Team:Sydney Australia/Notebook
-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.
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.
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.
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.
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!
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.
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!
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.
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.
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.
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.
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.
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
Wet Lab:
•Erica and Alex reattempted transforming pSB1C3::Ecotin Proinsulin and
pSB1C3::Ecotin Winsulin. These transformations worked!
•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.
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.
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!
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.
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.
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.
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.
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
•Jamie sent off pSB1C3::YncM Proinsulin for sequencing to verify the insert
Education and public engagement:
•The team attended the Synthetic Biology Australasia conference and Emaleen
and Jamie gave a short presentation on our project
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.
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
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.
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:
WB800 pUS258::YncM proinsulin
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
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.
•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.
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
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
