iGEM 2019 — Team St_Andrews

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# Team:St Andrews/Notebook

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  + [Team](/Team%3ASt_Andrews/Team)
  + [Collaborations](/Team%3ASt_Andrews/Collaborations)
  + [Attributions](/Team%3ASt_Andrews/Attributions)
* Project ▾
  + [Description](/Team%3ASt_Andrews/Description)
  + [Design](/Team%3ASt_Andrews/Design)
  + [Experiments](/Team%3ASt_Andrews/Experiments)
  + [Notebook](/Team%3ASt_Andrews/Notebook)
  + [Model](/Team%3ASt_Andrews/Model)
  + [Results](/Team%3ASt_Andrews/Results)
  + [Demonstrate](/Team%3ASt_Andrews/Demonstrate)
* Parts ▾
  + [Parts Overview](/Team%3ASt_Andrews/Parts)
  + [Improve](/Team%3ASt_Andrews/Improve)
* Human Practices ▾
  + [Human Practices](/Team%3ASt_Andrews/Human_Practices)
  + [Public Engagement](/Team%3ASt_Andrews/Public_Engagement)
* [Safety](/Team%3ASt_Andrews/Safety)
* [Judging Form](https://igem.org/2019_Judging_Form?team=St_Andrews)

Notebook

## January

Preliminary meetings were to think of potential projects and then narrow them down by considering feasibility and topics of research being done in the university. By the end of January we had narrowed the choices down to three projects: monoclonal antibodies, nerve agents, and the killer peptide. At the same time, we were also applying for funding from the BBSRC.

[January 3rd 2019](https://static.igem.org/mediawiki/2019/c/c5/T--St_Andrews--Meeting--3-1-19.pdf)

[January 7th 2019](https://static.igem.org/mediawiki/2019/3/3c/T--St_Andrews--Meeting--19-1-7.pdf)

[January 28th 2019](https://static.igem.org/mediawiki/2019/7/7b/T--St_Andrews--Meeting--19-1-28.pdf)

## February

This was quite a major month as it was when we chose the project we would go forward with, monoclonal antibodies. After choosing this we met Uli, a structural biologist at the university. He suggested we look into improving antibodies by trying to introduce a bond called an isopeptide bond into them that would hopefully make them more stable. To this end we started looking at the rational design aspect of our project to decide which proteins were most promising to be able to add an isopeptide bond to. We also started looking for lab space and supervisors to watch over us in the summer.

[February 13th 2019](https://static.igem.org/mediawiki/2019/b/ba/T--St_Andrews--Meeting--19-2-13.pdf)

[February 18th 2019](https://static.igem.org/mediawiki/2019/f/fb/T--St_Andrews--Meeting--19-2-18.pdf)

[February 18th (2) 2019](https://static.igem.org/mediawiki/2019/0/0c/T--St_Andrews--Meeting--19-2-18-2.pdf)

[February 19th 2019](https://static.igem.org/mediawiki/2019/6/64/T--St_Andrews--Meeting--19-2-19.pdf)

[February 25th 2019](https://static.igem.org/mediawiki/2019/3/37/T--St_Andrews--Meeting--19-2-25.pdf)

## March

In March we continued planning the project while looking for further funding and lab supervisors. We also planned an iGEM team retreat near Strathpeffer to get to know each other better and create a funding video. March was when we had our half term, so we had a two week break from iGEM.

[March 4th 2019](https://static.igem.org/mediawiki/2019/6/62/T--St_Andrews--Meeting030419.PDF)

[March 11th 2019](https://static.igem.org/mediawiki/2019/3/38/T--St_Andrews--Meeting110319.PDF)

## April

April began with our iGEM retreat which everyone really enjoyed. The rest of the month was spent looking into more specific tasks for the project. We divided the Wet Lab team into subgroups to allow different parts of the project to be worked on at the same time. We also focussed more on human practices by looking into making an impact analysis and researching companies and experts we could reach out to.

[April 1st 2019](https://static.igem.org/mediawiki/2019/3/35/T--St_Andrews--Meeting010419.PDF)

[April 8th 2019](https://static.igem.org/mediawiki/2019/d/df/T--St_Andrews--Meeting080419.PDF)

[April 15th 2019](https://static.igem.org/mediawiki/2019/8/85/T--St_Andrews--Meeting150419.PDF)

[April 22nd 2019](https://static.igem.org/mediawiki/2019/8/88/T--St_Andrews--Meeting220419.PDF)

## May

As everyone had exams in May, iGEM was put on hold until the official start of our project on 3rd June. However, the rational design was completed so that the Wet Lab students could begin experiments immediately.

## June

June marked the official beginning of our project and so progress from the different aspects of the project will be split into three parts: Wet Lab, Human Practices, and Modelling

### Wet Lab

After designing and ordering the first set of primers, the wet lab team started preliminary lab work by using cell lines provided by Marta to create competent cell aliquots to be used for the rest of the summer. The plasmids that would be the vector for the majority of our constructs (pHisTEV and Bal3 were used for cloning and protein expression respectively) was grown in E. coli and purified for use in future transformations. Once primers arrived, we attempted to clone the desired sequences into the pHisTEV plasmids.

### Modelling

The modelling team very quickly split into two sub-teams to focus on different modelling goals for the project. The first team (AI) focussed on learning how to fold proteins using various AI programs and machine learning software available. If they could fold their own proteins, then this would greatly help to see if a slightly edited protein had the potential to form an isopeptide bond. The first protein folding software they looked at was Rosetta, which led to PyRosetta as it allowed more selective folding. They learned how to use the Robetta server to create protein fragments for Pyrosetta and got access to the Edinburgh cluster which allowed them to run thousands of cycles of the protein folding script to see the best outputs much quicker than it would have taken. However, PyRosetta was not able to correctly fold proteins with isopeptide bonds, so the AI team found another AI by Dr M. AlQuraishi which involved training an AI on a list of a large amount of proteins. The team hoped that this would allow the AI to fold proteins correctly that had isopeptide bonds. The second team (Isopeptide Hunters) explored ways to detect isopeptide bonds already present in proteins, the idea being that if they improved it enough, they could use it to find isopeptide bonds in proteins that may have been missed in the pdb database. They started writing a python script that would give them a list of potential proteins with isopeptide bonds, but the initial results were of thousands of potential proteins, so they started looking into what actually made an isopeptide bond.

### Human Practices

Human practices made the impact analysis for our projects. This involved reaching out to antibody companies and experts, including Antibody Analytics from whom they were able to have a very insightful call with.

[June 3rd (1) 2019](https://static.igem.org/mediawiki/2019/2/21/T--St_Andrews--Meeting030619%281%29.PDF)

[June 3rd (2) 2019](https://static.igem.org/mediawiki/2019/6/64/T--St_Andrews--Meeting030619%282%29.PDF)

[June 3rd (3) 2019](https://static.igem.org/mediawiki/2019/e/e1/T--St_Andrews--Meeting030619%283%29.PDF)

[June 3rd (4) 2019](https://static.igem.org/mediawiki/2019/f/f7/T--St_Andrews--Meeting030619%284%29.PDF)

[June 3rd (5) 2019](https://static.igem.org/mediawiki/2019/1/1d/T--St_Andrews--Meeting030619%285%29.PDF)

[June 3rd (6) 2019](https://static.igem.org/mediawiki/2019/3/3d/T--St_Andrews--Meeting030619%286%29.PDF)

[June 3rd (7) 2019](https://static.igem.org/mediawiki/2019/8/8e/T--St_Andrews--Meeting030619%287%29.PDF)

[June 4th 2019](https://static.igem.org/mediawiki/2019/2/27/T--St_Andrews--Meeting040619.PDF)

[June 5th (1) 2019](https://static.igem.org/mediawiki/2019/0/03/T--St_Andrews--Meeting050619%281%29.PDF)

[June 5th (2) 2019](https://static.igem.org/mediawiki/2019/b/bf/T--St_Andrews--Meeting050619%282%29.PDF)

[June 6th (1) 2019](https://static.igem.org/mediawiki/2019/0/00/T--St_Andrews--Meeting060619%281%29.PDF)

[June 6th (2) 2019](https://static.igem.org/mediawiki/2019/f/f2/T--St_Andrews--Meeting060619%282%29.PDF)

[June 7th (1) 2019](https://static.igem.org/mediawiki/2019/a/aa/T--St_Andrews--Meeting070619%281%29.PDF)

[June 7th (2) 2019](https://static.igem.org/mediawiki/2019/1/13/T--St_Andrews--Meeting070619%282%29.PDF)

[June 10th (1) 2019](https://static.igem.org/mediawiki/2019/2/28/T--St_Andrews--Meeting100619%281%29.PDF)

[June 10th (2) 2019](https://static.igem.org/mediawiki/2019/f/fd/T--St_Andrews--Meeting100619%282%29.PDF)

[June 11th 2019](https://static.igem.org/mediawiki/2019/0/0d/T--St_Andrews--Meeting110619.PDF)

[June 13th (1) 2019](https://static.igem.org/mediawiki/2019/b/b4/T--St_Andrews--Meeting130619%281%29.PDF)

[June 13th (2) 2019](https://static.igem.org/mediawiki/2019/6/62/T--St_Andrews--Meeting130619%282%29.PDF)

[June 14th (1) 2019](https://static.igem.org/mediawiki/2019/a/a2/T--St_Andrews--Meeting140619.PDF)

[June 14th (2) 2019](https://static.igem.org/mediawiki/2019/9/96/T--St_Andrews--Meeting140619%282%29.PDF)

[June 17th 2019](https://static.igem.org/mediawiki/2019/7/7d/T--St_Andrews--Meeting170619.PDF)

[June 20th (1) 2019](https://static.igem.org/mediawiki/2019/8/87/T--St_Andrews--Meeting200619%281%29.PDF)

[June 20th (2) 2019](https://static.igem.org/mediawiki/2019/5/58/T--St_Andrews--Meeting200619%282%29.PDF)

[June 24th 2019](https://static.igem.org/mediawiki/2019/b/b0/T--St_Andrews--Meeting240619.PDF)

## July

### Wet Lab

Once sequences were successfully cloned into the cloning plasmid, constructs were transformed into the Bal3 cells to attempt expression. To screen for expression, we used a Nickel pull-down to bind and separate the desired protein. After several tests it was determined that the Bal3 may not be expressing as desired and so a new cell line was acquired. Simultaneously started small-scale expression tests of mutant CH3 domains in SoluBL-21 and Origami cells, and achieved successful expression of AB2, AB7, and the commercial antibody. Cloning of mOrange from the distribution kit for the part improvement medal criteria (yeast-optimised mOrange with SpyTag) was performed in parallel with the main project.

### Modelling

The isopeptide hunters spent the first half of July refining their python script to find isopeptide bonds from PDB files. They were continually looking for more parameters they could use to refine their list of potential proteins, such as distances between specific atoms, and the atom locations within the protein. One of the parameters they wanted to use was the fofc electron density maps. They found an expert online called Prof. Robert Hanson who was luckily nearby at the time. The isopeptide hunters were able to meet up with Prof. Hanson and learned how to use Jmol to quantify the electron density data for specific regions of a protein. They then started to incorporate this into their refining process in order to further narrow down the list. Meanwhile the AI team got access to a computer in one of the Computer Science labs, which had the needed software and after some corresponding with Dr AlQuraishi, as well as making a script to converter .tertiary files to .pdb, so the AI team went back to the original AI and managed to get folded proteins.

### Human Practices

Human Practices organized a discussion forum, inviting both community members and academics. They decided that this was a great way to discover first, the worries and interests of the general public regarding the project, and second, a way to educate members of the public on what the research entailed. The forum began with presentations by some of the academics, teaching those in attendance both about the specific iGEM project and about biology more generally. The public was engaged and asked many questions. Through the experience, the whole iGEM team took away multiple lessons on how best to communicate their project in the future, and how they could emphasize certain elements to further invigorate the curiosity of interested individuals.

[July 1st 2019](https://static.igem.org/mediawiki/2019/f/fe/T--St_Andrews--Meeting010719.PDF)

[July 2nd 2019](https://static.igem.org/mediawiki/2019/5/56/T--St_Andrews--Meeting020719.PDF)

[July 3rd (1) 2019](https://static.igem.org/mediawiki/2019/3/3b/T--St_Andrews--Meeting030719%281%29.PDF)

[July 3rd (2) 2019](https://static.igem.org/mediawiki/2019/3/35/T--St_Andrews--Meeting030719%282%29.PDF%20)

[July 3rd (2-2) 2019](https://static.igem.org/mediawiki/2019/c/c3/T--St_Andrews--Meeting030719%282-2%29.PDF)

[July 3rd (3) 2019](https://static.igem.org/mediawiki/2019/e/ef/T--St_Andrews--Meeting030719%283%29.PDF)

[July 8th 2019](https://static.igem.org/mediawiki/2019/b/b7/T--St_Andrews--Meeting080719.PDF)

[July 10th 2019](https://static.igem.org/mediawiki/2019/3/3b/T--St_Andrews--Meeting100719.PDF)

[July 11th 2019](https://static.igem.org/mediawiki/2019/6/6e/T--St_Andrews--Meeting110719.PDF)

[July 12th 2019](https://static.igem.org/mediawiki/2019/a/ae/T--St_Andrews--Meeting120719.PDF)

[July 15th 2019](https://static.igem.org/mediawiki/2019/9/9a/T--St_Andrews--Meeting150719.PDF)

[July 19th 2019](https://static.igem.org/mediawiki/2019/8/82/T--St_Andrews--Meeting190719.PDF)

[July 22nd (1) 2019](https://static.igem.org/mediawiki/2019/f/fb/T--St_Andrews--Meeting220719%281%29.PDF)

[July 22nd (2) 2019](https://static.igem.org/mediawiki/2019/0/00/T--St_Andrews--Meeting220719%282%29.PDF)

[July 23rd 2019](https://static.igem.org/mediawiki/2019/1/16/T--St_Andrews--Meeting230719.PDF)

[July 24th 2019](https://static.igem.org/mediawiki/2019/c/cd/T--St_Andrews--Meeting240719.PDF)

[July 25th (1) 2019](https://static.igem.org/mediawiki/2019/c/c0/T--St_Andrews--Meeting250719%281%29.PDF)

[July 25th (2) 2019](https://static.igem.org/mediawiki/2019/9/99/T--St_Andrews--Meeting250719%282%29.PDF)

[July 29th 2019](https://static.igem.org/mediawiki/2019/a/ab/T--St_Andrews--Meeting290719.PDF)

## August

### Wet Lab

In August expression trials of various antibody mutations continued, and expression of fragments were confirmed using mass spectrometry, however none of the mutants had the desired isopeptide bond as was suggested by prior SDS analysis of cell lysate. We also ran mOrange+SpyTag expression trials in BL21 cells, however it was shown that while the construct was being expressed, it was also being cleaved, resulting in the protein being about half the expected mass upon SDS analysis. To correct this the construct was incubated with OIPD (a SpyCatcher analogue), however results may have been hidden by the presence of an OIPD dimer.

### Modelling

The Isopeptide Hunters ran their newly finished code over all of the proteins in the databank. The utilization of JMol to extract information about the fo-fc maps allowed for complete automation of the process. The team found multiple candidates, both for possible pre-existing isopeptide bonds, and for possible proteins to easily insert isopeptide bonds into. They plan to continue this research after iGEM ends.

### Human Practices

The Human Practices team ran an outreach session at Edinburgh Zoo in collaboration with Edinburgh UG and OG teams. During the session, members of the public participated in the Microbiome of Scotland project, collecting soil samples, learning about synthetic biology and conducting some experiments.

[August 2nd 2019](https://static.igem.org/mediawiki/2019/9/93/T--St_Andrews--Meeting020819.PDF)

[August 5th 2019](https://static.igem.org/mediawiki/2019/1/1e/T--St_Andrews--Meeting050819.PDF)

## September

In September, our classes started back up for the year. The Human Practices team attended a Fife teachers training day to showcase the Microbiome of Scotland outreach project in the hopes of encouraging teachers to take part in the project by delivering the sessions themselves. The Modelling team spent the majority of their time creating the wiki in preparation for the wiki freeze. The Wet Lab team compiled the information on the work they had done over the summer and began to edit it into a wiki-ready form.

[September 15th 2019](https://static.igem.org/mediawiki/2019/9/9d/T--St_Andrews--Meeting150919.PDF)

## October

The entire team worked together tirelessly to ensure the wiki was complete and all information accounted for. We then finished the poster and the other last-minute preparations, and headed to Boston for the Jamboree!

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School of Biology

School of Chemistry

School of Mathematics

School of CS

School of Physics

School of Philosophy

Sir Kenneth Murray Endowment Fund

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iGEM St Andrews 2019


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# Team:St Andrews/Experiments

Template

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* Project ▾
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  + [Results](/Team%3ASt_Andrews/Results)
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* Parts ▾
  + [Parts Overview](/Team%3ASt_Andrews/Parts)
  + [Improve](/Team%3ASt_Andrews/Improve)
* Human Practices ▾
  + [Human Practices](/Team%3ASt_Andrews/Human_Practices)
  + [Public Engagement](/Team%3ASt_Andrews/Public_Engagement)
* [Safety](/Team%3ASt_Andrews/Safety)
* [Judging Form](https://igem.org/2019_Judging_Form?team=St_Andrews)

Experiments

* [Cell culturing](#miraprep)
* [PCR, agarose gels and DNA purifcation](#digest)
* [Plasmid preparation](#gel)
* [Protein purification](#ligation)
* [Protein analysis](#transformation)

### Cell culturing

## **1. Making chemically competent cells**

**Materials**

* Cells
* 0.1 M CaCl2
* 10% Glycerol
* LB media
* 250 mL conical flasks
* 50 mL Falcon tubes
* Ice bucket
* Pipette / Pipette gun

**Protocol**

1. Prepare 0.1 M CaCl2, filter-sterilize or autoclave and pre-chill to = or <4deg; you will need ~30 ml of that per 50ml cells so prepare 100-200 ml
2. In a sterile tube (e.g. a Falcon or a white-capped tube) mix the above solution with sterile glycerol to obtain final glycerol concentration of 10% (e.g. 8ml CaCl2 and 2ml sterile 50% glycerol), keep that chilled as well. This is the solution for storing competent cells.
3. In the afternoon/evening inoculate a small (5-10ml) LB media vial with cells from a colony or a glycerol stock (don’t thaw the glycerol stock, just scrape some frozen cells off the surface) and incubate overnight in the shaking incubator at 37deg C overnight
4. Next day use the culture to inoculate 50-100 ml LB in a 250ml conical flask (~1 ml per 50ml media) and keep the flask in the shaking incubator at 37 deg
5. After about an hour start measuring the optical density at 600nm, if it is less than 0.2 check again in half an hour, if it is getting closer to 0.3 then check every 15-20 min after that (they are entering exponential phase so will accelerate!)
6. When the optical density is 0.5-0.8 transfer the cells into a fresh 50ml Falcon tube, or several tubes if you have more than 50ml, and spin for 10 min at >3000g (if they go above 1 they are entering stationary phase, either abandon ship or dilute them in a fresh flask with fresh media 10x and then keep checking OD again)
7. Pour off the media from the Falcon (ideally back in the conical flask) and you will see a pellet at the bottom. From now on KEEP CELLS ON ICE.
8. Resuspend the cells in pre-chilled 0.1M CaCl2 using the long sterile pipette and pipette gun, use ~30 ml per Falcon tube
9. Incubate the resuspended cells on ice for 1-2 hours
10. Spin the cells at 4deg, >3000g for 10 min
11. Resuspend the cells in 0.1M CaCl2/10%glycerol solution by gently pipetting them up and down and occasionally swirling them in the tube, use ~1-2 ml of the storage solution per Falcon tube (see comment below)
12. Keeping all the tubes and cells on ice, aliquot the cells into sterile eppendorfs and freeze the cells at -80deg C
13. For BL21/soluBL21 cells I would use 2ml of storage solution to resuspend make lots of 50-100μl aliquots, plus a few larger volumes (e.g. 200 or 300 or 500 μl). You will need 25-50 μl cells per transformation of a plasmid and these are the “easy” transformations. Larger volumes can save you space in the freezer boxes, and if you are transforming several plasmids in one go you can defrost 1 larger aliquot and transfer smaller aliquots from that into separate tubes.
14. For cloning strains like DH5alpha I resuspend cells in 1 ml storage solution and aliquot 100μl (or multiples of that, as explained for BL21 cells) into sterile tubes. For each ligation reaction/mutagenesis reaction transformation I would use 100μl cells. You can also make a few smaller aliquots, say 50μl, which can be used for plasmid transformations -e.g. when you want to make more of that plasmid for further cloning.

## **2. Transformation**

**Materials**

* Cells
* Plasmid
* LB media
* Ice bucket
* Eppendorf tubes 1.5 mL
* Pipette
* Water bath

**Protocol**

1. Thaw an appropriate volume of cells on ice, it can take 5-20 min. you can speed it up by occasionally flicking the tube. After they thaw they may look a bit clumpy – then stir them with pipette tip and if necessary (see above), aliquot them into separate sterile tubes.
2. For standard plasmid transformation of either cell type (clean plasmid, nothing difficult) I would add 0.5-1μl plasmid per cell aliquot, for the more complex things I would add 10μl of ligation/mutagenesis reaction per 100μl DH5alpha cells
3. After adding plasmid/reaction, flick the tube a couple of times to ensure mixing
4. Incubate the cells on ice for at least 10 min (for the challenging transformations perhaps 20 or so), it won’t hurt cells if you leave them much longer than that
5. Heat shock by putting the cells in 42deg C water bath for 30 sec (you can use a floater if you have many tubes)
6. Put the cells back on ice for a couple of minutes; for standard transformations you can shorten or even skip this step completely
7. Add ~200-300μl fresh LB media per tube for standard transformation; for challenging ones use 0.9-1 ml, preferably pre-warmed to 37degC, and you can use SOC or other rich media which would enhance the cell growth. Some people even use 14 ml round-bottom Falcon tubes, not eppendorfs, for these transformations to ensure good aeration…
8. Recovery step: incubate the cells in the shaking incubator at 37deg for 30-60 min (standard transformation) or 90-120min (challenging ones); I either put the tubes at the bottom of the beaker or tape them down to the bottom of the incubator. Importantly, for better aeration they should be lying flat rather than vertically.
9. For plating standard transformations I plate 50-100μl cells on a plate. For challenging ones I spin the tubes for a couple of minutes at ~6000g, discard most of the supernatant, and resuspend the pellet in whatever remains (100-200μl) or just add some more fresh media. That maximises the cell concentration. To further maximise the chances of success you can pre-warm the plates to 37degC and use a couple of plates per transformation (each covered with 50-100μl cells).
10. Incubate the cells overnight at 37degC INVERTED to prevent condensation on the actual plate.
11. Next day (preferably don’t leave them growing for longer than 16hours) inspect the plates and put them in the fridge or keep on the bench (inverted!) if you are going to use them later in the day to inoculate
12. If it is Friday and you want to transform then instead of 37deg C you can leave the plates on the bench, at room temp for 2-3 days and they should grow at the same rate as overnight at 37deg. I would then normally inoculate cultures on Sunday evening. For expression strains I would never use a plate older than a few days to inoculate an overnight culture for overexpression as cells may be slowly mutating even in the cold!

## **3. Measuring bacterial transformation efficiency**

**Protocol**:

1. 1 Transform 1 aliquot of competent cells with 1 ng of a control plasmid
2. Plate 5 µl, 50 µl and 300 µl of in plates with the appropriate antibiotic
3. Incubate overnight at 37ºC
4. Count the colonies on the plate where you can find 30 to 300 isolated colonies
5. Use this equation to calculate the number of CFUs per micrograms of DNA:

µg CFU = Volume plated Colonies in the plate·(Volume of cells+Volume of SOC +Volume of DNA)

CFU/g=Colonies in the plate(Volume of cells+Volume of SOC +Volume of DNA)/ Volume plated +1000

## **4. Bacterial glycerol stock**

For storing different strains or cells that contain plasmids; instead of transforming fresh cells every time to re-prep plasmids you can just scrape some cells from the glycerol stock to inoculate an overnight culture. Glycerol stocks are made of expression strains (e.g. soluBL21 DE3) containing different expression plasmids (e.g. pHisTevM3110C) so that they can be directly inoculated overnights for expression. Some people are opposed to that idea, and always do a fresh transformation.

**Materials:**

* Overnight cell culture
* Glycerol
* MilliQ water

**Protocol:**

1. As glycerol is very viscous, make 100 ml of 50% glycerol (mixed with Milli Q water) and autoclave that in advance.
2. To prepare a glycerol stock, mix ~0.5 ml of cells from an overnight culture with 0.5 ml of 50% glycerol in a sterile Eppendorf, invert a few times to ensure proper mixing and put the tube in the -80deg freezer.

## **5. Growing up E.Coli essential procedures**

### **Liquid medium preparation**

**Materials:**

* Agar
* LB broth salt-free
* NaCl
* Distilled water
* 500 ml autoclavable bottle
* Autoclave
* Magnetic stirrer

**Protocol:**

1. Add 10g of Agar, 7.5 g of yeast extract and 5g of NaCL.
2. Mix them.
3. Add the magnetic stirrer.
4. Add distilled water until liquid reaches 500 mL.
5. Stir solution.
6. Put bottle into autoclave.

### **Solid medium preparation**

For 500 ml of medium for 20 plates:

**Materials:**

* Agar
* LB broth salt-free
* NaCl
* Distilled water
* 500 ml autoclavable bottle
* Autoclave
* Magnetic stirrer
* Petri dishes
* 1000x antibiotic

**Protocol:**

1. Add 10g of Agar, 7.5 g of yeast extract and 5g of NaCL.
2. Mix them.
3. Add the magnetic stirrer.
4. Add distilled water until liquid reaches 500 mL.
5. Stir solution.
6. Put bottle into autoclave.
7. Set LB medium to cool to 50 degrees while stirred
8. Pour LB media into plates.
9. Set plates to cool.
10. After1 hour set plates upside down and leave on the bench overnight
11. Store plates in a cold room.

### **Liquid medium culture (inoculation)**

After plating transformed competent cells with a ligation product, single colonies are picked and inoculated into liquid medium.

**Materials**

* Shaker
* Vials
* LB broth liquid medium
* Antibiotic (1000x)

**Protocol:**

 Add 10 ml of LB broth and 10 µl of the antibiotic into a vial per colony that will be picked. A single colony is picked up and inoculated into the liquid medium with a pipette tip and this culture is then incubated overnight (12-16 h) at 37 ºC with vigorous shaking (220 rpm).

### **Petri dish culture**

Cells are grown in solid agar medium in order to isolate specific colonies. Agar petri dishes must contain the antibiotic for the resistance carried by the transformed strain.

**Materials:**

* LB agar plates
* Sterile spreaders

**Protocol:**

1. Spread the bacteria culture all over the plate using the spreader.
2. Invert plates and incubate overnight at 37ºC.

### PCR, agarose gels and DNA purifcation

## **1. Re-suspension of iGEM DNA**

Re-suspending dried DNA from the iGEM distribution kit. Protocol taken from the iGEM website.

**Protocol:**

*Note: There is an estimated 2-3ng of DNA in each well. When following this protocol, assume that you are transforming with 200-300pg/µL*

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be **red**, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic\* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock.

## **2. PCR Clean- up**

### **NEB Monarch® PCR & DNA Cleanup Kit (5 μg)**

DNA Cleanup and Concentration: for the purification of up to 5 μg of DNA (ssDNA > 200 nt and dsDNA > 50 bp) from PCR and other enzymatic reactions.

Oligonucleotide Cleanup: for the purification of up to 5 μg of DNA fragments ≥ 15 bp (dsDNA) or ≥ 18 nt (ssDNA). Expected recovery is > 70%. When purifying ssDNA of any size, recovery can be increased by using this protocol; however, it is important to note that this protocol shifts the cutoff for smaller fragments to 18 nt (rather than 50 nt for the DNA Cleanup and Concentration Protocol).

General Guidelines:

Input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume to the recommended volume range. Centrifugation should be carried out at 16,000 x g in a standard laboratory microcentrifuge at room temperature.

**Protocol:**

1. Add ethanol to Monarch DNA Wash Buffer prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer).
2. Always keep all buffer bottles tightly closed when not in use
3. All centrifugation steps should be carried out at 16,000 x g. (~13K RPM in a typical microcentrifuge). This ensures all traces of buffer are eluted at each step.
4. Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume. For diluted samples larger than 800 μl, load a portion of the sample, proceed with Step 2, and then repeat as necessary.

|  |  |  |
| --- | --- | --- |
| **SAMPLE TYPE** | **RATIO OF BINDING BUFFER: SAMPLE** | **EXAMPLE** |
| **dsDNA > 2 kb (plasmids, gDNA)** | **2:1** | **200μl:100 μl** |
| **dsDNA < 2 kb(some amplicons, fragments)** | **5:1** | **500 μl:100 μl** |
| **ssDNA > 200 nt\*** | **7:1** | **700 μl:100 μl** |

1. Insert column into collection tube and load sample onto column and close the cap. Spin for 1 minute, then discard flow-through.
2. To save time, spin for 30 seconds, instead of 1 minute.
3. If using a vacuum manifold\* instead of centrifugation, insert the column into the manifold and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.
4. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
5. If using a vacuum manifold, add 200 μl of DNA Wash Buffer and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.
6. Repeat wash (Step 3).
7. \* Make sure to follow the manifold manufacturer's instructions to set-up the manifold and connect it properly to a vacuum source.
8. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
9. If using a vacuum manifold: Since vacuum set-ups can vary, a 1 minute centrifugation is recommended prior to elution to ensure that no traces of salt or ethanol are carried over to the next step.
10. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
11. Note: Typical elution volumes are 6–20 μl. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield.
12. Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency. To save time, spin for 30 seconds, instead of 1 minute.

### **DNA Clean- up and Concentration System (ReliaPrep)**

**Materials:**

* 100% isopropanol, RNase-free
* 95–100% ethanol, RNase-free
* 1.5ml microcentrifuge tubes • agarose gel (standard or low-melt; only for gel purification)
* 1X TAE or TBE electrophoresis buffer (only for gel purification)
* 50–65°C heating block (only for gel purification)
* microcentrifuge capable of maintaining at least 10,000 × g (14,000rpm)
* Kit

**Protocol:**

Note: Perform all centrifugation steps at 10,000 × g (14,000rpm).

1. Pipet 25–400µl of a PCR amplification or reaction pool into a 1.5ml microcentrifuge tube.
2. Add an equal volume of Membrane Binding Solution and vortex 5 seconds.

|  |  |  |
| --- | --- | --- |
| **PCR Volume (µl)** | **Membrane Binding Solution (µl)** | **Total Load (µl)** |
| 25 | 25 | 50 |
| 50 | 50 | 100 |
| 100 | 100 | 200 |
| 150 | 150 | 300 |
| 200 | 200 | 400 |
| 250 | 250 | 500 |
| 300 | 300 | 600 |
| 350 | 350 | 700 |
| 400 | 400 | 800 |

1. Load sample onto a ReliaPrep™ Minicolumn seated in a Collection Tube and centrifuge for 30 seconds.
2. Remove column and discard the contents of the Collection Tube. Reseat the minicolumn into the same Collection Tube.
3. Add 200µl of Column Wash Solution (CWE) and centrifuge for 15 seconds. Remove column and discard the contents of the Collection Tube. Reseat the minicolumn into the same Collection Tube.
4. Wash with 300µl of Buffer B (BWB) and centrifuge for 15 seconds. Repeat wash with 300µl of Buffer B (BWB) and centrifuge again.
5. Remove minicolumn, and discard the contents of the Collection Tube. Reseat the minicolumn into the same Collection Tube and centrifuge for 1 minute to dry the minicolumn; then transfer minicolumn to an Elution Tube.
6. Pipet 15µl of Nuclease-Free Water or TE buffer (not provided) to the center of the minicolumn, then centrifuge for 30 seconds. Note: Touch the pipette tip to the column bed surface before dispensing Nuclease-Free Water or TE buffer to completely wet the column matrix. The color should change from light to dark tan.
7. For maximum recovery, repeat elution with an additional 15µl of Nuclease-Free Water or TE buffer.

### **Wizard® SV Gel and PCR Clean-Up System**

**Protocol:**

1. Amplify target of choice using standard ampliﬁcation conditions.
2. Add an equal volume of Membrane Binding Solution to the PCR ampliﬁ cation (Notes 1–4 below).
3. Notes:

* The maximal capacity of a single SV Minicolumn is approximately 1ml of PCR ampliﬁ cation added to 1ml of Membrane Binding Solution (2ml total). For PCR volumes >350µl, continue to pass the sample through the column until all of the sample has been processed.
* The maximum binding capacity is approximately 40µg per column, and as little as 10ng has been successfully puriﬁed.
* Mineral oil does not interfere with puriﬁcation.
* For ampliﬁcation reactions that do not produce a single product or where ampliﬁcation has been ineﬃcient and there is highly visible primer dimer, gel puriﬁcation of the band of interest is recommended. Alternatively, an 80% ethanol wash solution can be substituted for the supplied Membrane Wash Solution to reduce primer-dimer carryover.

1. Place one SV Minicolumn in a Collection Tube for each dissolved gel slice or PCR ampliﬁ cation.
2. Transfer the dissolved gel mixture or prepared PCR product to the SV Minicolumn assembly and incubate for 1 minute at room temperature.
3. Centrifuge the SV Minicolumn assembly in a microcentrifuge at 16,000 × g (14,000rpm) for 1 minute. Remove the SV Minicolumn from the Spin Column assembly, and discard the liquid in the Collection Tube. Return the SV Minicolumn to the Collection Tube. Note: Failure to spin at 16,000 × g (14,000rpm) can result in reduced yield. !
4. Wash the column by adding 700µl of Membrane Wash Solution, previously diluted with 95% ethanol (Section 4.A), to the SV Minicolumn. Centrifuge the SV Minicolumn assembly for 1 minute at 16,000 × g (14,000rpm). Empty the Collection Tube as before, and place the SV Minicolumn back in the Collection Tube. Repeat the wash with 500µl of Membrane Wash Solution, and centrifuge the SV Minicolumn assembly for 5 minutes at 16,000 × g.
5. Remove the SV Minicolumn assembly from the centrifuge, being careful not to wet the bottom of the column with the ﬂowthrough. Empty the Collection Tube, and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or oﬀ ) to allow evaporation of any residual ethanol.
6. Carefully transfer the SV Minicolumn to a clean 1.5ml microcentrifuge tube. Apply 50µl of Nuclease-Free Water directly to the center of the column without touching the membrane with the pipette tip. Incubate at room temperature for 1 minute. Centrifuge for 1 minute at 16,000 × g (14,000rpm).
7. Discard the SV Minicolumn, and store the microcentrifuge tube containing the eluted DNA at 4°C or –20°C. Note: The volume of the eluted DNA will be approximately 42–47µl. If the DNA needs to be further concentrated, perform an ethanol precipitation. Alternatively, the DNA may be eluted in as little as 15µl of Nuclease-Free Water without signiﬁ cant reduction in yield. If using an elution volume of 15µl, verify that the membrane is completely covered with Nuclease-Free Water before centrifugation. Elution volumes less than 15µl are not recommended*.*

|  |  |
| --- | --- |
| **Elution Volume** | **Percent Recovery compared to 50** **L** |
| 10 L | 35 % |
| 15 L | 98% |
| 25 L | 98% |
| 50 L | 100% |
| 75L | 100% |
| 100L | 100% |

## **3. PCR - NEB Q5-HF Kit**

**Materials:**

* 50uL PCR Tubes
* Ice Box
* Nuclease-Free dH2O
* 10uM Forward Primer
* 10uM Reverse Primer

+ Primers should be at 100um, but check

* Template DNA
* Q5 High-Fidelity 2X Master Mix
* PCR Machine

**Protocol:**

1. Get out primers from -20C freezer (4th drawer down, on the left), and keep on ice
2. Get the Q5 High 2X Master Mix kit from the -20C freezer (4th drawer down, on the left), and keep on ice
3. Add the following to PCR Tube, adding the Q5 High-Fidelity 2X Master Mix Last:

|  |  |
| --- | --- |
| **Q5 High-Fidelity 2X Master Mix** | **25uL** |
| **Template DNA** | **Variable (~0.2uL for Plasmids)** |
| **10uM Forward Primer** | **2.5uL** |
| **10uM Reverse Primer** | **2.5uL** |
| **Nuclease-Free dH2O** | **Make up to 50uL** |

4. Keep the above mixture on ice until you are ready to put it into the machine
5. Turn on PCR machine at the back and wait for it to boot. Once booted, select the program you intend to run, or create a new program. The program should have:

|  |  |  |
| --- | --- | --- |
| Initial Denaturation | 98C | 30s |
| Denaturation | 98C | 15-30s |
| Annealing | 45-68C | 15-60s |
| Extension | 72C | 20s per kb of template DNA |
| Final Extension | 72C | 5 minutes |
| Hold | 4-10C | - |

6. The annealing temperature should be ~3C below the lowest melting temperature of the two primers.
7. If you want to keep the PCR product in the machine overnight, set the time to infinite on the machine and the final hold temperature to 4C. Steps 2-4 in the table above should be repeated for 30 cycles.

## **4. Agarose Gel**

### **Gel preparation and running**

Makes 1L of 50X TAE Buffer for running DNA gels.

**Materials:**

* Tris Base
* Disodium EDTA
* Glacial Acetic Acid
* 1L Beaker
* 1L Shop Bottle (Empty and Clean)
* Magnetic Stirrer (Optional)

**Protocol:**

1. Weigh out 242g of Tris and add to 800mL of dH2O in the 1L beaker. Stir until all the Tris has dissolved.
2. Once Tris dissolved, add 18.61g of Disodium EDTA to the mixture. Stir until dissolved.
3. Once EDTA dissolved, add 57.1mL of Glacial Acetic Acid and stir.
4. Make the mixture up to 1L with dH2O. Transfer to 1L shop bottle and cap.

### **Making 50x TAE buffer for gel**

Making Agarose gels to run Nucleic acid samples. Makes circa 10 1% Agarose gels.Wear gloves throughout as risk of burns from Agarose, and to avoid skin contact with SYBR Safe.

**Materials:**

* Agarose Powder
* 1x TAE Buffer (Diluted from 50x Buffer)

* 500mL Shop Bottle
* Gel Cast
* Gel Comb
* Gel Clamp

**Protocol:**

Making the Gel Stock:

1. Weigh out 5g of Agarose Powder, and transfer with x1 TAE Buffer to a clean 500mL Shop Bottle.
2. Top up the shop bottle to 500mL with x1 TAE Buffer.
3. Microwave the mixture in the bottle until the Agarose powder is dissolved. This takes circa 6 mins of microwave time, but ensure to swirl and mix the mixture every 2 mins or so. Mix upon boiling.
4. The gel can now be left to cool before pouring or left to cool on the bench top. Once solidified, the cap is placed on the bottle.
5. You can now microwave the agarose shop bottle to prepare more gels.

#### Casting the Gel

1. Place the Gel cast in the gel clamp and clamp tight.
2. Once the molten agarose bottle is just cool enough to hold, pour in the agarose to the cast.
3. If you are unsure about what volume to use, measure out 40-50mL of molten Agarose in a measuring cylinder.
4. Add 1 uL of SYBR Safe and stir round the molten agarose using the pipette tip.
5. Place the comb in the cast. Leave the gel to set.

#### Running the Gel

1. Remove Gel + Cast from clamp and place in the gel tank, ensuring the gel is covered in x1 TAE buffer.
2. Remove the comb from the gel gently, once submerged in x1 TAE Buffer
3. Load samples into the gel
4. Run the Gel for 1hr at 90V in most conditions, but you can use a lower voltage and longer time if needs be.

## **5. DNA gel extraction**

### **NEB Monarch kit**

The input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). DNA fragments were excised from an agarose gel and are diluted by the addition of four volumes of Gel Dissolving Buffer. For a typical 100 mg (100 μl) gel slice, 400 μl of Gel Dissolving Buffer is added. Centrifugation should be carried out at 16,000 x g in a standard laboratory microcentrifuge at room temperature.

**Buffer Preparation:**

1. Add ethanol to Monarch DNA Wash Buffer prior to use (4 volumes of ≥ 95%ethanol per volume of Monarch DNA Wash Buffer).
2. For 50-prep kit add 20 ml of ethanol to 5 ml of Monarch DNA Wash Buffer
3. For 250-prep kit add 100 ml of ethanol to 25 ml of Monarch DNA Wash Buffer. Always keep all buffer bottles tightly closed when not in use.

**Protocol:**

1. Excise the DNA fragment to be purified from the agarose gel using a razor blade, scalpel or other clean cutting tool. Use care to trim excess agarose. Transfer it to a 1.5 ml microcentrifuge tube and weigh the gel slice.
2. Note: Using UV light to visualize the slice is common, but the exposure time should be kept as short as possible to minimize damage to the DNA. Use long-wave UV when possible, as shorter wavelengths induce greater damage. Also, trim off excess agarose from the perimeter of the band to minimize the amount of dissolving buffer needed, and to reduce the time necessary to extract the DNA.
3. Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the slice.
4. Note: If the volume of the dissolved sample exceeds 800 μl, the loading of the sample onto the column should be performed in multiple rounds to not exceed the volume constraints of the spin column.
5. Incubate the sample between 37–55°C (typically 50°C), vortexing periodically until the gel slice is completely dissolved (generally 5–10 minutes).
6. Note: For DNA fragments > 8 kb, an additional 1.5 volumes of water should be added after the slice is dissolved to mitigate the tighter binding of larger pieces of DNA (e.g., 100 μl gel slice: 400 μl Gel Dissolving Buffer: 150 μl water). Failure to dissolve all the agarose will decrease the recovery yield due to incomplete extraction of the DNA and potential clogging of the column by particles of agarose.
7. Insert the column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
8. To save time, spin can be reduced to 30 seconds.
9. If using a vacuum manifold\* instead of centrifugation, insert the column into the manifold and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.
10. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
11. If using a vacuum manifold, add 200 μl of DNA Wash Buffer and switch the vacuum on. Allow the column solution to pass through the column, then switch the vacuum source off.
12. Repeat wash (Step 5).
13. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If in doubt, re-spin for 1 minute before placing into clean microfuge tube.
14. If using a vacuum manifold: Since vacuum set-ups can vary, a 1 minute centrifugation is recommended prior to elution to ensure that no traces of salt and ethanol are carried over to the next step.
15. \* Make sure to follow the manifold manufacturer's instructions to set-up the manifold and connect it properly to a vacuum source.
16. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA.
17. Note: Typical elution volumes are 6–20 μl. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield. Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency. To save time, spin can be reduced to 30 seconds.

## **6. Ethanol precipitation of plasmid DNA**

DNA is polar due to its highly charged phosphate backbone. This polarity, based on the principle of "like dissolves like", makes it soluble in water, which is also highly polar. Ethanol is much less polar than water, its dielectric constant is 24.3 (at 25 °C). This means that adding ethanol to solution disrupts screening of charges by water. If enough ethanol is added electrical attraction between phosphate groups and any positive ions present in solution becomes strong enough to form stable ionic bonds and precipitate DNA. This usually happens when ethanol makes around 64% of the solution. As the mechanism suggests solution has to contain positive ions for precipitation to occur, usually Na+, NH4+ or Li+ play this role.

Optimal incubation time depends on the length and concentration of DNA. Smaller fragments and lower concentrations will require longer times to achieve the same recovery. For very small lengths and low concentrations overnight incubation is recommended. In such cases the use of carriers like tRNA, glycogen or linear polyacrylamide can greatly improve recovery. During incubation DNA and some salts will precipitate from solution, in the next step this precipitate is collected by centrifugation in a microcentrifuge tube at high speeds (~12,000g). Time and speed of centrifugation has the biggest effect on DNA recovery rates. Smaller fragments and higher dilutions require longer and faster centrifugation. Centrifugation can be done either at room temperature or in 4 °C or 0 °C. During centrifugation precipitated DNA has to move through ethanol solution to the bottom of the tube, lower temperatures increase viscosity of the solution and larger volumes make the distance longer, so both those factors lower efficiency of this process requiring longer centrifugation for the same effect. After centrifugation the supernatant solution is removed, leaving a pellet of crude DNA. Whether the pellet is visible depends on the amount of DNA and on its purity (dirtier pellets are easier to see) or the use of co-precipitants. 70% ethanol is then added to the pellet, and the sample gently mixed to break the pellet loose and wash it. This removes some of the salts present in the leftover supernatant and bound to DNA pellet making the final DNA cleaner. This suspension is centrifuged again to once again pellet DNA and the supernatant solution is removed. This step is repeated once. Finally, the pellet is air-dried and the DNA is resuspended in water or other desired buffer. It is important not to over-dry the pellet as it may lead to denaturation of DNA and make it harder to resuspend. Isopropanol can also be used instead of ethanol; the precipitation efficiency of the isopropanol is higher making one volume enough for precipitation. However, isopropanol is less volatile than ethanol and needs more time to air-dry in the final step. The pellet might also adhere less tightly to the tube when using isopropanol.

**Materials:**

* Sodium Acetate (3M, pH 5.2)
* Ethanol (>95 %)
* Ethanol (70 %)
* Water as buffer
* Sample

**Protocol:**

1. Add 1/10 volume of Sodium Acetate (3 M, pH 5.2).
2. Add 2.5-3.0 X volume (calculated after addition of sodium acetate) of at least 95% ethanol
3. Incubate on ice for 15 minutes. In case of small DNA fragments or high dilutions overnight incubation gives best results, incubation below 0 °C does not significantly improve efficiency.
4. Centrifuge at > 14,000 x g for 30 minutes at room temperature or 4 °C.
5. Discard supernatant being careful not to throw out DNA pellet which may or may not be visible.
6. Rinse with 70% Ethanol (see note above for details)
7. Centrifuge again for 15 minutes.
8. Discard supernatant and dissolve pellet in desired buffer. Make sure the buffer comes into contact with the whole surface of the tube since a significant portion of DNA may be deposited on the walls instead of in the pellet. o This last step should be done with sterile TE.

### Plasmid preparation **1. Miniprep** **NEB Monarch Miniprep** **Materials:** * Monarch Plasmid Resuspension Buffer * Monarch Plasmid Lysis Buffer * Monarch Plasmid Wash Buffer 1 * Monarch Plasmid Wash Buffer 2 * Monarch DNA Elution Buffer * Monarch Plasmid * Miniprep Columns * Cell Culture * 15mL Falcon Tubes * 1.5mL Eppendorf Tubes **Protocol:** **Culture preparation:** 1. Divide culture evenly between 15mL Falcon Tubes. Try to have no more than 5mL per tube, and no less than 1mL. 2. Centrifuge cultures in the falcon tubes at max speed for 30s and discard supernatant 3. Resuspend pellet in 1mL of dH2O and transfer to 1.5mL eppendorf 4. Centrifuge culture again at max speed for 30s in 1.5mL eppendorf, discard supernatant Miniprep: Before beginning the miniprep, ensure that Ethanol **has been** added to Plasmid Wash Buffer 2, in the correct amount. Correct amount is 4 Ethanol : 1 wash buffer but it should say on the bottle how much to add. If it has already been added, don't add more! 1. Resuspend the pellet in the eppendorf using 200uL Monarch Plasmid Re-suspension Buffer. Gently pipette up and down to ensure the pellet is completely re-suspended (vortex if necessary) 2. Add 200uL of Monarch Plasmid Lysis Buffer. Invert tube immediately and gently 5-6 times. Colour should change from light to dark pink. Incubate at room temperature for 1 min. Avoid incubating for longer than 1 min. 3. Add 400uL of Monarch Plasmid Neutralisation Buffer and gently invert until colour becomes uniformly yellow and precipitate has formed. 4. Spin the mixture in the eppendorf down at 16000xg for 2-5 mins. 5. Transfer supernatant to Plasmid Miniprep Spin column and spin at 16000xg for 1 min. Discard flow-through. 6. Add 200uL of Plasmid Wash Buffer 1 to column and centrifuge at 16000xg for 1 min. You can discard the flow-through at this step but it is only necessary if you think the tip of the column will come into contact with the flow-through - at no point allow this to happen! If it has, spin at 16000xg again for 1 min and proceed as normal. 7. Add 400uL of Plasmid Wash Buffer 2 to the column and centrifuge at 16000xg for 1 min. Discard flow-through. 8. Transfer column to **clean** 1.5mL eppendorf tube. 9. Add >30uL of DNA Elution Buffer to the mixture. Adding more will increase yield slightly but decrease product concentration. Spin the column at 16000xg for 1 min. Remove column. **Do not discard flow-through**, this is your DNA product. 10. Store DNA product **labelled** at -20C. **2.Eznyme digestion** **Restriction enzyme plasmid digestion** **Materials:** * Plasmid * CutSmart buffer * dH2O * DNA inserts * Restriction enzymes * Water bath **Protocol:** 1. Digestion of plasmid: Combine 50 uL plasmid, 2 uL of restriction enzyme (BamHI-HF), 2 uL of restriction enzyme (NcoI-HF), 8 uL of CutSmart buffer and 20 uL dH2O.Leave at 37°C (water bath) for 1 and a half hours. 2. Digestion of insert: 8 uL of H2O, 2 uL BamHI, 2 uL NcoI, 2 uL Cutsmart, 8 uL of insert DNA left at 37°C (water bath) for 1 and a half hours. **Double digestion of plasmid/ inserts** **Materials:** * Restriction enzymes (BamHi-HF, Ncol-HF) * CutSmart buffer * Plasmid * DNA inserts **Protocols:** 1. BamHI and NcoI can be used together in digestion, make sure you use the right buffer (presumably CutSmart for the HF versions of these enzymes). Ensure you have quite a lot of plasmids at hand, e.g. Miniprep plasmid from 2x10ml overnight LB cultures using two separate spin columns, and check concentration using Nanodrop – preferably you would have more than 100ng/μl. 2. Inserts which you amplified by PCR need to be **cleaned up first**, e.g. by PCR purification kit. 3. To ensure each enzyme actually works prepare two separate single digests of the plasmid (you won’t see any difference with the inserts), assuming you have at least 100μl plasmid at hand prepare the following using either NcoI or BamHI: 4. 5μl buffer + 44μl plasmid + 1μl enzyme (added last) = final volume of 50ul 5. Spin the reactions briefly to make sure everything is at the bottom of the tubes. 6. From each single digest take 44μl and transfer to a separate tube. Mix the two reactions which will give you now the double digest. 7. Incubate all three tubes (3 reactions per 1 construct) at 37 degC for a couple of hours; for the gene inserts I sometimes incubate for several hours or even overnight. 8. Digested plasmid should be gel-extracted; to ensure each enzyme cut properly run a few μl of each single digest and of the double digest on the same gel (using smaller wells) or before you attempt to gel extract. As long as you see just one vector band in each reaction at the appropriate size then it must have been successfully linearised and thus the enzymes work. 9. After gel extraction it is likely your vector will be at quite a low concentration (e.g. 10ng/μl or less) and the intercalating agents like Sybr Safe or ethidium bromide trailing from gel may interfere as well. If you want to be sure you can run the gel-extracted plasmid on the gel to check it is there. 10. Digested inserts can be purified by something like “PCR cleanup” – e.g. Qiagen have a kit where you mix the reaction with 5 volumes of buffer PB and you clean that on a spin column and then elute the purified DNA. You just want to remove cleaved bits, buffer and the enzymes, and you can as well gel extract the bands but you always lose more DNA by gel extraction. 11. Ellute the gel-extracted plasmid and purified linear DNA in the smallest volume possible to concentrate the DNA. **3.DNA ligation reaction** **Materials:** * Digested insert * Digested plasmid * T4 DNA ligase * Ligase buffer **Protocols:** 1. You want to use an excess of the digested insert over the digested plasmid, usually people use 3-5-fold excess. Remember that your inserts are considerably shorter (say 500bp) than the plasmid backbone (say 5000bp) so if Nanodrop/agarose gel shows similar concentration of plasmid and insert, if fact you have 10x more insert. 2. As long as you know there is some plasmid from gel extraction and the purified inserts have some reasonable concentration in Nanodrop (above 20ng/μl for short, <500bp constructs, for longer constructs it would be expected even more) Set up a reaction like this, using NEB T4 DNA ligase: 2μl ligase buffer + 13μl digested insert + 4μl digested plasmid + 1μl ligase (added last). 3. After spinning the tube briefly, incubate the reaction at room temperature for a few hours or even overnight before transforming into DH5 alpha cells. **4. Colony PCR** Colony PCR is a method for determining the presence of insert DNA in a plasmid construct. This procedure was carried out in order to check the presence of a domesticated DNA part or transcriptional unit. **Materials:** * 250 µL tubes * Ice box * Thermocycler * Pipettes and pipette tips * Nuclease-Free dH2O * Standard Taq buffer (10X) NEB * Primers forward and reverse (10 uM) * dNTPs 2.5 mM * Taq polymerase enzyme - NEB * 5 µl diluted colony **Protocol:** Colony PCR protocol from New England Biolabs (NEB). For each PCR reaction, primers annealing temperature was determined using the New England Biolabs online calculator tool (NEM TM calculator). 1. Colonies are picked and smirred inside the PCR tubes. 2. On ice, add all components in a 250 µL tube, making up to a 25 µl volume reaction. If checking the presence of a plasmid in many colonies, a Master Mix is recommended to be done by including all reagents except colonies.

### Protein purification **Nickel column****Dialysis and reverse nickel purification** **Materials:** * Buffer A: 50mM Tris-HCl pH 7.5-8, 500mM NaCl * Buffer B: 50mM Tris-HCl pH 7.5-8, 500mM NaCl + 500mM imidazole * Dialysis buffer: 20mM Tris pH 7.5-8, 200mM NaCl **Protocols:** 1. Prepare 5 litres of dialysis buffer (20mM Tris pH 7.5-8, 200mM NaCl) 2. Thaw out TEV: use about 1-2mg TEV per 50mg protein 3. Take a sample of the elution for gel and then add the appropriate amount of TEV 4. Set up a dialysis sausage with the elution+TEV (ensure you use the right cut-off, for proteins 20kDa or smaller use SnakeSkin with cut off 3 kDa!) 5. Leave the dialysis stirring slowly overnight in the cold room 6. Next day transfer the dialysate into a falcon tube and spin 15 min at max speed to pellet precipitate, then filter with 0.45um syringe filter (there will probably still be a lot of crap, TEV is notorious…) 7. Load the cut protein slowly onto the column – the slower the better, you want to remove TEV/HisTag/uncut protein; again you can discard the first 5 ml that go through 8. When all protein went through, pass another 10ml of dialysis buffer (or just diluted buffer A) through to column to ensure all the cut protein has gone out of the column 9. To remove TEV/HisTag/uncut protein from the column pass 3-4 column volumes of Buffer B ( 50mM Tris-HCl pH 7.5-8, 500mM NaCl + 500mM imidazole), collect that bulk elution in case cleavage was not efficient – you can dialyse that to remove imidazole, add more TEV and do the reverse nickel again! 10. To ensure the column is clean, pass some strip buffer (containing 50mM EDTA that chelates nickel ions) until the column is white, then a couple CVs of water, then the green nickel solution to recharge the column, and again some water. [If it looks dirty or you had huge amounts of crashing out protein on the column, you may want to wash it with a couple of CVs of 0.5M NaOH – ideally after stripping and water wash, before recharging with nickel.] Then the column can be stored in 20% EtOH in the fridge. Make sure the peristaltic pump tubing is washed with WATER before the tube is TAKEN OFF THE WHEEL – that makes the tube relax and less prone to cracking. **Large scale cell lysis and Ni purification** **Materials:** * Buffer A: 50 mM Tris-HCl (pH 7.7), 500 mM NaCl * Buffer B: 50mM Tris-HCl pH 7.5-8, 500mM NaCl + 500mM imidazole **Protocols:** 1. Use 1 L overnight culture 2. Pellet at 7000 rpm, 4°C for 7 minutes 3. Take the pellet, add some (10-20ml) Buffer A. 4. In a small beaker with a stirrer dissolve 1 tablet of complete protease inhibitors (Roche) and add DNase (1 aliquot for pellet up to 2L, 2 aliquots if more) 5. Add the contents of the beaker to the Falcon tube with the pellet and swirl it around; you can use a spatula to scrape pellet off the sides 6. Transfer the buffer with chunks of pellet into the beaker, try to take all the pellet out 7. Keep stirring until pellet is properly resuspended (it can take 20 minutes or so, you can leave the beaker on the stirrer in the cold room for longer) 8. Switch the cell disruptor and the chiller on (ideally >10 min before you start lysis) 9. Pass 200ml water through the disruptor at >15 kpsi, then some Buffer A. [Stop the disruptor ideally when the liquid disappears from the cup but no air has yet entered the system] 10. Pour the resuspended pellet into cup VIA METAL SIEVE; if the solution looks very viscous or clumpy leave it stirring for longer and perhaps add some more DNase to it 11. Change pressure to 30 kpsi and pass the sample through, collecting it in a beaker 12. You can do a second round of lysis – stop the disruptor again when the liquid just went down, pour the lysate back in the cup and lyse again 13. At the end you can pass some (10-20ml) of buffer A to rinse out the remaining lysate 14. Reduce the pressure back to around 15 kpsi, fill the cup with water and pass it all through 15. Switch the CHILLER OFF 16. Pass 200ml 70% EtOH through the disruptor; the foam is tough to get rid of so you can use the EtOH in a squirty bottle to remove the foam from the sides 17. Remove the cup and the underlying little sieve piece and wash them in the sink to remove any crap, then put them back on 18. Remove any waste, refill water/EtOH bottles if necessary, log your usage in the book and switch the machine off (button on the side) 19. Transfer the lysate into cream round-bottom tubes with caps, balance them 20. Spin the tubes in the JA20 rotor, 20 min at 20 000 rpm, 4 deg 21. In the meantime equilibrate nickel column on the peristaltic pump: first 1-2 volumes of water, then a few column volumes of buffer A 22. Pour the supernatant from the cream tubes into a small Duran bottle, supplement it DROPWISE with imidazole, final concentration 20mM (calculate how much of 5M imidazole stock you need to add). If lysate looks dense either dilute it down with buffer or filter with 0.45um filter 23. If the lysate looks clear, load it onto the nickel column – preferably slowly as this enhances the binding. Collect the flow-through in a beaker 24. When all lysate is loaded, pass a few column volumes (20-30ml) of Buffer A containing 40-50mM imidazole, collect the wash 25. Elute in 3-4 column volumes of buffer A containing 250mM imidazole; when you start to elute you can discard the first 5ml that go through as the elution buffer wouldn’t have reached the bottom of the column by then anyway 26. After elution pass a few column volumes of Buffer B through the column, then at least 5 column volumes of Buffer A – the column can then be ready for the next day 27. Measure the absorbance of the elution using elution buffer as a blank to estimate the protein content **Protocol for small scale Ni pull-down** **Materials:** * Sample * 1x PBS Buffer * 0.1 mm glass beads * Ni- Nta resin * milliQ water * 5 mM imidazole **Protocols:** 1. Pellet cells by centrifugation for 20-30 mins at maximum speed. 2. Discard media. 3. Resuspend cells in 500uL 1x PBS buffer until homogeneous. 4. Lyse cells by bead beating, add a scoop or “a bit” of 0.1mm beads and vortex on the vortex genie (chemical hood) for 10-20 mins. 5. Centrifuge for 20-30 mins at 14000 rpm (4 degrees C) to separate soluble/insoluble proteins. 6. While centrifugation is happening, take 100uL of Ni-Nta resin slur and pipette it in the column that will be used for pull-down. Centrifuge for 1-3 minutes to remove residual EtOH from storage. Discard Flow through. 7. Wash the resin slur with at least 500uL milliq H2O. Centrifuge for 1-3 minutes. Discard Flow through 8. After centrifugation from step 6 is done, take the supernatant containing soluble proteins and add it to the equilibrated resin (KEEP 20 uL of protein before mixing to the Ni resin, this is your LOADED sample for the gel later). Pipette up and down to mix and then incubate then for 30mins-1h at 4 degrees in the spinning wheel to allow protein binding to Nickel resin. 9. After proteins are bound to Nickel resin, centrifuge for 1-3 minutes or until all liquid has passed through the filter. KEEP the flow through as this are the proteins that did not bind to Nickel. This is the FLOW THROUGH sample. 10. Add 500uL of PBS+5mM imidazole to the resin to wash residual unbound proteins. KEEP the flow through as this are the proteins that did not bind to Nickel strong enough and came out in the wash. This is the WASH sample. 11. Add 100uL of PBS+250mM imidazole to the resin to elute proteins bound to Nickel. KEEP the flow through as this potentially has your protein of interest. – ELUTED. 12. Run a gel: 3-5 uL of the LOADED and FLOW-THROUGH samples, 10-25uL of the WASH sample and 25uL of the ELUTED sample.

### Protein analysis **Protein gel preparation and running (Amersham ECL™ gel system)** **Electrophoresis** **Protocol:** 1. Prepare 1×running buffer by diluting 19ml of Amersham ECL Gel Running Buffer, 10× in 171 ml water.190ml buffer is sufficient for one electrophoresis gel. 2. Add 90ml of 1×running buffer to each tank of Amersham ECL Gel Box. 3. Cut open the gel package and gently remove Amersham ECLGel from the package. 4. Rinse the gel cassette with distilled water.Peel off the tapes from the two legs of the cassette. 5. Place Amersham ECL Gel in Amersham ECLGel Box so that the well side of the cassette faces toward the cathode(-) and the other cassette leg faces toward the anode(+). 6. Place the safety lid on top of Amersham ECL Gel Box. 7. Connect AmershamECL Gel Box to the power supply (EPS301) and pre-run the gel for 12 minutes at 160V. 8. Do not exceed the maximum operating voltage of 200V . 9. Note: Expected start current approx.45mA. 10. Once the pre-run is finished,switch off the power. 11. Remove the safety lid. 12. Wigglethecombbackandforth,andbringitstraightupfrom thecassettetomakethewellsavailableforsampleloading. 13. Note: Small gel pieces can be detached from the well container.This will not affect gel performance. Do not discard the comb. 14. Add 6ml of 1×running buffer to the well container. 15. Prepare the samples by adding sample and 2×sample buffer in a 1:1 mixture. 16. Note: For native conditions: use a sample buffer without SDS and DTT. 17. Heat the samples at 95°C for 5 minutes. 18. Spin down the samples quickly in a microcentrifuge and load the samples directly into the wells in the gel. Note: A maximum of 0.5μg /band per well of sample can be loaded. Overloading may cause smearing and distortion. 19. Note: To ensure uniform mobility ,load an equal volume of 1×SDS sample buffer into any unused well. 20. Place the safety lid on top of Amersham ECL Gel Box. | | | | --- | --- | | **Parameter** | **Value** | | Voltage | 160 V | | Expected current | Start:approx.45mA End:23to33mA | | Run time | 60 minutes | **Removing the gel from the cassette** **Protocol:** 1. Once the run is completed,shut off the power, disconnect the electrodes, remove the safety lid and finally, remove the gel cassette from Amersham ECL Gel Box. 2. Open the cassette by inserting the edge of the comb in the slot opposite the sample wells, and twist. 3. Remove the top plate from the gel cassette and allow the gel to sit on the bottom plate. 4. Cut the stacking gel with the end of the top plate approximately 2cm downstream of the wells. Repeat the procedure on the other side of the gel to remove the front. 5. Hold the gel cassette bottom plate over a container with suitable buffer, with the gel facing downwards. Gently push a tweezer between the gel and the cassette bottom plate until the gel is removed from the bottom plate. **Post-staining of the gel** *250ml of each solution is used in each step.* | | | | --- | --- | | **Solution** | **Preparation** | | Fixing solution | 1. Mix 400ml of ethanol and 100ml of acetic acid with distilled water. 2. Make up to 1.0l with distilled water. | | Staining solution | 1. Mix 1 table to PhastGel™ BlueR-350 with destaining solution. 2. Make up to 400ml with destaining solution.Heat to 60°C , stirring constantly, and filter before use. | | Washing solution | Distilled water | | Destaining solution | 1. Mix 250 ml ethanol and 80 ml acetic acid. 2. Make up to 1.0 l with distilled water. | | Preserving solution | 1. Mix 25ml of (87%v/v) glycerol with distilled water. 2. Make up to 250 ml with destaining solution. | 1. Fixation: Immediately after electrophoresis, immerse the gel in fixing solution for 30 minutes.This solution precipitates the proteins and allows the SDS to diffuse out of the gel. 2. Staining: Discard the fixing solution.Stain the gel for 10 minutes in staining solution. Cover the staining dish. 3. Washing: Rinse the gel once in distilled water. 4. Destaining: Destain the gel by changing the destaining solution several times until the stained protein bands are clearly visible against the clear background. 5. Preserving: Soak the destained gel in preserving solution for 30 minutes. **Western blotting** **Protocol:** 1. Run an SDS-Page gel with samples - any gel running system can be used. 2. Stack the gel between sponges, filter paper and a nitrocellulose membrane using the following order: 3. Negative electrode - sponge - filter paper - protein gel - nitrocellulose - filter paper - sponge - positive electrode 4. Run the transfer to the membrane at 90 V for 90 minutes using transfer buffer (Tris-glycin with 10% methanol) 5. Remove the membrane and block with milk powder dissolved in 50 mL PBS.A tween 20 for 1 hour 6. Pour off used blocking solution 7. Prepare 25 mL of fresh milk powder in PBS.A tween 20 for 1 hour and add 5 uL of anti-His antibody 8. Leave for shaking for 1 hour. 9. Wash the membrane with excess PBS A Tween 20 then at least 3x50 mL of PBS.A Tween 20 and leave for 5 minutes each wash 10. Spread 1 mL of ECL (the substrate solution for the horse-radish peroxidase) in order to visualize the blot 11. Use the ChemiDoc stain free blot setting to visualize the blot

## **Laboratory Notes**

### Our laboratory work was documented using Benchling and the laboratory notes are presented here.

1. Basic laboratory preparations: Made competent cells, agar plates and plasmids that can be digested and used for cloning.

2. Preparing cells containing the correct plasmids for antibody expression: Cloned in the antibody fragment DNA of all our wild-types and mutant antibody CH3 domains into pEHISTEV expression vector. Transfomed SoluBL21 and Origami cells with expression plasmids.

3. Expressing and purifying antibody fragments: Expressed antibody fragments in SoluBL21 and Origami cells, purified these proteins via nickel affinity chromatography and reverse nickel chromatography. Analysed expressed proteins via mass-spectrometry

4. Expressing a commercially available antibody fragment as an antibody expression test in E. coli.:

5. Expressing the Bal-3 isopeptide domain, as a positive control for bacterial isopeptide domain expression:

6. Expressing wild-type and mutant Thermotoga maritima enzyme mutants:

7. Characterizing the mOrange-SpyTag conjugate.:

8. Neomycin resistance characterization in DH5Alpha cells:

9. Characterizing T7 promoter using in vitro transcription:

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# Team:St Andrews/Results

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Results

Antibody Expression Results

Table 1. Abbreviations and classifications for each antibody fragment studied during our project

|  |
| --- |
| Antibody 1 (AB1) = 4B53, Human IgG CH3 domain edit 4 |
| Antibody 2 (AB2) = 4B53, Human IgG CH3 domain edit 5 |
| Antibody 3 (AB3) = 1CQK, Mouse IgG CH3 domain edit 4 |
| Antibody 4 (AB4) = 1CQK, Mouse IgG CH3 domain edit 5 |
| Antibody 5 (AB5) = Human IgE CH3 domain edit 4 |
| Antibody 6 (AB6) = Human IgE CH3 domain edit 5 |
| **Antibody 7 (AB7) = 4B53, Human IgG CH3 domain wild type** |
| Antibody 8 (AB8) = 4B53, Human IgG CH3 domain edit 1 |
| Antibody 9 (AB9) = 4B53, Human IgG CH3 domain edit 2 |
| **Antibody 10 (AB10) = 1CQK, Mouse IgG CH3 domain edit wild type** |
| Antibody 11 (AB11) = 1CQK, Mouse IgG CH3 domain edit 1 |
| Antibody 12 (AB12) = 1CQK, Mouse IgG CH3 domain edit 2 |
| **Antibody 13 (AB13) = Human IgE CH3 domain wild type** |
| Antibody 14 (AB14) = Human IgE CH3 domain edit 1 |
| Antibody 15 (AB15) = Human IgE CH3 domain edit 2 |

### Cloning Antibody Fragments into pEHISTEV

The antibody wild-type CH3 domains and the four mutant edits for each antibody-fragment were successfully cloned into the pEHISTEV plasmid. The pEHISTEV plasmid is designed to express proteins with a His-tag that is removable by the TEV protease. The expression is under a T7 promoter and control by the lac repressor. The plasmids were first transformed into SoluBL21 E. coli cells and the success of the cloning was determined by digestion with restriction enzymes and colony-PCR.

The re-digestion of AB3, AB4, AB5 and AB6 by BamHI and NcoI restriction enzymes that were used for the initial double-digest showed the correctly sized fragments at ~350 base pairs and 6.5 kilobases.

![](https://static.igem.org/mediawiki/2019/c/cf/T--St_Andrews--Re-run_of_gel_with_well_3.jpg)

Figure 1. Digestion of the antibody fragments cloned into pEHISTEV using the restriction enzymes that were used of the initial double digest. All four cloned fragments showed correctly sized inserts, so the cloning was successful.

The cloning was confirmed by colony-PCR for AB3, AB4, AB5 and AB6 as well, where primers on the two ends of our inserts were used to PCR-up ~350 base-pair long fragments.

![](https://static.igem.org/mediawiki/2019/3/37/T--St_Andrews--Colony_PCR_for_AB_3_4_5_and_6.jpg)

Figure 2. Colony PCR for the four cloned antibody fragments, showing correct colonies for AB3 in lane 2, for AB5 in lane 6 and for AB6 in lane 8. AB4 has no correct colonies based on this colony PCR.

The colonies used for inoculation for 3, 5, and 6 were correct for these fragments based on colony-PCR, but AB4 showed no amplified product.

![](https://static.igem.org/mediawiki/2019/f/f9/T--St_Andrews--More_colony_PCR_for_AB4.jpg)

Figure 3. AB4 colony PCR showing multiple amplified products, including ones around 1500 base pairs, while the expected product is around 350 base pairs. It is possible that none of the screened colonies contain correct ligations but nonspecific primer binding can also be an issue.

More colonies were screened, buts Since no colonies showed only one amplified product of the correct size, the extracted plasmids were digested by an restriction enzyme (AflII) which should produce three fragments (4 kilobases, 1.5 kilobases and 470 bases) for our plasmids to see if the extra amplified fragment of 1.5 kilobases is made due to improper ligation or non-specific primer-binding.

![](https://static.igem.org/mediawiki/2019/0/06/T--St_Andrews--AB4_plasmid_digestion_on_right_of_the_gel_AflII_-_4kb%2C_1.5_kba%2C_470_ba.jpg)

Figure 4. AFlII digestion for AB4 cloned into pEHISTEV. The digestion produced three fragments of the correct sizes (4000, 1500 and 470 base pairs, so the ligation was correct.

The digestion showed correctly sized inserts, so these plasmids were used for future transformations.

The cloning success was determined by colony PCR for all the other antibody fragments, where similar primers annealing to the end of our inserts were used to asses the presence of a correct insert.

![](https://static.igem.org/mediawiki/2019/e/e4/T--St_Andrews--Colony_PCR_for_antibody_fragments-tile.jpg)

Figure 5. Colony PCRs for AB7, AB10, AB1, AB2, AB8, AB9, AB11, AB12, AB13, AB14 and AB15. All antibody fragments have at least one colony with the correct cloning, based on the correctly amplified ~350 bas pairs long fragments, these will be used for future inoculation steps.

All the antibody fragments were cloned correctly in pEHISTEV so expression was carried out using SoluBL21 cells that express sequences under the T7 promoter.

The cloning success was determined by sequencing for AB7 as well, where the T7 promoter was used as a sequencing primer to examine the sequences outside the coding sequence as well. The sequencing results showed no point mutations and the AB7 plasmid showed a 100% correct sequence.

### Expressing Human IgG Wild Type (AB7)

After initial expression tests were unsuccessful due to probable problems with cell lines or IPTG, we focused our attention on expressing the Human IgG wild-type (AB7) and its mutants.

The ideal expression conditions were determined for the wild-type, using induction with IPTG at different time-intervals, temperatures and IPTG concentrations.

![](https://static.igem.org/mediawiki/2019/0/08/T--St_Andrews--Expression_test_by_Ulis_research_group.jpg)

Figure 6. Expression tests showing for the wild type (iGEM7) under different conditions. The protein produced the strongest band, hence expressed the best at 25°C overnight expression with 0.2 mM IPTG used for induction.

The expression test showed the highest amount of protein for AB7 at 25°C with 0.2 mM IPTG concentration at overnight so the one-litre protein-expressions were carried out using these conditions.

![](https://static.igem.org/mediawiki/2019/f/f0/T--St_Andrews--Human_IgG_wild_type_large_scale_expression.jpg)

Figure 7. Purification of the human IgG wild type using nickel affinity chromatography and TEV cleavage. The expressed protein is highly soluble and was purified well via this method. The reverse nickel purified fraction only contains one band at the expected 12 kDa mass, so pure recombinant protein was obtained.

The wild type (AB7) was expressed in a high amount in the one litre culture and most of the protein is in the soluble fraction, with the protein being only partially insoluble. The elution fraction contained a high amount of recombinant protein at 15 kDa, and the cleavage was successful, although partial, as the 3 kDa size decrease is visible in the post-cleavage fraction. The reverse nickel purified fraction contains pure, recombinant protein at 12 kDa. The identity was confirmed by electrospray mass-spectrometry with an expected mass of 12373.8 Da.

![](https://static.igem.org/mediawiki/2019/1/1a/T--St_Andrews--AB7_mass-spectrometry_results.jpg)

Figure 8. Mass-spectrum for the expressed and purified wild type human IgG fragment. The spectrum shows a very strong and clear signal at the expected molecular mass (12373 Da) which correspond to the recombinant protein.

The correct recombinant protein was expressed based on the SDS-Page gel and the mass-spectrometry results.

### Expressing Mutant Antibody Fragments

![](https://static.igem.org/mediawiki/2019/4/49/T--St_Andrews--AB_1_2_8_and_9_large_scale_culture_whole_cell_lysates.jpg)

Figure 9. Whole cell lysates showing clear bands at around 15 kDa for all mutant antibody fragment expressions. All four mutants expressed, but the solubility cannot be assessed based on this gel alone.

four IgG mutants (AB1, AB2, AB8 and AB9) expressed well and produced a double band at around 15 kDa. Edit 2 shows the strongest band, and hence probably the most protein. This culture was induced at a lower optical density (0.22 instead of >0.6) since the cells were not growing over a long period of time.

### Expressing Human IgG Edit 1 (AB8)

The mutant protein was induced under the same condition and the purified protein sample was analysed via mass-spectroscopy with an expected mass of 12303.64 Da.

![](https://static.igem.org/mediawiki/2019/c/cb/T--St_Andrews--AB8_mass-spectrometry_results.jpg)

Figure 10. Mass-spectrometry analysis of purified edit 1 sample. The spectrum showed no signal around the expected 12 kDa molar mass, only a contamination at 43 kDa that cannot be related to a dimeric state of the recombinant fragment based on molar mass.

Mass-spectrometry analysis only showed a contamination and no mutant protein present, even though the gel showed a correctly sized band.

### Expressing Human IgG Edit 2 (AB9)

AB9 was induced under the same condition and purified using the same procedures.

![](https://static.igem.org/mediawiki/2019/9/9f/T--St_Andrews--AB9_reverse_Ni-purification_fractions_gel.jpg)

Figure 11. Reverse nickel affinity purification for edit 2 recombinant protein expression. The elution fraction shows a band at around the expected 15 kDa molar mass, while the concentrated reverse nickel purified fraction shows an expected band at 12 kDa. Edit 2 expressed and was successfully purified by this method, although some contaminations are still present in the final fraction, around 40 kDa and 70 kDa.

Edit 2 was mostly insoluble and only a fraction of the expressed protein was in the soluble fraction, while the elution contained less protein than the wild type as well. The TEV cleavage is still visible, with a 3 kDa decrease in size from 15 kDa to 12 kDa between the elution fraction and the concentrated reverse Ni-purified sample. An impurity around 60 kDa is also present in the purified sample.

The mutant expressed in a smaller quantity but it was still analysed via mass-spectrometry, with an expected mass of 12261.56 Da.

![](https://static.igem.org/mediawiki/2019/5/58/T--St_Andrews--AB9_mass-spectrometry_results.jpg)

Figure 12. Mass-spectra for edit 2 showing a clear signal at the expected molecular mass at 12258.8. corresponding to a fragment with a disulphide bond. No secondary signal is present that would indicate isopeptide bond formation.

Mass-spectrometry confirmed the expression but the contamination is not visible so the sample was submitted to mass-spectrometry again the next day. The mass-spectrometry analysis required more sample than expected based on the concentration calculated from the absorption.

![](https://static.igem.org/mediawiki/2019/f/f5/T--St_Andrews--AB8_mass-spectrometry_results_2.jpg)

Figure 13. Second submission of the same sample for edit 2, on this spectrum the original signal corresponding to the recombinant protein is not visible. New signal for contamination is picked up around 68 kDa, but this cannot be corresponded to any multimeric state of the recombinant protein.

The contamination is visible on the spectrum but the mutant protein is no longer present in the same sample.

### Expressing Human IgG Edit 4 (AB1)

AB1 was induced under the same condition and purified using the same procedures.

![](https://static.igem.org/mediawiki/2019/b/b3/T--St_Andrews--Ab_1_reverse_Ni-purification_fractions_gel.jpg)

Figure 14. Reverse nickel purification of edit 4, including the nickel affinity purified elution fraction and the concentrated solutions after TEV protease cleavage. The gel shows no band in the 12-15 kDa range, so edit 4 might not have expressed at all.

The gel showed no clear band at the right size in any of the purified fraction, but the insoluble fraction contains a strong band of the right size. Edit 4 might be entirely insoluble so no mass-spectrometry sample was run.

### Expressing Human IgG Edit 5 (AB2) – Cysteine-free mutant

![](https://static.igem.org/mediawiki/2019/2/26/T--St_Andrews--AB2_Ni_purification_protein_gel.jpg)

Figure 15.Nickel affinity chromatography purification fractions for edit 5 expression. A clear and strong band is present in the soluble fraction at around 15 kDa, which is also present in a smaller quantity in the elution fraction. Edit 5 expressed well and Is soluble, although less so than the wild type.

The cysteine-free mutant was expressed under the same conditions, and the band corresponding to the mutant protein at 15 kDa is strong in the soluble fraction and present in the elution too. AB2 is soluble but it is expressed in a smaller quantity than the wild type.

![](https://static.igem.org/mediawiki/2019/a/ac/T--St_Andrews--AB2_reverse_Ni-purification_fractions_gel.jpg)

Figure 16. Reverse nickel purification for the expressed edit 5 solutions. No strong band associated with recombinant protein is visible in either of the purified or elution fractions, even though the elution showed clear bands previously. The 10x concentrated final sample shows a band at around 12 kDa where the recombinant protein is expected.

Edit 5 was only present in a very small quantity after TEV cleavage and reverse nickel chromatography, but the concentrated elution fraction still showed a correctly sized band at 12 kDa so a mass-spectrometry sample was run with an expected mass of 12229.52 Da.

![](https://static.igem.org/mediawiki/2019/8/87/T--St_Andrews--AB2_mass-spectrometry_results.jpg)

Figure 17. Mass-spectrometry result for edit 5. No signal is present around 12 kDa where our protein is expected, only one clear signal at 43320 kDa is present, which is most likely an E. coli protein.

The spectrum shows no recombinant protein present only a contamination at 43 kDa. Even though the gel clearly showed a band, no protein is picked-up by mass-spectrometry.

### Expressing the Human IgG Wild-Type and its Mutants

![](https://static.igem.org/mediawiki/2019/4/41/T--St_Andrews--SDS-Page_gel_with_all_the_purified_Human_IgG_fragments.jpg)

Figure 18. Expressed and purified wild type and mutant human IgG CH3 domains. The wild type is present in a high quantity, containing a small amount of un-cleaved protein, while only edit 1 (AB8) and edit 2 (AB9) show very weak bands at the correct size. Edit 4 (AB1) and edit 5 (AB2) show no band corresponding to recombinant protein. The gel also shows no difference for AB7 between a sample buffer with or without reducing agent.

A gel was run for all the expressed protein solutions. The wild type (AB7) produced a strong band corresponding the recombinant protein before TEV cleavage (~15 kDa) and after cleavage (~12 kDa). The mutant proteins were expressed in a significantly smaller quantity and this final gel only shows very weak bands for AB8 and AB9 while the AB1 and AB2 fractions contain no band at the correct size.

The mutant proteins expressed less than the wild type under the same conditions and were also less soluble.

Table 2. Concentrations for each expressed antibody fragment for the initial elution step after nickel affinity chromatography and for each concentrated reverse-nickel purified protein solutions. The wild type expressed in a much greater quantity while edit 2, edit 4 and edit 5 produced a similar amount of protein after nickel purification. Protein concentration is slightly higher for edit 1, but still lower than the wild type.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | **Wild type (AB7)** | **Edit 1 (AB8)** | **Edit 2 (AB9)** | **Edit 4 (AB1)** | **Edit 5 (AB2)** |
| **Elution (20-25 mL)** | 5.3 mg/mL | 0.70 mg/mL | 0.27 mg/mL | 0.21 mg/mL | 0.23 mg/mL |
| **2.5 mL concentrated** | - | 0.21 mg/mL | 0.30 mg/mL | 0.23 mg/mL | 0.15 mg/mL |
| **300 µL concentrated** | - | 0.17 mg/mL | 0.37 mg/mL | - | 0.21 mg/mL |
| **150 µL concentrated** | - | - | - | - | 0.32 mg/mL |

The mass-spectrometry analysis proved to be inconclusive for the mutants as only one mutant protein (AB9) was visible on the mass-spectra and only for during the first analysis. The AB8 and AB2 sample, as well as the second AB9 sample, contained correct bands on the SDS-Page gels that were not visible on the mass-spectra.

The cysteine-free edit (AB2) seemed highly soluble based on the first gel run after nickel chromatography but subsequent gels showed less and less protein after each purification or concentration steps as this protein precipitated from the solution.

No data supports the presence of an isopeptide bond in any of the human IgG CH3 domain edits based on SDS-Page gels and mass-spectrometry analysis.

### Expressing Mouse IgG CH3 Domain Wild-Types and Mutants in 10 mL Cultures

The mouse IgG CH3 domain wild-type and edit 2 showed no soluble protein expression based on small scale (10 mL) E.coli cultures with 0.2 mM IPTG overnight expression at 25°C.

The wild type expressed well, but the protein is insoluble based on small scale SDS-page gels (Figure 19.)

![](https://static.igem.org/mediawiki/2019/2/2c/T--St_Andrews--Antibody_10_Expression_Test.jpg)

Figure 19. Mouse IgG wild-type expression duplicates showing insoluble recombinant protein at 15 kDa and no soluble expression based on the soluble fraction.

Edit 2 is not visible on the gel even in the insoluble fraction, so if this protein expresses, it only does so in a very low quantity.

![](https://static.igem.org/mediawiki/2019/c/ca/T--St_Andrews--Antibody_12_Expression_Test.jpg)

Figure 20. Small-scale mouse IgG CH3 domain edit 2 expression, showing no clear band corresponding to a recombinant protein.

### Expressing Human IgE CH3 Domain Wild-Types and Mutants in 10 mL Cultures

Human IgE CH3 domain wild-type expression showed strong bands corresponding to insoluble protein expression, while no soluble recombinant protein is observable.

![](https://static.igem.org/mediawiki/2019/3/30/T--St_Andrews--Antibody_13_Expression_Test.jpg)

Figure 21. Human IgE wild-type expression, showing clear bands corresponding to insoluble recombinant protein for the induced fraction. Soluble recombinant protein expression is not visible on this gel.

None of the IgE CH3 domain mutants showed clear soluble protein expression based on the small-scale expression. All of the mutants expressed well insolubly, as strong bands are visible in the insoluble factions at 15 kDa corresponding to recombinant protein. As these proteins are insoluble no isopeptide bond formation is predicted.

![](https://static.igem.org/mediawiki/2019/5/55/T--St_Andrews--Antibody_5_Expression_Test-tile.jpg)

Figure 22. Human IgE CH3 domain mutant expressions. All mutant expressed well, but no soluble protein is visible on these gels. The strong band at 15 kDa corresponds to the recombinantly expressed protein in the insoluble fraction.

Thermatoga Results

### Background/Idea

To test the idea that substitution of an inert amino acid into a double salt bridge could induce isopeptide bond formation, we identified a protein with a double salt bridge motif, the beta-glucosidase enzyme from the thermophile *Thermatoga maritima* (figure 1), which we referred to as TMBG.

![](https://static.igem.org/mediawiki/2019/b/bb/T--St_Andrews--Thermotogamod.jpg)

Figure 1:Triple salt bridge motif in the beta-glucosidase from Thermatoga maritima (PDB ID: 2J77). Hydrogen bonds, forming salt bridges between residues, are shown as yellow dashed lines. Figure made using PyMol.

To induce and isopeptide bond, we created the substitutions R371M and R413M, with the intention that Methionine would occupy a similar space in the protein but not form Hydrogen bonds with any residues, preventing isopeptide bond formation. We also introduced the mutant R371M, R413Y to shield the bond from solvent with a Tyrosine.

### SDS-PAGE

As we supposed the correctly folded protein was soluble, we surmised that checking the mutant’s solubility would give a quick indicator of their ability to form isopeptide bonds. As a positive control, we expressed the wild type protein to check its solubility in our system.

Samples were prepared as outline in Methods. The wild-type protein was given to us in a pET28a vector, mutant sequences were ordered in from IDT then cloned into pEHisTEV vectors (see Methods). Expression was induced using 1mM iPTG.

![](https://static.igem.org/mediawiki/2019/0/0e/T--St_Andrews--ThermotogaWT.jpg)

Figure 2. shows soluble expression for the wild-type protein, the pET28a vector was known to have a leaky promoter so the bands at ~55kDa (expected mass 52kDa) indicate expression, as they are absent in lanes used for mOrange and Antibody expression (lanes 2-10).

Figures 3 shows expression tests for the TMBG mutants. There is no visible expression for TMBG R371M R413M (Edit 1) with no bands around the 55kDa mark. In both, expression for TMBG R371M R413Y (Edit 2) appears to be dominantly insoluble, with no discernible difference at 55kDa between the soluble bands but a large 55kDa band in the insoluble fraction.

![](https://static.igem.org/mediawiki/2019/5/57/T--St_Andrews--Thermotogamuta.jpg
)

Figure 3: Expression tests for TMBG Edit 1 (Lanes 2-5) and TMBG Edit 2 (Lanes 6-9). **Lane 1** = Ladder, **Lane 2** = Edit 1 uninduced soluble fraction, **Lane 3** = Edit 1 uninduced insoluble fraction, **Lane 4** = Edit 1 induced soluble fraction, **Lane 5** = Edit 1 induced insoluble fraction, **Lane 6** = Edit 2 uninduced soluble fraction, **Lane 7** = Edit 2 uninduced insoluble fraction, **Lane 8** = Edit 2 induced soluble fraction, **Lane 9** = Edit 2 induced insoluble fraction. Expression of TMBG Edit 2 is highlighted in the red circle.

Thus, as the wild-type TMBG is soluble but TMBG R371M R413Y is dominantly insoluble, we predict the mutant to not be capable of forming isopeptide bonds. Absence of expression for TMBG R371M R413Y means we can draw no conclusions as to its solubility.

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iGEM St Andrews 2019