](https://2019.igem.org/Team%3ANottingham/Basic_Part)
[Contribution](https://2019.igem.org/Team%3ANottingham/Contribution)

![](https://static.igem.org/mediawiki/2019/1/17/T--Nottingham--human.png)

# Human Practices

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[Human Practices](https://2019.igem.org/Team%3ANottingham/Human_Practices)
[Outreach](https://2019.igem.org/Team%3ANottingham/Public_Engagement)