iGEM 2017 — Team Sydney_Australia

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Sydney Australia/Notebook

* [Team](https://2017.igem.org/Team%3ASydney_Australia/Team)
  + [About Us](https://2017.igem.org/Team%3ASydney_Australia/Team)
  + [Attributions](https://2017.igem.org/Team%3ASydney_Australia/Attributions)
* [Design](https://2017.igem.org/Team%3ASydney_Australia/Design)
  + [Project Design](https://2017.igem.org/Team%3ASydney_Australia/Design)
  + [Applied Design](https://2017.igem.org/Team%3ASydney_Australia/Applied_Design)
  + [Integrated Human Practices](https://2017.igem.org/Team%3ASydney_Australia/HP/Gold_Integrated)
  + [Entrepreneurship](https://2017.igem.org/Team%3ASydney_Australia/Entrepreneurship)
  + [Patent Law](https://2017.igem.org/Team%3ASydney_Australia/Patent_Law)
* [Project](https://2017.igem.org/Team%3ASydney_Australia/Description)
  + [Description](https://2017.igem.org/Team%3ASydney_Australia/Description)
  + [Protocols](https://2017.igem.org/Team%3ASydney_Australia/Experiments)
  + [Safety](https://2017.igem.org/Team%3ASydney_Australia/Safety)
  + [Notebook](https://2017.igem.org/Team%3ASydney_Australia/Notebook)
  + [Results](https://2017.igem.org/Team%3ASydney_Australia/Results)
  + [Demonstrate](https://2017.igem.org/Team%3ASydney_Australia/Demonstrate)
* [Model](https://2017.igem.org/Team%3ASydney_Australia/Model)
  + [Our Modelling](https://2017.igem.org/Team%3ASydney_Australia/Model)
* [Communications](https://2017.igem.org/Team%3ASydney_Australia/Human_Practices)
  + [Human Practices](https://2017.igem.org/Team%3ASydney_Australia/HP/Silver)
  + [Engagement](https://2017.igem.org/Team%3ASydney_Australia/Engagement)
  + [Collaborations](https://2017.igem.org/Team%3ASydney_Australia/Collaborations)
  + [Interlab](https://2017.igem.org/Team%3ASydney_Australia/Interlab)
  + [Rate My Gel](https://2017.igem.org/Team%3ASydney_Australia/Rate_My_Gel)
* [Parts](https://2017.igem.org/Team%3ASydney_Australia/Parts)
  + [Our Parts](https://2017.igem.org/Team%3ASydney_Australia/Parts)
  + [Our Composite Parts](https://2017.igem.org/Team%3ASydney_Australia/Composite_Part)
  + [Our Basic Parts](https://2017.igem.org/Team%3ASydney_Australia/Basic_Part)
  + [Part Improvement](https://2017.igem.org/Team%3ASydney_Australia/Improve)
* [Medals](https://2017.igem.org/Team%3ASydney_Australia/Medals)
  + [Medals](https://2017.igem.org/Team%3ASydney_Australia/Medals)

![](https://static.igem.org/mediawiki/2017/9/93/T--Sydney_Australia--notebook_banner.png)

#### [Week 1: 24/04/2017-30/04/2017](#collapse1)

-The 2017 University of Sydney iGEM team met up for the first time (on a
public holiday)

-The project was introduced to us by our PI Nick Coleman as well as team
member Alex Kelly, who introduced the project to Nick at a BioFoundry
meeting.

#### [Week 2: 1/05/2017-7/05/2017](#collapse2)

Project design:

•This week the team discussed various insulin analogues on the market as
well as a new type of analogue which is starting to appear in the
literature. This ‘Single chain insulin’ keeps the C-peptide incorporated,
resulting in greater stability.

•We also discussed various expression systems for expressing our insulin.
Steph suggested expressing insulin in yeast. This was thought to be too
difficult as no one had much experience working with yeast.

•Alex referred to several scientific papers that summarised the expression
of insulin in the periplasm of E.coli - allowing for simpler purification
of insulin. He also suggested purification using a His-tag fused to the
protein.

•Various assays to test our insulin were compared. We compared antibody
assays (ELISA), cell culture glucose uptake assays, and an alternative C.
elegans assay where the bioactivity of the insulin can be monitored through
phenotype.

•The team agreed that we wanted to stay away from mice convulsion assays
due to ethical reasons.

#### [Week 3: 8/05/2017-14/05/2017](#collapse3)

Project design:

•This week, lead by our team legal expert Steph, the team looked at various
patented single chain and other insulin analogues and summarised their
features. We also looked through the patents for each of these insulins and
to determine which features are covered in the patent.

•We also discussed expression systems for our insulin analogues. Steph
suggested using Bacillus subtilis to secrete our insulin if the protein was
fused to a secretion tag.

-

Wet Lab:

•This week our PI Nick inducted us into the lab. He introduced us to the
other Honours and PhD students in the lab. We also met our team advisor
Mark.

#### [Week 4: 15/05/2017-21/05/2017](#collapse4)

Project design:

•Each member of the team received a free SnapGene license

•Our Secondary PI and protein expert, Jacqui Matthews, helped us design our
own single chain insulin analogue which we called Winsulin.

•The team narrowed down our research on insulin analogues and expression
and finalised which parts we plan to submit into the registry and/or
express.

•We planned to express 3 different insulins: Human Proinsulin, a single
chain insulin found in a paper called SCI-57, and our own single chain
insulin Winsulin.

•We plan to express each of these insulins in the cytoplasm of E. coli, the
periplasm of E. coli and to be secreted by Bacillus subtilis.

•Our parts each included a 6x His tag for purification. Insulins that will
be expressed in the periplasm will have an Ecotin ‘periplasmic-expression’
tag, and insulins that will be to be secreted will be fused to a YncM
‘Bacillus-secretion’ tag.

•This leaves 9 parts to be designed and synthesised. As a team of 8, each
team member was assigned a part. Our lab guru Jamie was assigned two of
these gBlocks to design as he was especially enthusiastic.

•We hope to order them next week.

#### [Week 5: 22/05/2017-28/05/2017](#collapse5)

Project design:

•The team worked really hard last week to finalise our gBlock parts. Rough
draft of the parts were made. Unfortunately, these still needed work. The
team spent the week fixing up the minor details such as codon optimisation
for E. coli and Bacillus subtilis and restriction enzyme recognition sites
in our gBlocks.

Education and Public Engagement:

•Courtney started up our team social media pages (Facebook, Twitter, and
Instagram) as well as our team email.

•The team agreed to participate in National Science Week at the Australian
Museum later in the year in cooperation with BioFoundry.

•Fortnightly Skype meeting with Open Insulin Team!

#### [Week 6: 29/05/2017-04/06/2017](#collapse6)

Project design:

•Our PI Nick suggested designing our own E.coli/Bacillus shuttle
replicative expression vector. However, we do not think that we will have
time to construct it. This could be a plasmid we design for future USYD
iGEM teams to construct.

•Instead due to time constraints, our B.subtilis expression vector ended up
being an integrative plasmid called pUS258, designed by Yanwei - a PhD
student in our lab. Nick decided to use pET15b as our expression vector in
E. coli.

Human practices:

•The team explored various human practices approaches for the project,
including researching various charity organisations that focus on insulin
accessibility that we could get in contact with over the course of the
project.

•Emaleen suggested Health Action International and the 100 Campaign.

•Alex suggested getting in contact with Insulin for Life.

•Courtney suggested contacting the Australian government to discuss the
Pharmaceutical Benefits Scheme which currently covers most of the insulin
costs in Australia.

#### [Week 7: 05/06/2017-11/06/2017](#collapse15)

Project design:

•This week Alex prepared a flow chart as an overview of our project. This
can be seen below.

•Steph brought forward the idea of testing the functionality of our insulin
using adipocyte cells in a glucose uptake assay and consulted a few insulin
experts in other labs.

-

Human practices:

•Sent emails to Health Action international and the World Health
Organisation in attempts to hear their opinions on insulin inaccessibility
world wide.

•Fortnightly Skype meeting with Open Insulin Team!

#### [Week 8: 12/06/2017-18/06/2017](#collapse7)

Project design:

•This week we finally placed the order with IDT for synthesis of our
designed gBlocks!

Wet lab:

•This week we finally got started in the wet lab. With gBlocks still on
the way, the 8 of us spent time making up the essential media needed for
future lab work.

•This included LOTS of LB broth, LB Agar, M9 minimal media, RF1 and RF2
etc.

#### [Week 9: 19/06/2017-25/06/2017](#collapse8)

Wet lab:

•Erica and Emaleen did plasmid preps on JM109 cells which contained our
expression vector pET15b.

•Jamie and Steph digested the pET15b stock and ran it on an agarose gel in
order to quantify the concentration.

Human practices:

•Fortnightly Skype meeting with Open Insulin Team! We were getting excited
to show them our first results.

#### [Week 10: 26/06/2017-02/07/2017](#collapse9)

Wet lab:

•Our gBlocks finally arrived!!!

•Alex and Jamie digested our pSB1C3 stock and ligated the cytoplasmic and
Ecotin-tagged gBlocks into them.

•Erica, Courtney and Grace transformed these vectors into TOP10 E.coli
cells – colonies grew for each of the transformations.

#### [Week 11: 03/07/2017-9/07/2017](#collapse10)

This was the start of our winter break – however, the team took advantage
of the break and planned to get a lot of lab work done.

Wet lab:

•Grace and Emaleen performed a screening colony PCR on the transformations
from last week for verification.

•Ruby and Courtney ligated our Bacillus secretion gBlocks (YncM tagged)
into pSB1C3.

•Before transforming these vectors into TOP10 E.coli, we had to make a new
batch of these cells competent. Alex was in charge of this.

#### [Week 12: 10/07/2017-16/07/2017](#collapse11)

Wet lab:

•Jamie redid the colony screening PCR on the TOP10 E.coli transformed with
our cloned pSB1C3 vectors from last week in order to get a clearer gel. The
results showed that Cytoplasmic Winsulin, Ecotin Winsulin and Ecotin
Proinsulin in pSB1C3 need to be retransformed into TOP10 E.coli.

•Steph transformed the pSB1C3 vectors with the YncM gBlocks inserted last
week into TOP10 E.coli. These transformations did not work and we believe
that it is due to a problem with the ligation of the gBlock into pSB1C3.

#### [Week 13: 17/07/2017-23/07/2017](#collapse12)

Wet lab:

•Jamie redid the ligation of the YncM gBlocks into pSB1C3 and transformed
these vectors into TOP10 E.coli.

•Cytoplasmic Winsulin, Ecotin Winsulin and Ecotin Proinsulin were
re-ligated into in pSB1C3 and transformed into TOP10 E.coli by Ruby. Ecotin
Proinsulin and Ecotin Winsulin still didn’t appear positive on the agarose
gel.

•Steph did plasmid preps on TOP10 E.coli transformed with pSB1C3::RFP to
regenerate our stock of this plasmid

#### [Week 14: 24/07/2017-30/07/2017](#collapse13)

Wet Lab:

•Erica and Alex reattempted transforming pSB1C3::Ecotin Proinsulin and
pSB1C3::Ecotin Winsulin. These transformations worked!

#### [Week 15: 31/07/2017-06/08/2017](#collapse14)

•This week was the first week of semester 2 and the team were all back from
holidays and ready to continue work both in and out of the lab.

Wet lab:

•We received the WB800 strain of Bacillus subtilis which has 8 proteases
knocked out. This was kindly donated to us by Sui-Lam Wang’s lab from the
University of Calgary, Canada.

•Now that it appears that all 9 gBlocks in pSB1C3 have been transformed
into TOP10 E.coli, Steph and Emaleen performed a colony screening PCR on a
few colonies of each transformation to verify presence of the gBlock.

•Alex and Grace cloned all 9 Ecotin and Cytoplasmic gBlocks into pET15b.

Human practices:

•Courtney emailed Insulin for Life for more detail on what they do and what
they perceive to be the biggest problems contributing to insulin
inaccessibility.

•Fortnightly Skype meeting with Open Insulin Team started back up again! We
had some excellent progress to show them.

#### [Week 16: 07/08/2017-13/08/2017](#collapse100)

Lab work:

•Based on the colony PCRs from last week, Emaleen and Steph performed
plasmid preps on those TOP10 E.coli which showed a positive result in the
colony PCR last week.

Human practices:

•Dr David Beran from Health Action International replied to our email
asking for further information on the current issues with insulin
accessibility. Alex organised a time to skype him later this month.

-

Education and Public Engagement:

•Grace wrote up an Insulin Accessibility Survey and made it public on our
social media and the iGEM collaborations page.

•The team took shifts at the Joint Academic Microbiology Seminars (JAMS)
stall at the Australian Museum for National Science week.

#### [Week 17: 14/08/2017-20/08/2017](#collapse101)

Wet lab:

•Emaleen, Ruby and Grace performed double digests on the prepped pSB1C3
plasmids using the restriction enzymes EcoRI and Pst1. They then ran them
on an agarose gel to confirm that the plasmid contained an insert before
sending the plasmids off for sequencing. This had to repeated many times
until we finally got a good gel.

•Erica transformed the Ecotin and Cytoplasmic gBlocks that were ligated
into pET15b in Week 13 into pET15b. These transformations were successful.

•Steph and Courtney performed colony screening PCRs on these pET15b
transformations. Bands were not correct size

Education and Public Engagement:

•On the 18th of August, Steph participated in a webinar over Skype hosted
by the Uppsala iGEM team. In this webinar, five teams discussed the ethics
behind intellectual property and open-source science within the context of
the iGEM competition.

•The team continued doing shifts at the JAMS stall at the Australian Museum
for National Science week.

Human practices:

•Fortnightly Skype meeting with Open Insulin Team – this time joined by our
new collaborator in Ghent!

#### [Week 18: 21/08/2017-27/08/2017](#collapse102)

Wet Lab:

•Emaleen and Jamie amplified the gBlock insert in these prepped plasmids.
The gel showed correct bands following the double digest and PCR with high
fidelity polymerase, so they were finally sent off for sequencing. We will
need to re-ligate the other inserts in to pSB1C3 (AGAIN!)

•pSB1C3::Ecotin Proinsulin and pSB1C3::Cytoplasmic Proinsulin came back
with good sequencing results and these parts are now ready for submission
into the parts registry!!

•Alex and Courtney did plasmid preps on the positive TOP10 E. coli pET15b
transformations from the colony PCR performed last week.

Education and public engagement:

•The team set up a microbiology stand at the University of Sydney Open Day
where we spoke to future students about our experience doing science
degrees.

Human practices:

•Organised a Skype call with Dr David Beran on the 30th August

•This week we received advice from the university that we were at risk of
infringing patents if we went on to express SCI-57 and Winsulin. We
scrapped any work that had been done with these gBlocks.

•Steph came up with a design for ‘Winsulin 2.0’ that would not infringe any
patents. It was based on the design of Prof. Peter Arvan from the
University of Michigan. We contacted him and he gave us permission to
continue working with the sequence for Winsulin 2.0 in an open-source
setting as he had no patent claims on it.

•Courtney contacted a patent attorney, Len Mancini, who works at Maxwell's
for advice about our newly designed Winsulin 2.0.

#### [Week 19: 28/08/2017-03/09/2017](#collapse103)

Wet lab:

•We performed double digests on the prepped pET15b plasmids from last week
to verify insertion. We amplified the inserts of plasmids which, according
to our double digests, contained the correctly sized inserts. These
amplified inserts were then sent off for sequencing.

•In the meantime, we transformed the pET15b plasmids into BL21 cells to
start expression. These transformations were unsuccessful and will need to
be done again.

•

Human Practice:

•Alex Skyped Dr David Beran from Health Action International to discuss the
ACCISS reports and his research on insulin accessibility. It was an
extremely productive and informative discussion.

#### [Week 20: 4/09/2017-10/09/2017](#collapse105)

Wet lab:

•Sequencing from last week was entirely bad quality. We determined to send
off PCRs rather than whole plasmid, so Jamie amplified the inserts in the
pET15b vector and sent them off for sequencing again.

•Courtney also redid the transformations of pET15b-gBlock into BL21, as
they didn’t work last week. This time we finally got some successfully
transformed cells!

•Emaleen performed colony PCR on these transformants and correctly sized
bands were seen. We hope to start induction next week.

Education and Public Engagement:

•Courtney, Grace, Emaleen and Jamie gave a talk about iGEM and our project
at an event hosted by the USYD Biomod team. These excellent scientists are
entered in the Biomod competition which focuses on biomolecular design.

#### [Week 21: 11/09/2017-17/09/2017](#collapse106)

Wet Lab:

•Alex made competent DH5α cells to use in the Interlab study.

•Erica started transforming the test constructs into DH5α and performing
colony PCRs on them for verification of transformation

•Emaleen amplified the YncM proinsulin gBlock cloned into pSB1C3 for
sequencing

•We received new primers that bind either side of the gBlock inserts in
pET15b.

•Alex did IPTG induction on BL21 cells transformed with pET15b containing
Ecotin and Cytoplasmic gBlocks.

-

Human Practices:

•Len Mancini, the patent attorney Courtney contacted a few weeks ago has
agreed to assist us with looking through patents and our Winsulin 2.0
sequence

•Courtney called Neil from Insulin for Life to discuss what goes on behind
the scenes at their organisation and to learn about the struggles of
sending insulin to impoverished countries with poor distribution systems.

#### [Week 22: 18/09/2017-24/09/2017](#collapse1012)

Wet Lab:

•Erica and Emaleen finalised the transformation of Interlab study test and
control plasmids into DH5α cells and performed a colony PCR to verify this
transformation

![](https://static.igem.org/mediawiki/2017/3/32/T--Sydney_Australia--InterlabcolonyPCR1.png)

#### [Week 23: 25/09/2017-1/10/2017](#collapse107)

Wet lab:

•Emaleen and Erica performed the fluorescence and OD600 readings on the
cells for the Interlab study and submitted last minute on Friday!

•Courtney and Alex cloned Ecotin-Winsulin 2.0, YncM-Winsulin 2.0 and
Cytoplasmic-Winsulin 2.0 into pSB1C3 and transformed them into E.coli
TOP10, while simultaneously performing the ligation into pET15b.

•Steph and Grace transformed pET15B::Cytoplasmic-Winsulin 2.0 and
pET15b::Ecotin-Winsulin 2.0 into TOP10 E. coli.

•We ordered precast SDS-PAGE gels as our own gel casting skills left a lot
to be desired.

#### [Week 24: 2/10/2017-8/10/2017](#collapse108)

Wet lab:

•Erica and Alex performed colony PCRs on the pSB1C3 and pET15b plasmids
transformed into TOP10 E.coli last week.

•Many many gels were run and eventually we saw at least one positive result
for each colony PCR. Below is just two of these gels loaded with the PCRs
performed on the pET15b with Ecotin proinsulin inserted

![](https://static.igem.org/mediawiki/2017/5/5d/T--Sydney_Australia--wk24.png)

#### [Week 25: 9/10/2017-15/10/2017](#collapse109)

Wet lab:

•Jamie sent some pSB1C3 submission vectors, some pET15b E. coli expression
vectors, and the pUS258 B. subtilis expression vectors off to sequencing to
verify correct gBlock insertion. Got verification of correct insertion so
that we can progress with submission into the parts registry and also
verification that we have the correct inserts in our expression vectors.

•Alex performed autoinduction on our BL21 cells transformed with our pET15b
expression vectors with with our Cytoplasmic and Ecotin gBlock inserts.

•He then did periplasmic chloroform extraction for the cells transformed
with the Ecotin constructs and cytoplasmic extraction for those with the
cytoplasmic constructs and ran them on an SDS-PAGE gel. We did not get
conclusive results from this method, partly due to a disputable negative
control. Will go back to IPTG induction.

Human Practices:

•Grace, Alex and Emaleen met with USYD bioethics expert Dr Narcyz Ghinea
who specialises in the ethics behind high cost medicines. We discussed
issues with how medicines are priced and the costs of going through
clinical trials.

Education and Public Involvement:

•We were visited in the lab by a group of year 10 girls from Mt St
Benedicts school. Our PI Nick spoke to them about microbes in our
environment and Ruby, Grace and Emaleen taught them how to gram stain and
use a microscope.

#### [Week 26: 16/10/2017-22/10/2017](#collapse1011)

Wet lab:

•Emaleen and Grace made competent B. subtilis WB800 cells.

•Erica transformed our bacillus expression vectors pUS258::Yncm-Winsulin
and pUS258::Yncm-Proinsulin into our competent WB800 B. subtilis cells.
These transformants were patched to a starch plate to identify where the
gene had been correctly integrated into the genome:

![](https://static.igem.org/mediawiki/2017/b/b2/T--Sydney_Australia--wk26.jpeg)

WB800 pUS258::YncM proinsulin

![](https://static.igem.org/mediawiki/2017/9/9a/T--Sydney_Australia--wk26YncMWinsulin.jpeg)

WB800 pUS258::YncM Winsulin

•Erica and Jamie transformed our sequence verified pET15B with Ecotin
tagged gBlocks into E.coli expression vectors into BL21.

•We decided to express the cytoplasmic gBlocks in the SHUFFLE strain as
this strain will aid in the folding of cytoplasmic proteins.

•Alex and Courtney IPTG induced transformed B. subtilis and E.coli cells
transformed with our pET15B and pUS258 expression vectors. Grace and Alex
extracted the proteins and His-tag purified them, then and ran non-His tag
purified and His-tag purified samples on SDS-page gels. This didn’t give
any indication of protein expression. Alex and Grace then ran whole cell
lysates of these induced cells on a gel. It was still difficult to see
whether any protein expression has been induced

![](https://static.igem.org/mediawiki/2017/a/a8/T--Sydney_Australia--wk26SDSpage.png)

#### [Week 27: 23/10/2017-29/10/2017](#collapse1000)

Wet Lab:

•Grace continued to run SDS-PAGE gels on our induced cell lysates for
insulin detection.

•We received the last of the sequencing data which correctly verified our
gBlocks were correctly inserted into pSB1C3.

•Jamie did one final double digest for our parts and found that 5/6 of them
were able to be submitted. YncM proinsulin could not be submitted as the
double digest resulted in multiple bands. The parts were sent off on the
27th of October.

![](https://2017.igem.org/File:T--Sydney_Australia--wk27doubledigestparts.png)

•Jamie, Emaleen, Ruby and Steph performed an ELISA and glucose uptake assay
on our cell lysates.

•The sandwich ELISA test detected insulin in the induced cell lysates
expressing parts Cytoplasmic Proinsulin, Ecotin Winsulin, Cytoplasmic
Winsulin and YncM Winsulin. The visible colours that appeared at the end of
this ELISA was single handedly the most exciting moment of this project.

•The Glucose uptake assay showed some increased activity, however we were
hesitant to draw a strong conclusion from this before showing to some
experts.

Integrated Human practices/Entrepreneurship:

•The team met with Mike Nicholls at the USYD start up hub Incubate as well
as Main Sequence Ventures to discuss the future of our project.

#### [Week 28: 30/10/2017-05/11/2017](#collapse10002)

Day and night (what, what)
I toss and turn, I keep stressing my mind, mind (wiki, wiki)
I look for peace but see I don't attain (wiki, wiki)
What I need for keeps this silly game we play, play
Now look at this (wiki, wiki)
Madness to magnet keeps attracting me, me (wiki, wiki)
I try to run but see I'm not that fast (wiki, wiki)
I think I'm first but surely finish last, last

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# Team:Sydney Australia/Experiments

* [Team](https://2017.igem.org/Team%3ASydney_Australia/Team)
  + [About Us](https://2017.igem.org/Team%3ASydney_Australia/Team)
  + [Attributions](https://2017.igem.org/Team%3ASydney_Australia/Attributions)
* [Design](https://2017.igem.org/Team%3ASydney_Australia/Design)
  + [Project Design](https://2017.igem.org/Team%3ASydney_Australia/Design)
  + [Applied Design](https://2017.igem.org/Team%3ASydney_Australia/Applied_Design)
  + [Integrated Human Practices](https://2017.igem.org/Team%3ASydney_Australia/HP/Gold_Integrated)
  + [Entrepreneurship](https://2017.igem.org/Team%3ASydney_Australia/Entrepreneurship)
  + [Patent Law](https://2017.igem.org/Team%3ASydney_Australia/Patent_Law)
* [Project](https://2017.igem.org/Team%3ASydney_Australia/Description)
  + [Description](https://2017.igem.org/Team%3ASydney_Australia/Description)
  + [Protocols](https://2017.igem.org/Team%3ASydney_Australia/Experiments)
  + [Safety](https://2017.igem.org/Team%3ASydney_Australia/Safety)
  + [Notebook](https://2017.igem.org/Team%3ASydney_Australia/Notebook)
  + [Results](https://2017.igem.org/Team%3ASydney_Australia/Results)
  + [Demonstrate](https://2017.igem.org/Team%3ASydney_Australia/Demonstrate)
* [Model](https://2017.igem.org/Team%3ASydney_Australia/Model)
  + [Our Modelling](https://2017.igem.org/Team%3ASydney_Australia/Model)
* [Communications](https://2017.igem.org/Team%3ASydney_Australia/Human_Practices)
  + [Human Practices](https://2017.igem.org/Team%3ASydney_Australia/HP/Silver)
  + [Engagement](https://2017.igem.org/Team%3ASydney_Australia/Engagement)
  + [Collaborations](https://2017.igem.org/Team%3ASydney_Australia/Collaborations)
  + [Interlab](https://2017.igem.org/Team%3ASydney_Australia/Interlab)
  + [Rate My Gel](https://2017.igem.org/Team%3ASydney_Australia/Rate_My_Gel)
* [Parts](https://2017.igem.org/Team%3ASydney_Australia/Parts)
  + [Our Parts](https://2017.igem.org/Team%3ASydney_Australia/Parts)
  + [Our Composite Parts](https://2017.igem.org/Team%3ASydney_Australia/Composite_Part)
  + [Our Basic Parts](https://2017.igem.org/Team%3ASydney_Australia/Basic_Part)
  + [Part Improvement](https://2017.igem.org/Team%3ASydney_Australia/Improve)
* [Medals](https://2017.igem.org/Team%3ASydney_Australia/Medals)
  + [Medals](https://2017.igem.org/Team%3ASydney_Australia/Medals)

![](https://static.igem.org/mediawiki/2017/f/f9/T--Sydney_Australia--ProtocolBanner.gif)

General Protocols

E. Coli Protocols

B. Subtilis Protocols

#### [Making Agar Plates](#collapse1)

1. Figure out the total number of plates you will need, then multiply
   x 25 to get the approx. amount of agar you will need (in ml). Its
   better to make more than you think you need ! Alternatively, just
   make up the agar in multiples of 400 ml, this a standard amount
   that will make ~16 plates (this is about right for one ‘sleeve’ of
   sterile petri dishes, which have 20 dishes in them).
2. Add all the required media ingredients (eg. for LB agar, this is 10
   g tryptone, 5 g yeast extract and 5 g of NaCl per litre) – make
   sure you correct these ingredient weights for the amount of agar
   you are making (e.g. multiply by 0.4 if you are only making 400
   ml).

1. DON’T ADD THE AGAR YET ! (this doesn’t dissolve, it settles
   out)
2. DON’T ADD ANTIBIOTICS, TRACE METALS, or TWEEN yet, if these
   ingredients are needed.

3. Dispense the media into multiple lots of 400 ml in 500 ml media
   bottles. Make sure that the media bottles have a plastic pouring
   ‘lip’ on them, not just a naked glass top; the lip is important to
   be able to pour the agar neatly. You could also make multiples of
   200 ml in 250 ml bottles, but don’t make >400 ml in a 1 L bottle
   – this is too heavy to easily pour plates with.
4. Once you have dispensed the media into the bottles, now you can add
   the appropriate amount of agar to each bottle. This is 17 grams per
   litre (=6.8 grams per 400 ml). DON’T USE AGAROSE!
5. Add bottle caps. Don’t screw these on all the way, leave them a bit
   loose. Hold the caps in place with a small piece of autoclave tape.
6. Arrange bottles of agar in a wire rack, add a piece of masking tape
   on one corner with your name and the lab number (566), then take to
   the autoclave. If the agar media will not be autoclaved on the same
   day, keep it in the cold room until the next day (microbes will
   start to grow within a few hours, especially in rich media like LB)
7. When the agar comes out of the autoclave, if its still molten and
   very hot, put it in the hot water bath (~60°C) until it cools down
   to pouring temperature (~15 min). If you don’t want to use it
   immediately, leave on the bench until it solidifies (if it hasn’t
   already), then tighten the cap and it can be stored indefinitely at
   room temp. If agar has solidified and you want to use it straight
   away, microwave it (lid loose!) for approx 10 min on 50% power
   (these settings are for 400 ml of agar at room temp, adjust
   accordingly!), then put in hot water bath for ~15 min.
8. Prepare laminar flow hood for plate pouring as follows: give it a
   blast of UV for ~15 min (switch in upper position, with the doors
   closed!), then turn off UV light (switch in middle position), take
   out the doors, then turn on the light and fan (switch in bottom
   position). Swab down with 80% ethanol.

1. (optional). If you are working with very slow-growing microbes
   and/or you are making rich medium non-selective plates, you can
   add an extra layer of sterility to the proceedings by wearing
   gloves, and rinsing the gloves thoroughly in 80% ethanol before
   you start pouring the plates.
2. (optional). If you don’t have access to the laminar flow hood,
   you can make plates on your regular lab bench. This is usually
   fine, but swab down the bench with ethanol first, and work
   close to the Bunsen flame. (if you swab your gloves with
   ethanol, be careful to let this evaporate before turning on the
   Bunsen burner!)

9. Collect one bottle of molten agar from the waterbath, wipe down the
   outside with paper towel, and take to your bench. Add any
   “after-autoclaving” additions at this stage, from filter-sterilised
   stock solutions (this may be antibiotics, or trace metals solution,
   or Tween etc). Mix by swirling for 10-20 seconds, don’t shake the
   bottle as this will cause persistent bubbles. Its good practice to
   write all the additions on the bottle, then tick them off after
   each addition. Forgetting these is bad!
10. Take the final agar to the laminar flow hood. Open up one sleeve of
    plates (at the ‘base’ end), and slide them out. Keep the sleeve!
    Label all the plates with the type of medium (e.g LB-Cm25). Pour
    the agar into the first plate; use a single smooth pouring motion.
    Stop when it reaches all the edges. Put the lid on. Do the next
    plate. Repeat. Don’t stack up the plates in big towers, they will
    take longer to cool.
11. Leave the plates for about 10 min to alow the agar to solidify.
    Then invert the plates with the lids off to allow them to dry for
    20-30 min. The setup should look like this:

![](https://lh6.googleusercontent.com/XuHm7_IRDa0Fzbst5RfRPn0jt9uSttbewv6KtI8FgZbzoH2kvaMFceXTNgtLTlU0EyFQuEBFh1p6IyoBbOblaEaQKqCcS1AHNYpDMt0GAMOrbeOI_-SPT6_VCJaMZ820zQMo3Sjz)

12. If you don’t dry the plates, you won’t get nice spread-plates, and
    the risk of contamination is greater from other microbes ‘climbing’
    into the plate over the edge.
13. Pack the plates back into the sleeve, then label the sleeve with
    the type of agar and the date of manufacture and your name. (don’t
    label the individual plates with the date, this is confusing, since
    the date written on an agar plate is usually the date it was
    inoculated).
14. Store plates in the cold room. They should stay good for >6
    months if you have been careful!

#### [Restriction Enzyme Digest](#collapse2)

NOTES: First quantify the plasmid (by gel comparison, not nanodrop!), and
quantify the insert DNA (usually a column-purified PCR product; you can use
nanodrop for this) then set up digests, as below. A typical setup would be
250 ng plasmid and 250 ng insert in digest volume of 100 ul (these numbers
used below), but range may be 100-1000 ng of each DNA, in a volume of 30 –
200 ul.

Check which restriction buffer is appropriate. For double digests, it’s OK
to use a buffer which gives 100% activity for one enzyme, and 75% activity
of the other, but lower than this is not good. Also check the appropriate
digest temperature, its usually 37°C, but not always.

DIGEST OF PLASMID

* Calculate how much volume of plasmid you need to use to get 250 ng
  – call this ‘X’.
* Calculate how much water to add to the digest; this is (100 – 10 –
  2 – X) – call this ‘Y’.
* Retrieve 10 x restriction buffer from freezer, thaw completely, and
  vortex to mix. Ensure the buffer is fully thawed. The same tube of
  buffer can be used many times, if you are careful with your aseptic
  technique. The buffer doesn’t have to be kept cold.
* Retrieve the plasmid from the freezer, allow to thaw, (e.g. in 37°C
  waterbath, or rub in your hands, or on bench etc), then put it on
  ice when it is thawed. Its not good to leave the plasmid stock at
  room temp or above for prolonged periods or it may degrade due to
  traces of nucleases.
* Retrieve the restriction enzyme(s) from the freezer, put
  IMMEDIATELY on ice. These are heat-sensitive and you need to look
  after them. Do not leave them at room temp. Keep on ice while
  setting up the reaction, then immediately put back in freezer.
  These don’t need to be thawed, they are in a glycerol solution
  which doesn’t freeze at -20°C.
* Label your tube(s), then set up the digest(s) by adding the
  ingredients in the following order. Make sure you use excellent
  aseptic technique, and change tips every time. It's OK to set this
  up at room temp, the reaction tube doesn’t have to be on ice.

+ 10 ul of 10x restriction buffer
+ ‘Y’ ul of sterile MQ water
+ ‘X’ ul of plasmid DNA
+ 2 ul of (each) restriction enzyme

------------------------------------------------

Total 100 ul

* Mix by flicking, then tap on bench to get liquid to bottom of tube.
  Incubate at correct temperature for approx. 2 hours. (1 - 4 hr is
  OK, but overnight digest is too long; this can lead to ‘raggedy
  ends’ of the plasmid even if it looks OK on a gel; this is due to
  non-specific nuclease activity)

NOTE: 100 ul seems like a large volume to use, but this helps dilute any
impurities in the plasmid prep. Increasing the total volume of the digest
and/or reducing the volume of plasmid added often help to improve the
quality of a poor digest. If the digested plasmid is for the purpose of
ligation, we can use a large volume, since it will be column-purified &
concentrated anyway.

DIGEST OF INSERT DNA

1. Calculate how much volume of insert DNA you need to use to get 250
   ng – call this A.
2. Calculate how much water to add to the digest; this is (100-10-2-A)
   – call this B.
3. Label your tube(s). Set up digest by adding ingredients in the
   following order. Make sure you use excellent aseptic technique, and
   change tips every time. Its OK to set this up at room temp.

* 10 ul of 10x restriction buffer
* ‘A’ ul of sterile MQ water
* ‘B’ ul of insert DNA
* 2 ul of (each) restriction enzyme

------------------------------------------------

Total 100 ul

4. Mix by flicking, then tap on bench to get liquid to bottom of tube.
   Incubate at correct temperature for approx. 2 hours. (1 - 4 hr is
   OK, but overnight digest is too long)
5. Dephosphorylate the vector by adding 11 ul of 10x phosphatase
   buffer and 2 ul of antarctic phosphatase enzyme, incubate 30 min at
   37°C. Note that you don’t need to purify the DNA between digestion
   and dephosphorylation, but you need to top up the buffer with the
   specific phosphatase buffer, which adds Zinc. This step prevents
   the vector religating to itself, and is essential for cloning when
   a single restriction enzyme is used to cut on both sides; it is not
   required (in theory!) when you are cloning with two different
   restriction enzymes.
6. 6. Purify both vector and insert DNA using Qiaquick columns (or
   similar), elute in 15 ul EB.

#### [Purification of DNA samples](#collapse3)

1. Mix your DNA sample with the appropriate buffer, in the appropriate
   ratio:

* DNA < 4 kb: Mix 1 vol sample with 3 vol of QG buffer
* DNA > 4 kb: Mix 1 vol sample with 3 vol of QG buffer + 1 vol
  isopropanol\*\*\*
* PCR products: Mix 1 vol sample with 5 vol PB buffer.

\*\*\* For large DNA fragments in TAE-agarose, DON’T add the isopropanol
immediately here. First melt the agarose in the QG with heating (60 C for 5
min is usually. enough, mix occasionally), then when it’s all dissolved,
allow to cool to room temp, then add the isopropanol.

2. Load the mixture onto a Qiaquick spin column (purple) and spin 30
   sec 10,000g.
3. Discard the flow-through, and replace spin column in the catch
   tube.
4. The spin column will hold a max. of 800μl sample and has a max.
   Binding capacity of approx 10 μg DNA. You can wash through multiple
   800 μl aliquots of DNA+QG if you have a lot of sample, so long as
   the total amount of DNA added doesn’t exceed approx 10 μg per
   column. (if it does, this is not a tragedy, it just means you will
   lose some of your DNA)
5. Add 750 μl of buffer PE to the column, allow to sit for ~2 min, then
   spin 30 sec, discard flow-through, replace spin column in catch tube.
6. Spin again for 30 sec to remove all traces of PE from the column.
   Discard both the flow-through and catch tube, and transfer the spin
   column onto a clean Kimwipe. Leave the column lid open. Transfer
   Kimwipe to 50°C incubator box, and allow to dry for 10 min.
7. Transfer spin column to a sterile 1.5 ml Eppi tube, and add 50 μl\* of
   EB buffer (5 mM Tris, pH 8) to the centre of the spin column – ie on
   the membrane, not the walls of tube. Allow to sit for 5 min. Spin 30
   sec, retain Eppi tube with DNA solution in EB, discard spin column.

\* Can reduce this to as little as 15 μl EB to give a more concentrated DNA
solution (you will lose approx 3-5 μl EB during the procedure). Using
larger volumes of EB will increase the overall yield of DNA, while using
smaller volumes will increase the concentration of DNA. Which one is better
depends on what you are using the DNA for…usually for ligations,
concentration is more important than yield, so go with the 15 µl amount.

#### [Ligation](#collapse4)

1. Retrieve one 10x ligase buffer aliquot from the -20°C freezer. This
   should be a small amount of buffer (e.g. 10 µl) in a small generic
   tube, not the large tube from the manufacturer with ~1 ml of
   buffer. If there are no ligase buffer aliquots prepared, make these
   by aliquoting multiple 10 µl lots into small Eppi tubes, and freeze
   at -20°C. The ligase buffer (unlike restriction buffer) cannot be
   repeatedly frozen and thawed, it starts to ‘die’ after even one
   freeze/thaw cycle.
2. Retrieve the T4 ligase enzyme from the -20°C freezer and put
   IMMEDIATELY on ice. This reagent is VERY heat sensitive, and must
   be handled with care.
3. Put your labelled tube(s) on ice, then set up the ligase reaction
   in this tube on ice. Be careful not to get ice or melted ice in the
   tube - this is not sterile! Add reagents in this order (aseptic;
   change tips each time) :

* 2 µl of 10 x ligase buffer
* 8 µl purified insert DNA
* 8 µl purified plasmid DNA
* 2 µl of T4 DNA ligase enzyme

------------------------------------------------

Total 20 µl

4. Mix by flicking (brief! 5 sec), then tap on bench to get liquid to
   bottom of tube (brief! 5 sec). Incubate at 4°C overnight. You can
   do this instead at room temp for 1 hour, but it doesn’t work quite
   as well. Supposedly 16°C overnight is ideal, but this a tricky
   temperature to achieve in practice.
6. Return ligase immediately to -20°C freezer. Throw out any unused
   thawed ligase buffer.

#### [Screening Recombinant Clones](#collapse15)

1. The diagram shows some PCRs that can be used to determine if a plasmid clone is:
    A. The desired recombinant
    B. Some other kind of recombinant
    C. The vector only
   ![](https://static.igem.org/mediawiki/2017/a/a3/T--Sydney_Australia--screening-recombinant-clones.png)
2. The PCR using primers F1-R1 is the best starting point for screening– this is the ‘left junction’ PCR. If you get a product of the expected size in this PCR, this is a good indication that you have the desired recombinants. The PCR using primers F2-R2 (right junction PCR) can then be done on clones that are positive in the F1-R1 PCR. If the clones are also positive in this PCR, this is further evidence that they are the right thing.
3. The PCR using primers F1-R2 is known as a ‘spanning’ PCR or ‘insert spanning’ PCR – this will amplify the whole insert region. This PCR can be used to determine if the clones have an insert of the expected size, but note that this is not an all or nothing PCR, unlike the junction PCRs. In the spanning PCR, the clones will give a small product if they have no insert, and a large product if they have an insert. This PCR is a bit ambiguous though, since if you have a mixed population of cells in your template DNA, you may see only the small product (this is amplified preferentially), even if you do have some positive clones in there too. The spanning PCR is most useful in preparation for sequencing the insert DNA region, rather than for initial clone screening.
4. The primers used for amplification of the original DNA insert (not shown in diagram) can also be used for screening PCRs. e.g. these primers used on their own can tell you if the insert DNA is present. (but they wont tell you which way around it has been cloned, unlike the junction PCRs). You can also do a junction PCR using one of the original primers, and a primer in the vector on the other side of the insert DNA, although this may give large products, and a less reliable PCR compared to using dedicated junction screening primers.
5. The positioning of the screening primers is important for later steps (sequencing). Make sure that the primers F1, R1, F2, R2 are at least 50 bp away from the ligation join – this is because the first 20-30 bp of sequence read are usually ‘junk’. Also ensure that these primers are not \*too\* far from the ligation join, otherwise you are amplifying (and later sequencing) stuff that you don’t need to, and it makes the PCR take longer to thermocycle. Placing the primers about 100 – 150 bp from the ligation join is good, which gives an overall PCR product size of 200-300 bp.

#### [Patch Plates and Colony PCR](#collapse20)

1. Choose 20 well-isolated colonies from your transformation plates.
   Circle these and number them on the base of the plate.
2. Label three PCR strip tubes with numbers 1- 20, leaving three
   blanks (one in each strip) as negative controls. (so your first
   strip should be clones 1-7, the next one should be clones 8-14, and
   the last one should be clones 15-20)
3. Using an agar plate containing the appropriate antibiotic, divide
   the plate into patches, using a square grid (5 x 5). Label twenty
   of the squares with numbers 1 to 20. (see diagram)
4. Prepare a PCR master mix with the appropriate left junction PCR
   primers, enough for ~30 PCRs, aliquot this into 3 x 8 lots of 25 µl
   in the labelled PCR strip tubes. Taq polymerase is good for this
   job (it is very robust to ‘mess’ in the PCR) but other polymerases
   can also be used.
   ![](https://lh5.googleusercontent.com/FL3cNdUVilhgLHv_lSkqFiv9aUba9uvPKVOD5bNE18XSop9ICEs2zjwJfTo3cGXkby00G7jFCTxC7wnnq_J2UnNBBfubym1lTpShvQhdhXxwOlrL5EGrWyoxC9FM1RNsypcRZ68E)
5. Using a white pipette tip, pick up some growth from the colony of
   your first clone of interest. Make sure you can see some cells on
   the tip, but you don’t need a lot (approx 1 mm3 = 1 µl). Dip the
   tip in and out of the mix in the first PCR tube five times. You
   don’t need to dislodge the whole chunk of growth, enough cells will
   fall into the mix to give you a product, if the gene of interest is
   there. (this is called a ‘colony PCR’)
6. With the same tip, transfer the remaining growth onto the first
   patch on your agar plate, by scratching the tip across the surface
   of the agar a few times (e.g. make a set of three closely-spaced
   parallel lines – see diagram). Stay well clear of the borders of
   each square, we don’t want the clones to touch each other when they
   grow up. Then discard the tip.
7. Repeat steps 3 and 4 with the remaining 19 clones, being very
   careful to keep track of which colony, which tube, and which patch
   you are up to (labelling before you start is important!)
8. Put the PCRs on the thermocycler and incubate the agar plate at
   37°C overnight.
9. Run out the PCRs on an agarose gel, and note which clones give a
   positive result.

#### [Secondary PCR Screen](#collapse21)

1. Examine the patch plates prepared above. Choose up to seven of the
   clones that gave a positive PCR in the left junction screen (at
   least three is recommended).
2. Set up a PCR master mix using the right junction primer set.
   Remember to make enough master mix so that there is at least 10%
   more than you need, so you don’t run out.
3. Screen the putative positive clones as described above, by colony
   PCR. Again, be careful to label everything before you start. Pay
   close attention to which clone patch is going into which PCR tube.
4. After thermocycling, run out the PCRs on agarose, and note which
   are positive for the right junction. Choose one of these for a
   plasmid prep; use this clone to inoculate 100 ml LB-antibiotic
   broth (see other protocol for details of plasmid prep).
5. Keep the patch plate! (wrap in parafilm, and store in cold room) –
   you may need to come back to this plate to plasmid prep a different
   clone if there are problems with the first one.

#### [Validation of Recombinant Plasmid by Restriction Digestion](#collapse22)

NOTES: When making recombinant clones, we need to have several lines of evidence that the new plasmid has the correct structure. It is not sufficient to rely solely on the junction PCRs. For example, if the same insert has been cloned twice (head to tail) into the vector, of if there is a deletion in the middle of the insert DNA, the junction PCRs will look fine, but the overall structure will be wrong. Doing digests of the new plasmid provides another line of evidence for the correct structure, and also it allows us to quantitate the plasmid accurately, which is usually needed in any case for further work.

1. Using the plasmid prep from your chosen recombinant clone (above), set up a number of restriction digests which will be diagnostic of the correct structure. For example, you can choose an enzyme that cuts in the insert DNA but not in the vector DNA (this is ideal), and/or you can use an enzyme that cuts in the vector only (this is still OK, if the insert is quite large i.e. >500 bp). You want to be generating between 1 and 3 restriction fragments in total, and you want the recombinant clones have a clearly distinct digest pattern to ‘vector only’. You want to be doing two or three different kinds of digests to give maximum confidence in the structure of the plasmid, not just one digest.
2. See other protocol “Protocol for quantitation and restriction digestion of plasmid (gel analysis)” for details of how to set up these plasmid digests.
3. If the chosen plasmid clone looks correct based on both junction PCRs and restriction digests, then you can proceed to sequencing (below).
4. The primers used for amplification of the original DNA insert (not shown in diagram) can also be used for screening PCRs. e.g. these primers used on their own can tell you if the insert DNA is present. (but they wont tell you which way around it has been cloned, unlike the junction PCRs). You can also do a junction PCR using one of the original primers, and a primer in the vector on the other side of the insert DNA, although this may give large products, and a less reliable PCR compared to using dedicated junction screening primers.

#### [Sequencing Preparation](#collapse23)

1. Add an equal volume of SureClean Plus to nucleic acid solution and
   mix thoroughly.
2. Incubate at room temperature for at least 10 min.
3. Centrifuge at maximum speed (best results at 14,000x g) in a
   bench-top centrifuge for 10 min and carefully remove the
   supernatant by aspiration. (Note: Centrifuging for longer time
   leads to better DNA recovery)
4. Add a volume of 70% ethanol equal to 2x the original sample volume
   and vortex for 10s. (Note: For sensitive applications an optional
   second ethanol wash can be performed)
5. Centrifuge at maximum speed (best results at 14,000 x g) in a
   bench-top centrifuge for 10 min, remove the supernatant and airdry
   to ensure complete removal of ethanol. (Note: Do not overdry the
   pellet)
6. Resuspend pellet in desired volume of TE, water or any other
   appropriate buffer for downstream procedures.

#### [SDS-PAGE (Sample Preparation and PAGE Creation)](#collapse25)

Prepare beadbeating tubes as follows:

1. Get glass beads plus the scoop and funnel from the chemicals
   cabinet (under ‘G’ for Glass) and put 2 large beads and two scoops
   each of the medium and small size beads into each tube.
2. Sterilise by autoclaving (prob. not essential if you are in a hurry
   or forget, but that would be ‘best practice’).
3. Pellet your cells from broth or scrape off a plate and resuspend in
   500µL of TE and 5µL of protease inhibitor cocktail.

1. How much culture to use is tricky to know, more concentrated is
   usually better, but too many cells and suspension may be gloopy
   and hard to work with.
2. The pellet from ~ 2 ml of broth culture or a large loopful from
   a plate is a good starting point.

4. Take your tubes over to the bead beater and unscrew the
   tube-holder. Space your tubes around the disc (make sure the caps
   are on tight!) and replace the cover as tight as you can.
5. Beat 3 times for 30 seconds with 1 minute on ice in between.

1. You may need 5 x 30 sec beatings to fully lyse tough cells like
   mycobacteria.

6. Centrifuge at ~10,000 g for 1 minute (ideally at 4C) to remove
   beads and any remaining whole cells.
7. Retain supernatant on ice.
8. Check the absorbance on the Nanodrop using the protein setting.
   Ideally we want around 5 mg/ml for nice PAGE gels. (between 2 and
   10 mg/mL may be OK)

1. If it’s less than 2 mg/ml, either repeat with more cells or
   bead beat for more cycles.
2. If it’s more than 10 mg/ml (unlikely!) dilute down to 5 mg/ml.
   Keep in mind this Nanodrop reading is only an estimate of the
   protein concentration, since there is other junk in the cell
   extract that will interfere.
3. For some applications like enzyme assays, you may need to use a
   different protein quantitation methods.

#### [SDS-PAGE Stain and Destain](#collapse24)

1. Remove SDS-PAGE gel from glass and rinse once in ddH2O in a suitable container with a lid. Try not to use a container much larger or much smaller then the gel.
2. Add enough Coomassie Stain to cover the gel by 1/2 inch (~ 1.5 cm).
3. Microwave on high power for 40 seconds to 1 minute (until the Coomassie Stain boils)
4. Incubate the gel in the Coomassie stain for 5 to 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour.
5. Pour off the Coomassie Stain. The Coomassie Stain can be recycled a couple of times by filtering it.
6. Rinse twice in ddH2O or used Destain solution to remove Coomassie Stain from the container.
7. Add fresh Destain solution to cover the gel by 3/4 inch (~ 2 cm).

   - Tie Kimwipes in a simple knot and place 4 of them in the Destain solution around the gel. Try to avoid laying the Kimwipes on the gel as this will cause an uneven destaining..
   - Microwave on high power for 40 seconds to 1 minute (until the Destain boils).
   - Incubate the gel in the Destain solution for 10 minutes on a rocking table. If you did not microwave the Coomassie/gel, incubate for at least 1 hour.
   - Discard the stained Kimwipes and replace with fresh knotted Kimwipes.
   - Incubate a second time for 10 minutes to overnight on a rocking table. Stop whenever the level of destaining is sufficient for you. Microwave again to speed up the process.
   - The used Destain solution can be recycled a couple of times by storing it in a sealed container with sponges or Kimwipes to remove all traces of Coomassie Stain.

#### [His-Tag Purification](#collapse26)

**Preparation for His Tag Purification**

For E. Coli / Bacillus - refer to the preparation protocols in the other subheading

**Solutions:

*Wash Buffer* - 50mM sodium phosphate (pH8.0), 300mM NaCl, 10mM imidazole (autoclaved)

*Elution Buffer* - 50mM sodium phosphate (pH8.0), 300mM NaCl, 250mM imidazole (autoclaved)

*Storage Buffer* - 50 mM sodium phosphate buffer (pH 8.0), 100 mM NaCl, 10 mM lysine\* (autoclave).

\*Only add lysine in the specific case of insulin purification - lysine apparently stabilises insulin solutions, and stops them aggregating.**

1. Centrifuge for 2 min at ~3000 g (in the cold if possible). Resuspend pellet in 10 ml Wash Buffer by vortexing/shaking. Centrifuge again. Repeat the wash steps 2 more times (3 washes total)
2. Add 1 ml of Elution Buffer, vortex to resuspend pellet. Mix gently on platform shaker in cold room for ~10 min to allow His-tag proteins to elute from resin.

   - Centrifuge for 2 min at ~3000 g (in the cold if possible). Transfer supernatant (contains purified His-tag protein) to new tube. Discard pellet. Note that this ‘purified protein’ contains lots of imidazole, which may interfere with downstream reactions. Continue purification as follows:
   - Assemble an Amicon ‘centrifugal filter unit’ (put inner tube into outer tube), and add purified protein solution to the top (max 500 µl). Spin for 15 min in the cold at max speed in microfuge. Discard flow-through, and add remaining protein solution to the top part, repeat 15 min spin. Note that most of the liquid will flow through, but not all of it.
   - Add 400 µl of Storage Buffer, spin for 15 min in the cold at max speed in microfuge. Discard flow-through. Repeat this step twice more (three rinses with storage buffer in total).
   - Pipette the remaining solution (~100 µl?) up and down vigorously several times to remove proteins from the filter, then pipette into a new tube. This is the final purified protein solution. Quantify by A280 and/or SDS-PAGE. Compare by SDS-PAGE to crude lysates stored earlier.
   - Can add TEV protease or trypsin at this stage to cleave off secretion tags: Add 5 µl of the protease stock solution (at ~1 mg/ml) to ~100 µL protein prep, incubate 1 hour at 30°C (TEV) or 4 hours at 37°C (trypsin). These proteases (and the tags) will remain in the final protein prep, but shouldn’t interfere with most kinds of activity assays. (with our insulin constructs, we could add these proteases earlier, to leave the tags bound to nickel column, but that approach is non-standard and risky…proteases might not cut column-bound proteins?)
   - Pipette the remaining solution (~100 µl?) up and down vigorously several times to remove proteins from the filter, then pipette into a new tube. This is the final purified protein solution. Quantify by A280 and/or SDS-PAGE. Compare by SDS-PAGE to crude lysates stored earlier.
   - May need to dilute the protein for biochemical/physiological tests. Use storage buffer for this (not water). Ensure that appropriate negative controls are included in these tests. These could include:

     1. storage buffer with no protein added.

     2. Mock purification of protein from an induced-but- vector-only culture (will contain some proteins)
   - Continue with general His-Tag purification protocol.

#### [ELISA](#collapse264)

Sample Prep: Dilute samples 1:10 to a total volume of 50 µL (5 µL sample +
45 Assay Buffer). Note: We recommend performing several different sample
dilutions with Assay Buffer to ensure the readings fall within the standard
curve;

* Standard wells = 50 µL standard dilutions.
* Control sample wells (no insulin treatment) = 1 - 50 µL diluted
  1:10 sample/well (adjust to 50 µL/well with Assay Buffer).
* Treated sample wells: 1 - 50 µL diluted 1:10 sample/well (adjust to
  50 µL/well with Assay Buffer

Assay:

1. Prepare 10 µL of Reaction Mix A (NADPH generation) for each
   reaction. Mix enough reagents for the number of assays (samples and
   controls) to be performed. Prepare a master mix of the Reaction mix
   to ensure consistency.

1. Assay Buffer: 8 µL
2. Enzyme Mix: 2 µL

2. Add 10 µL of Reaction Mix into each well of standard, control and
   sample wells.
3. Mix and incubate at 37°C for 1 hour.
4. Add 90 µL Extraction Buffer to each well, seal the microplate with
   a sealing tape and heat it at 85°C for 40 minutes to degrade any
   unused NADP left in the sample.
5. Cool plate on ice for 5 minutes and neutralize reaction by adding
   12 µL of Neutralization Buffer.
6. Prepare 38 µL of Reaction Mix B (recycling amplification reaction)
   for each reaction. Mix enough reagents for the number of assays
   (samples and controls) to be performed. Prepare a master mix of the
   Reaction Mix to ensure consistency;

1. Glutathione Reductase - 20 µL
2. Substrate DRNB - 16 µL
3. Recycling Mix - 2 µL

7. Add 38 µL Reaction Mix B to each well (standard, control and
   treated cells) and mix well by pipetting up and down.
8. Measure output at OD 412 nm on a microplate reader in a kinetic
   mode, every 2 – 3 minutes, at 37°C protected from light, until
   Standard #6 (100 pmol/well) reaches OD = 1.5 – 2.
9. Take an additional endpoint reading of all samples and standards

Source:
<http://www.abcam.com/glucose-uptake-assay-kit-colorimetric-ab136955-protocols.html>

#### [Glucose Uptake Assay](#collapse2632)

1. Seed adherent cells (2-5x104 cells/well) one day before starting
   the assay.
2. After 8-12 hrs, remove regular culture medium (10% FBS) and treat
   cells with test compound or vehicle control in 400 µl cell culture
   medium with 0.5% FBS.
3. Incubate cells at 37⁰C with 5% CO2 for 1 hr or desired time period
   depending upon the test compound.
4. To use Phloretin as a control, treat cells with 4 µl Phloretin
   (final concentration 1X) in 400 µl of cell culture medium with 0.5%
   FBS at 37⁰C with 5% CO2 for 1 hr.

1. Note: Cell seeding is not required for suspension cells. Use up
   to 1-2x105 suspension cells/well in 400 µl of cell culture
   medium with 0.5% FBS to treat with test compound or vehicle
   control.

5. Glucose Uptake: Prepare 400 µl glucose uptake mix for each well as
   following:

1. Cell culture medium (0.5% FBS) - 376 µl
2. 2-NBDG Reagent - 4µl
3. Glucose Uptake Enhancer - 20µl
4. Test compound or 1X Phloretin same conc. as in step 1

6. Mix well.
7. After the incubation in Step 1, spin down the plate at 400 x g for
   5 min. and carefully remove the medium without disturbing cells.
8. Gently add glucose uptake mix to each well and incubate cells at
   37⁰C with 5% CO2 for 30 min.
9. After incubation, collect cells from the plate and keep on ice.
10. Wash once with 1 ml ice-cold 1X Analysis Buffer.
11. Spin down at 400 x g for 5 min. and resuspend cell pellet in 400 µl
    of 1X Analysis Buffer.
12. Cells are ready to be analyzed on flow cytometer (488 nm excitation
    laser). For flow acquisition and analysis, select the main cell
    population in the FSC vs SSC plot to exclude dead cells and
    cellular debris.
13. Within the main cell population, mean fluorescence intensity in FL1
    can be quantified and compared between cells treated with test
    compounds and untreated control cells.

#### [IPTG Induction of T7](#collapse27)

**Day 1**

1. Start 10 ml overnight culture in LB w. 100 µg/ml Amp

**Day 2**

1. Dilute culture 1:100 into 1000 ml LB with 100 µg/ml Amp, (prewarmed is better). Shake at 37°C and follow the A600.
2. At A600 between 0.4-0.5 (do not overgrow!!) transfer culture to room temperature shaker
3. After 10-15 min, add IPTG to 0.2 mM final concentration.
4. Take 20-200ml aliquot at 6 hour timepoint and after overnight shaking. Size of aliquot can vary depending on downstream requirements.

(Temperature, time and IPTG concentration may have to be optimized for individual proteins).

#### [Auto-Induction of E. Coli](#collapse28)

Filter Sterilise and add to 960mL Broth

 :60 % v/v Glycerol - 10mL

 :10 % w/v Glucose - 5 mL

 :8 % w/v Lactose - 25mL

**Day 1**

1. Start 10 ml overnight culture in LB w. 100 µg/ml Amp

**Day 2**

1. Dilute culture 1:100 into 1000 ml LB with 100 µg/ml Amp, (prewarmed is better). Shake at room temperature or 37°C (room temperature should result in more natively folded protein).
2. Take 20-200ml aliquot at 6 hour timepoint and after overnight shaking. Size of aliquot can vary depending on downstream requirements.

#### [Preparation of Chemically Competent Cells](#collapse54)

Use excellent aseptic technique at all times. All materials must be
sterile.

Protocol can be scaled up or down as required. 100mL of E. coli culture
produces about 50 x 220 µL aliquots of competent cells. Each individual
aliquot of cells is sufficient for 4 transformations (4 x 50µl, with a bit
left over as a safety margin).

The competent cells can also be used fresh, without making frozen aliquots,
this actually works even better, but it is tedious to do over and over,
thus we typically make frozen stocks. These freezer stocks don’t survive
multiple freeze/thaw cycles well, so throw out unused cells if there are
leftovers after your transformation experiment.

After step 6, work on ice and in the cold room to increase the quality of
the final cell prep. Keep RF1 and RF2 solutions in cold room/fridge, so
they are always ready to use. At the beginning of the day, place centrifuge
bottles in cold room or in the -20°C freezer, ready to use.

PROTOCOL:

1. Streak E. coli strain (e.g. JM109, TOP10, DH5α) from glycerol stock
   or other source onto plain LB agar (no antibiotics). Incubate
   overnight at 37°C.
2. Check that culture looks pure. Inoculate a 5mL plain LB broth with
   growth (several colonies) from the LB plate. Incubate overnight
   (16-24 h) at 37°C, with shaking
3. Aseptically inoculate 100mL plain LB broth (in a 500mL Schott
   bottle or Erlenmeyer flask) with 3 ml of overnight culture; this
   should give an initial OD600 of ~0.05. (check this to be sure)
4. Grow cells at 37°C with shaking, until the culture reaches OD600 of
   ~0.5 (anywhere from 0.3-0.7 is OK, but try to get close to 0.5).
   This should take 2-3 hours, so check the OD600 every 15 minutes or
   so after the first hour of incubation.
5. Aseptically transfer (pour) the cells into sterile centrifuge tubes
   or bottles (one 250 ml Sorvall bottle or 2 x 50 ml Falcon tubes)
   and balance these, either by transferring culture aseptically
   between the tubes/bottles or by adding sterile water or LB to the
   lighter tube/bottle.
6. Centrifuge at 4000 rpm (~3000 g in Centaur/Centurion machine) at
   4°C for 10 minutes. Note: centrifuge needs to be cold. Turn on and
   set temperature beforehand. You can spin faster, up to say 7000 g,
   but above this point, faster is not better, and cell pellets will
   be hard to resuspend.
7. Working in the cold room with the cells on ice, pour off the
   supernatant into culture waste (don’t let the centrifuge
   tube/bottle actually touch the edge of the culture waste bottle).
   Try to remove as much of the liquid as possible – give it a shake /
   tap to assist this.
8. Resuspend the pellet gently in 33mL RF1 solution by vortexing
   and/or shaking the tube/bottle. It's OK to be rough with the cells
   at this stage in the process, but you shouldn’t need to shake or
   vortex for more than 10 sec or so.
9. Incubate on ice for 1 hour, then pellet the bacteria again at
   4000rpm, 4°C for 10 minutes.
10. Working in the cold room, pour off the supernatant into culture
    waste. As before, try to remove as much of the residual liquid as
    possible.
11. Resuspend the pellet in 8mL RF2 solution by vortexing or shaking.
    At this stage, the cells have become more fragile due to the RF1
    treatment, so its important not to shake/vortex any longer than ~10
    sec (this shouldn’t be necessary).
12. Incubate on ice for 15 minutes. While the cells are incubating, set
    up all your Eppi tubes (~50) on ice with the lids open, so they are
    pre-chilled, and ready to receive cells. Label these tubes on top
    with the strain name before putting them on ice (labelling becomes
    difficult with cold and wet tubes!). Be careful to only push the
    tubes only about 2/3 of the way into the ice. If they are pushed in
    too far (right up to the lip of the tube), you risk getting ice or
    melted ice (not sterile!) into your cell aliquots
13. Working quickly (but still carefully!), aliquot 220 µL of cell
    suspension into the pre-chilled Eppi tubes. Once dispensed, close
    tube lids tightly, and collect all tubes into a bag/box with a
    clear and prominent label and store it immediately in the -80°C
    freezer. It’s a good idea to label both the outside of the bag/box
    AND place a label written on paper inside.
14. Test the transformation efficiency of the freshly-prepared
    competent cells using a known amount of a plasmid standard (see
    protocol for heat shock transformation).
15. Streak out a loopful of the cells onto plain LB medium (using
    either a sample of a frozen aliquot or some residual cells
    remaining in the large centrifuge tube/bottle), and incubate at
    30°C for three days to allow any of the common types of
    contaminants (e.g. Staphylococcus) to grow. This is to check the
    purity of the cell stock. The streak-plate should look completely
    uniform, with colonies of only one type (E.coli), and no
    heterogeneity in the initial patch or the streaklines which would
    indicate a mixture of bacteria is present.

#### [Heat Shock Transformation of Chemically Competent Cells](#collapse63)

1. Remove one or more aliquots (as required) of chemically competent
   cells of your E.coli strain from the -80°C freezer. Thaw the cells
   e.g. by rubbing them in your hands or put them briefly in a 37°C
   waterbath, but don’t let them stay warm! As soon as they are
   thawed, put them onto ice.
2. Divide the cells into the appropriate number of 50 µl aliquots in
   separate Eppi tubes on ice. Add your DNA samples to each tube; you
   can use up to ~10 ul of ligation mixture or plasmid here, but note
   that typically 3 µl of ligation mix or 1 µl of plasmid would be
   standard.
3. Make sure you include both a positive control and a negative
   control in the transformation experiment. The positive control
   should be 1 µl of a plasmid with the correct antibiotic resistance
   (same resistance as the plasmid used for the ligation), and should
   also be a plasmid stock that you know is in good condition (based
   on agarose gel).The negative control is simply no DNA added.
4. Put the cells into a foam ‘floatie’ and put on ice. Ensure at least
   the bottom half of the tube (approx 2 cm) is embedded in the ice,
   don’t just rest them on top of the ice. Allow the cell/DNA mixtures
   to incubate on ice for 15-30 min.
5. Take your esky of ice over to the 42°C waterbath or 42°C heat
   block. Put the floatie into the waterbath. Allow 45 seconds for
   heat shock. (Plus or minus 10 seconds, this needs to be exact!).
   Then transfer the floatie straight back onto ice (embed into ice,
   as above, don’t just rest on top).
6. Allow transformation mixtures to sit for 2 min on ice, then add 1
   ml sterile LB broth to each tube. You can also use more fancy media
   (e.g. SOC or SOB), but there is not that much difference.
7. Incubate on 37°C shaker for 1 hour. Put the tubes horizontal so
   they get good shaking action. eg. put the tubes laying flat on the
   shaker platform and masking-tape into place. Make sure the lids are
   tight! You can incubate without shaking, and you can incubate for
   less time (30 min), but it won’t work as well in these cases.
8. Label the LB-antibiotic plates before starting the next bit; you
   need two plates for each ligation condition or plasmid type, since
   we will plate out two different cell concentrations of each to
   ensure we get countable/pickable numbers of colonies. Double check
   the plates to ensure you are using the correct type of
   antibiotic(s) for the type of plasmid(s) you are using.
9. Pipette 100 µl of the first cell suspension onto one LB-antibiotic
   plate (label ‘100 µl’ in addition to other info). Sterilise the
   glass spreader with ethanol and flame (CAUTION! READ THE SOP
   FIRST!), and spread the cells around the plate with the spreader.
   Do this by pushing the spreader with a back-and-forth motion, while
   turning the plate around in a circular motion. Be careful not to
   touch the spreader on your fingers! Keep spreading for approx 10
   seconds.
10. If the plates are properly dried, you should feel the spreader
    start to ‘stick’ to the agar, this means the liquid has been drawn
    into the agar. If this doesn’t happen after ~20 seconds, stop
    spreading, but next time, dry plates for longer! If the plates are
    incubated with a lot of liquid still on them, you may not get nice
    discrete colonies (the cells will swim around in the liquid, making
    a mess).
11. Spread 100 µl of the remaining samples, each onto a separate,
    appropriately-labelled plate.
12. Centrifuge all the tubes at ~15,000 rpm for 1 minute in a
    micro-centrifuge. Pour off most of the supernatant into culture
    waste (being careful not to touch the tubes on the edge of the
    culture waste bottle). Leave a little bit of liquid behind (about
    one or two drops).
13. Vortex the cells in the remaining liquid for about 10 seconds,
    until they are not sticking to tube anymore, and you have a nice
    smooth, even, cell suspension.
14. Pipette the cells from the first cell suspension onto the
    appropriate pre-labelled LB-antibiotic plate (label with ‘pellet’
    in addition to other info), and spread plate as described above,
    using the glass spreader. Repeat for the remaining samples
15. Incubate all plates at 37°C overnight. Note that for some plasmids
    and ligations, it may be beneficial to instead try room temp for
    2-3 days – this lowers the copy number of pUC type plasmids, and is
    useful to allow retrieval of clones that might be toxic to the
    host.
16. When examining your plates, first check your controls. The positive
    control should have thousands of colonies, perhaps even a confluent
    lawn of growth, especially on the ‘pellet’ plate. the negative
    control should have no colonies at all. If you don’t see these
    results with the controls, anything you see on your experimental
    plates is questionable. Common problems and their interpretation
    are summarised in the Table below. Detailed troubleshooting of the
    different ligation controls and their results is described in a
    different protocol.

![](https://lh5.googleusercontent.com/9frPUnBNJqw2v36gGkWqHZCF6AkLWzwVeVeW-yPR4uJwlgrh_v_KHV7dfFfBBHnxfknU-zA_80WVNAP4W4fbFJUf8clsZi1nJy4-6ugYSjycOleNnlSmSJ2uNcBJ5u2VjpLxVx-o)

![](https://lh6.googleusercontent.com/OrKq4riO13cif6m3pH5f0FmrcJSwsOm6gvwBMDRBi6qIElmEWBlJqYzL91IdPSyd9vEMY5mWz8Gxuem8wGkuKx1KFGMHU6HxBKMhp2EPDl4OhUO2mfbCHjL1jhaA7RjQUKsJ1rKM)

#### [Cell Lysis of E. Coli](#collapse29)

1. Harvest cells by centrifugation, and resuspend pellet(s) in 5 ml
   Wash Buffer (be careful not to mix pellets from different types of
   cultures!). Add 100 µl each of DNAse (10 mg/ml), lysozyme (10
   mg/ml), then freeze at -20°C.
2. Thaw cells, then allow ~10 min at room temp for lysozyme action.
   Divide cells into sterile beadbeater tubes containing glass beads
   (add 1.0-1.5 ml cells per tube). Allow to chill on ice (~5 min).
   Lyse by beadbeating (three cycles of 30 seconds, with 1 min on ice
   between beating steps)
3. Centrifuge in the cold for 5 minutes at ~10,000g to pellet beads
   and cell debris. Combine supernatants into a single 15-ml Falcon
   tube (be careful not to mix lysates from different cultures!!!).
   Set aside and freeze a sample of crude lysate (~100 ul) for later
   SDS-PAGE.

#### [Periplasmic Lysis with Chloroform](#collapse30)

1) Pellet cells 10 min x 1000g (in culture tube)

2) Decant supernatant thoroughly

3) Briefly vortex in remaining medium (ha, you weren't thorough enough!)

4) Add 20ul CHCl3 and briefly vortex

5) Leave at room temp 15 mins (coffee + bikkie break)

6) Add 0.2ml 0.01M Tris hydrochloride (pH 8.0)

7) Centrifuge 20 min x 6,000g (check large centrifuge goes this high before
doing this procedure in tubes)

8) Withdraw supernatant with a Pasteur Pipette. For quantitative gels,
withdraw only a portion (less is more!)

#### [E. Coli Preparation for His-Tag Purification](#collapse40)

1. Use colonies or patches from a fresh agar plate (<1 week old) to
   inoculate a starter culture in 5 ml LB-Amp100 – grow 16 h at 37°C
   with shaking. Early the next day, add this culture to 200 mL
   LB-Amp100 (preferably in a large conical flask with baffles) at a
   starting OD600 of ~0.02-0.1 and grow for ~3 h at 37°C with shaking
   until OD600 is ~0.5.
2. Move cultures to room temp., add IPTG to a final concentration of 1
   mM. Incubate shaking for 6 hours. Harvest cells by centrifugation,
   and resuspend pellet(s) in 5 ml Wash Buffer (be careful not to mix
   pellets from different types of cultures!). Add 100 µl each of
   DNAse (10 mg/ml), lysozyme (10 mg/ml), then freeze at -20°C.
3. Thaw cells, then allow ~10 min at room temp for lysozyme action.
   Divide cells into sterile beadbeater tubes containing glass beads
   (add 1.0-1.5 ml cells per tube). Allow to chill on ice (~5 min).
   Lyse by beadbeating (three cycles of 30 seconds, with 1 min on ice
   between beating steps)
4. Centrifuge in the cold for 5 minutes at ~10,000g to pellet beads
   and cell debris. Combine supernatants into a single 15-ml Falcon
   tube (be careful not to mix lysates from different cultures!!!).
   Set aside and freeze a sample of crude lysate (~100 ul) for later
   SDS-PAGE.
5. Find the Nickel-agarose resin (cold room), invert several times to
   resuspend the resin (this settles out quite quickly). Pipette out 2
   ml of resin into a 15 ml Falcon tube. Add 10 ml of Wash Buffer,
   mix, then centrifuge for 2 min at ~3000 g (in the cold if
   possible).
6. Pour off supernatant, add your cell lysate (assumes approx. 5-10 ml
   lysate). Vortex to resuspend the resin. Mix gently on platform
   shaker in cold room for ~20 min to allow His-tag proteins to bind
   to resin.

#### [B. Subtilis Competent Cell Preparation and Transformation](#collapse32)

1. Inoculate 5 mL sterileLB media with a small loopful of Bacillus
   subtilis colonies. Incubate overnight (18 hr) at 37°C. Measure its
   optical density OD 600 at the end of the growth.
2. Inoculate the overnight culture into 15 ml of Medium A in a sterile
   50 mL Falcon Tube to dilute to an optical density OD 600 of 0.1 -
   0.2.
3. Incubate at 37°C with vigorous aeration (200 rpm). Read the OD 600
   every 20 min, plotting ln(OD 600 ) against time (min) in excel.
   After a brief lag, the OD should increase logarithmically—that is,
   they should fall on a straight line. Note the point at the culture
   leaves log growth—the graph points fall below the straight line. In
   B. subtilis genetics, this point is known as t0. It should take
   60-90 minutes of incubation and occur at OD 600 ~ 0.9 - 1.
4. Continue incubation for 90 minutes after the cessation of log
   growth (t90). Transfer 1.7 ml of this culture into 15 ml of
   pre-warmed Medium B in a sterile 50 mL Falcon Tube.
5. Incubate the diluted cultures at 37°C with vigorous aeration for 90
   min. At this point, the cultures should be highly competent.
6. Aliquot 500 µL of this culture into sterile eppi tubes for
   transformation. This will make about 30 aliquots.
7. Add 1 μg of DNA to the competent cells and incubate at 37°C with
   aeration for 30 minutes.
8. Add sterile 50% glycerol (125 µL) of the rest of competent cells
   culture (500 µL) to a final glycerol concentration of 10%. Store at
   -80°C immediately. To transform glycerol stocks of competent cells
   retrieve from -80°C, add 1 μg of DNA to the aliquot and incubate at
   37°C with aeration for 30 minutes.
9. Plate aliquots of the transformed cells onto selective agar.

10× Medium A base:

* Yeast extract 1 g
* Tryptone 0.2 g
* Distilled water to 90 ml
* Autoclave, then add:

+ 50% glucose, filter-sterilized 10 ml

10× Bacillus salts

* (NH4)2SO4 2 g
* K2HPO4·3H2O 18.3 g
* KH2PO4 6 g
* Na+citrate 1 g
* MgSO4·7H2O 0.2 g
* Water to 100 ml

Medium A

* Sterile water 81 ml
* 10× Medium A base 10 ml
* 10× Bacillus salts 9 ml

Medium B

* Medium A 15 ml
* 50 mM CaCl2·2H2O 0.15 ml
* 250 µM MgCl2·6H2O 0.15 ml

#### [Retrieval of Bacillus Supernatant](#collapse14)

Label an equal number of falcon tubes exactly the same as the cultures in
the shaker

2. Pour the cultures into their corresponding labelled falcon tubes

3. Spin in the cold room centrifuge for 10 mins at the fastest speed
(should be around 3400)

4. Pour off supernatant in second set of labelled falcon tubes (probably
15ml falcon tubes – skinny ones)- MAKE SURE THESE ARE LABELLED REALLY WELL
AS WELL!!!!

5. Keep the pellets as well as the supernatant. Supernatant should contain
the expressed protein.

#### [IPTG Induction of Bacillus](#collapse33)

IPTG Induction of Bacillus

#### [B. Subtilis Preparation for His-Tag Purification](#collapse34)

1. Use colonies or patches from a fresh agar plate (<1 week old) to inoculate a starter culture in 5 ml LB-Spec100 – grow 16 h at 37°C with shaking. Early the next day, add this culture to 200 mL LB-Amp100 (preferably in a large conical flask with baffles) at a starting OD600 of ~0.02-0.1 and grow for ~3 h at 37°C with shaking until OD600 is ~0.5.

   - Move cultures to room temp., add IPTG to a final concentration of 1 mM. Incubate shaking for 6 hours. Harvest cells by centrifugation and keep supernatant, combine into a single large falcon tube.

     - Add imidazole and NaCl to the supernatant (10 mM imidazole, 300 mM NaCl). You can just add straight chemicals as powders, don’t worry too much about sterility in this case.

       - Shake up the bottle of nickel-agarose resin, and then add 5 ml resin to the culture supernatant (don’t worry about washing the supplied buffer off the resin in this case, since the total volume of the culture supernatant is so large, it will become insignificant)

         - Place the bottle on its side, and incubate with gentle shaking on platform shaker in cold room for 2 hours, to allow binding of His-tag protein to resin.

           - Separate into 4x50 ml, centrifuge 2 min in cold room, discard supernatants. Resuspend resin pellets in 1 x 10 ml Wash Buffer.

             - Continue with general His-Tag purification protocol

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# Evidence of Protease Knockout in *Bacillus subtilis* WB800

![](https://static.igem.org/mediawiki/2017/b/b9/T--Sydney_Australia--Results1.png)

###### **Figure 1:** Wild type (WT) B.subtilis 168 and WB800 B.subtilis grown on an LB milk plate. WB800 has 8 proteases knocked out of it and this test was used to determine whether in fact this was the correct strain with knocked out proteases. With 8 proteases removed, we did not expect to see clearing around the colony as a result of the proteolytic breakdown of the milk in the plate as you see in the 2 WT168 colonies on the plate. This result was evident on the plate as there was no clear zone around the WB800 strains, whereas substantial clearing is seen around the WT168 colonies.

# Evidence of Chromosome Integration in *Bacillus subtilis* WB800

![](https://static.igem.org/mediawiki/2017/0/0c/T--Sydney_Australia--Results2.png)

###### **Figure 2:** pUS258::YncM insulin transformed into *B.subtilis* WB800 patched on LB Agar + 1% starch + Spectinomycin 100.

#### pUS258 is an integrative plasmid, meaning when transformation occurs, the insert will integrate into the amylase genes of the chromosome of *Bacillus*, knocking them out. Therefore, by patching these transformants on starch plates, cells which have had integration of the YncM insulin gene into their amylase genes will be unable to digest starch on the plate. When flooded with iodine, the colonies in which integration have occurred are identified as showing no visible clearing. The colonies without clearing can therefore can be expected to contain the integrated plasmid, and may be induced to produce insulin.

# Confirmation of Inserts in pSB1C3

![](https://static.igem.org/mediawiki/2017/3/38/T--Sydney_Australia--Results3.png)

###### Figure 3: Double digest of constructs in pSB1C3 using EcoRI and PstI. Products were run on a 1.5% agarose gel in 0.5X TBE at 80V for 2 hours and post stained with GelGreen (Bio-rad) according to manufacturers protocol.

#### Our constructs were ligated into the submission vector, pSB1C3 to submit as composite parts to the iGEM registry. PCR was performed on the plasmid preps across the insert site using high fidelity polymerase and those PCR products were sequenced with the Australian Genomic Research Facility to ensure we would be submitting the correct sequence. The sequencing data showed some disparity in the YncM insulin construct compared to the G-block that was ligated. This is reflected in the gel in figure 3 that shows multiple digested bands. This could potentially be due to multiple partial inserts but we cannot confirm this. Based on this data, we made the decision not to submit this part to the registry and unfortunately, despite our attempts, we couldn’t redo the process due to a limited supply of our construct and time. On the other hand, our other constructs that can be seen in lanes 2-6 show clean single bands that correspond to their respective construct sizes (Cytoplasmic Winsulin: 306bp, Ecotin Winsulin: 867bp, YncM Winsulin: 522bp, Cytoplasmic insulin: 392, Ecotin insulin: 917bp, YncM insulin: 583). These were additionally confirmed with accurate sequencing data and so were able to be submitted.

# Evidence for ELISA Results

![](https://static.igem.org/mediawiki/2017/7/72/T--Sydney_Australia--Results4.png)

###### Figure 4: ELISA confirms expression of constructs, and the correct folding of Insulin and Winsulin constructs. ELISA was performed on cell lysates for all constructs and on the cell culture media of YncM Winsulin from B.subtilis, according to the manufacturer's protocol.

#### This showed the presence of expressed insulin or Winsulin in cells expressing Cytoplasmic expressed insulin, Cytoplasmic Winsulin, Ecotin expressed insulin and YncM Winsulin, as well as proving their ability to bind anti-insulin antibodies. 5µL of cell lysates were tested at multiple dilutions, shown here are the 1:1 dilutions. The inclusion of a whole-cell lysate control for YncM Winsulin proves the YncM secretion tag was effectively causing Bacillus secretion of Winsulin into the surrounding media, as it was not found in the cell lysate fraction. We performed an ELISA assay using capture antibodies specific to insulin (Figure 4). The purpose of performing this assay was: To identify if our insulin constructs had formed folded proteins of a shape similar enough to native human insulin that they would be bound by an anti-insulin antibody. Determine if we produced these constructs in our cells. Identify if the translocation tags used on some constructs (namely YncM Winsulin) transported the protein where we had predicted. From this assay we were able to confirm that our insulin analogues bound insulin antibodies. The parts tested, Cytoplasmic insulin, Cytoplasmic Winsulin, Ecotin insulin and YncM Winsulin, therefore all have a structure very similar to that of native insulin. The fact that we detected them in the ELISA assay also confirmed expression of our constructs. We also used this assay to identify the location of proteins with tags inducing translocation to different parts of the cell. Whilst we were unable to test the periplasmic fraction of Ecotin insulin due to time constraints, we did test our YncM Winsulin construct. YncM is an N-terminal tag which, when fused to a protein of interest and expressed in Bacillus subtilis, directs its secretion out of the cell and into the surrounding media. We proved that YncM does induce secretion by showing that whilst YncM Winsulin was present in the surrounding media, it was completely absent in the cell lysate fractions, as expected.

# Cell Culture Assay Results

![](https://static.igem.org/mediawiki/2017/f/ff/T--Sydney_Australia--Results5.png)

###### Figure 5: Expressed recombinant insulins stimulate glycogen synthesis. 4A and 4B: Comparison of mean glycogen synthesis rate in a glucose uptake assay using human HepG2 (4A) and murine AML12 cell lines (4B). 4C: Comparison of mean rate of glucose oxidation in HepG2 cell culture from glucose uptake assay. Conditions indicated on the graphs are: Basal (no insulin added); Ecotin Proinsulin (from whole cell lysate with trypsin treatment); Cytoplasmic Proinsulin (from whole cell lysate with trypsin treatment); YncM Winsulin (cell culture media only). Refer to protocols page for experimental details. Error bars represent SEM, horizontal bars indicate statistical significance (\* = p<0.05, \*\* = p<0.0001) calculated with GraphPad Prism using unpaired 1-tailed T-test, n=3.

#### While the ELISA provided evidence for whether or not our cells were producing insulin, and gave an indication that the protein fold was correct, the glucose uptake assay was essential for determining the functional capacity of our constructs. It is testing if our expressed human insulin and expressed Winsulin can stimulate an increase in the absorption of radioactive glucose by binding to the insulin receptor in vitro using human HepG2 and mice AML12 liver cells. The change is detected by measuring glycogen synthesis and glucose oxidation and the protocols for the experiment are available on our protocols page. We only had the materials and time available to use the samples that gave positive readings in the ELISA and were also restricted to using those that had high enough concentrations to fit the protocol, however we produced excellent preliminary data suggesting bioactivity of multiple insulin and Winsulin constructs.

## Ecotin Insulin

#### Because we were simply trying to show functionality, we used the fraction that would have overall the most Ecotin Insulin in it according to the ELISA experiment, so we used whole cell lysate in this assay that had been treated with trypsin protease to cleave insulin to the active form of human insulin. The results indicate that both the human (Figure 4) and murine (Figure 5) hepatocytes significantly increased their glycogen synthesis upon treatment with Ecotin Insulin, strongly indicating that there was successful expression and folding of the insulin to its correctly folded and functional form.

## Cytoplasmic Insulin

#### As with Ecotin Insulin, we used whole cell lysate to test Cytoplasmic Insulin function, however this was because of its intended expression in the cytoplasm. In the human hepatocyte cell line (HepG2), Cytoplasmic insulin significantly increased glycogen synthesis (Figure 4), and in the murine cell line (Figure 5), the result was highly significant (p<0.0001) compared to basal glycogen synthesis. Additionally, Cytoplasmic insulin showed a promising increase in glucose oxidation although the result was statistically insignificant. This data together is a very strong indication that this construct was successfully expressed and processed.

## YncM Winsulin

#### Because the ELISA showed successful secretion of YncM Winsulin into the cell culture media, we used that fraction in the glucose uptake assay. Unfortunately we only had a very small volume remaining and were only able to test on HepG2, and due to time constraints were unable to produce more for testing. However, it did appear to show an increase in glycogen synthesis, although not statistically significant, as illustrated in Figure 4.

#### Overall, these results are an incredibly exciting outcome. We have produced strong evidence that proinsulin and ecotin proinsulin isolated from the cytoplasm are functional following trypsin cleavage to active insulin. More importantly, we have a promising indication that our novel single chain insulin analogue, Winsulin, is functional. As we were limited by both supplies and time, we recognise that the data is of limited reliability, however we have certainly produced some promising preliminary results that warrant pursuing further investigation.

# Future Directions

## Improve reliability of preliminary data

#### We were limited in time and supplies to test all of our constructs rigorously and although we have good preliminary data, it would be insufficient to justify Winsulin for clinical trials at this point. Re-doing the expression process and assays with more samples would gather the data necessary to put it in the pipeline for large scale experimentation and eventually clinical trials.

## Optimise recombinant Insulin/Winsulin Yield

#### Increasing yield would be the most important aspect of improving our product to make large scale production viable and ultimately reduce the cost per dose. Here we have outlined a few ways in which we believe are realistic methods to approach this: Part of increasing yield would be to increase the amount of protein moved out of the cell in our Bacillus secretion method and into the periplasm for E. coli expression. This could potentially be achieved by increasing the efficiency of the respective targeting tags, YncM and Ecotin using random or targeted mutagenesis. While pET15-b and pUS258 are designed for high expression of recombinant protein, it may be the case that other vector/expression systems are better suited to expressing our particular constructs, and it would be naive to proceed to large scale production without trialling other options. Another option that some of the other big insulin producers have adopted is to modify their cell lines. While we already took the first steps towards this by using the existing protease knockout strain WB800, further optimisations could be made using CRISPR gene editing. We could experimentally target a number of genes including folding chaperones and proteins related to the secretory pathway. Another viable method would be to use whole genome CRISPR screening as a more random method of identifying knockouts that may enhance expression where we wouldn’t think of targeting. Characterise WinsulinBecause the Winsulin analogue is completely novel, it will need in depth characterisation, particularly if it continues to show functional capacity. Proteomic analysis to provide information about structure, charge, binding mechanism and stability are just some of the properties that are essential if it was to be considered as a therapeutic. Additional functional analysisWhile the glucose uptake assay using human hepatocytes is an excellent in vitro experiment to determine functionality of our recombinant protein, it simply won’t be sufficient evidence to prove that complies with the overall physiology of humans. We will need to perform a number of in vivo experiments using a diabetic mouse model to show that it can be viable as a hormone replacement therapy that won’t elicit an immune response. Scale-up ProductionOnce the expression efficiency has been optimised and the recombinant proteins have been well characterised, we will need to consider methods to scale up production to produce enough insulin to compete in the market. Our vision involves the use of large vats with bacteria secreting our insulin into the media, and figuring out a way to efficiently separate the protein directly giving a somewhat continuous production, rather than emptying the vats and starting from scratch.

# Considerations for replication of experiments

## Alternate vector system

#### pUS258 was a sufficient vector to use in our preliminary experiments to determine if we could express proinsulin and Winsulin in B. subtilis WB800. However, as an integrative plasmid with a limited multiple cloning site it added a number of steps to the process. Additionally, because it integrates into the chromosome, there is only a single copy of the recombinant gene in each cell which means less potential protein product produced. On our project design page, we outline an additional solution to this problem in the development of a novel replicative vector pUS270. Following construction and testing, we would opt for using this plasmid as an alternative for the reasons outlined on Project [Design.](https://2017.igem.org/Team%3ASydney_Australia/Design)

## Modify Purification Protocol

#### We encountered some trouble while trying to purify our proteins using the His column. Our SDS-PAGE gels were showing significant protein carry over that may be attributed to insufficient washing or possibly a large number of proteins binding to the column. To improve our purity, we could add in a dialysis step to remove larger proteins leaving the relatively smaller insulin behind. It also appeared as though we may have lost some insulin during purification. This may be attributed to insulins limited solubility in water. One option may be to elute the protein off the His column in HCl with Tris-HCL sitting in the collection tube to immediately neutralise the acidity and minimise damage to the protein with dialysis to dilute the HCl. Realistically, there are a number of small modification like this that could be made to optimise purification that we would repeat the experiment with.

## Assays

#### Due to the complications with purification, we were unable to use purified Proinsulin/Winsulin in either the ELISA or glucose uptake assay. We also didn’t have the opportunity to use test all of our expressed constructs in the glucose uptake assay either because of the low concentrations that resulted in from using whole cell lysates in the ELISA. In repeating the ELISA, we would ideally use a higher concentration of purified samples from different extractions of the appropriate cellular compartments as a comparison like originally planned, rather than the un-purified samples. This would provide a more accurate representation of the culture yield. In the glucose uptake assay we would use higher concentrations of a purified sample rather than just the unpurified extracts to prevent interference of other cellular proteins to give a better assessment of function.

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