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Commit 9fed04b8 authored by Lucy Hao's avatar Lucy Hao :moyai:
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components/Figure.js
+ 35
− 3
View file @ 9fed04b8
......@@ -44,17 +44,41 @@ const Img = styled.img`
}
`;
const RowImg = styled.img`
height: 40vh;
@media only screen and (max-width: 768px) {
width: 80vw;
height: auto;
}
`;
const Center = styled.div`
display: flex;
justify-content: center;
margin: 1%;
`;
export default function Figure({ src, long, num, caption, alternate, table }) {
export default function Figure({
src,
long,
num,
caption,
alternate,
table,
imgs,
}) {
return caption ? (
<figure style={{ textAlign: "center" }}>
<Center>
<Img src={src} />
{imgs ? (
<div>
{imgs.map((i) => (
<RowImg src={i} />
))}
</div>
) : (
<Img src={src} />
)}
</Center>
{alternate ? (
<AlternateCaption>
......@@ -78,7 +102,15 @@ export default function Figure({ src, long, num, caption, alternate, table }) {
</figure>
) : (
<Center>
<Img src={src} />
{imgs ? (
<div>
{imgs.map((i) => (
<RowImg src={i} />
))}
</div>
) : (
<Img src={src} />
)}
</Center>
);
}
pages/results.mdx
+ 232
− 89
View file @ 9fed04b8
......@@ -10,7 +10,7 @@ export const Page = ({ children }) => (
<ContentLayout meta={meta} children={children} />
);
<div className="padded-content">
<div className="alternate-colors-curved-background padded-content">
## Construct Assembly
On Benchling, the mock restriction digest feature was utilized to
......@@ -24,50 +24,96 @@ amplified out pSB1C3 from pSB1C3-amilCP in the 2021 distribution kit
(Plate 1 Well 19E, bba_k592009) and used parts of the amilC P insert
as primer binding sites (Figures 1 and 2).
<Figure src="https://static.igem.wiki/teams/4796/wiki/results/image7.png" />
<Figure num="1" caption="Diagnostic digest of pSB1C3-amilCP plasmids, run against
Froggabio 1kb+ plus ladders. Lanes 1 to 3 show pSB1C3-amilCP plasmids
miniprepped from separate *E. coli* culture replicates (A1, A2, A6)
and digested with BbsI. Diagnostic digest appears to match virtual
Benchling digest." src="https://static.igem.wiki/teams/4796/wiki/results/image17.png" />
<Figure num="2" caption="Diagnostic digest of pSB1C3 backbone DNA amplified out
<Figure
alternate
num="1"
caption={
<>
Diagnostic digest of pSB1C3-amilCP plasmids, run against Froggabio 1kb+
plus ladders. Lanes 1 to 3 show pSB1C3-amilCP plasmids miniprepped from
separate <i>E. coli</i> culture replicates (A1, A2, A6) and digested with
BbsI. Diagnostic digest appears to match virtual Benchling digest.
</>
}
imgs={[
"https://static.igem.wiki/teams/4796/wiki/results/image7.png",
"https://static.igem.wiki/teams/4796/wiki/results/image17.png",
]}
/>
<Figure
alternate
num="2"
caption="Diagnostic digest of pSB1C3 backbone DNA amplified out
from pSB1C3-amilCP plasmid using PCR. Run against Froggabio 1kb+ plus
ladder. Lane 1 shows undigested PCR-amplified pSB1C3 DNA. Lane 2 shows
PCR-amplified pSB1C3 DNA digested with Pstl and Xbal. Lane 3 shows
PCR-amplified pSB1C3 DNA digested with EcoRI and SpeI. Diagnostic
digest appears to match virtual Benchling digest." src="https://static.igem.wiki/teams/4796/wiki/results/image15.png" />
digest appears to match virtual Benchling digest."
src="https://static.igem.wiki/teams/4796/wiki/results/image15.png"
/>
We assembled our three main plasmid constructs (Staygold Expression
Vector T3C, Mdh-Fls, and PC-Dhak) using Gibson Assembly, and followed up
with colony PCRs and diagnostic digests to confirm expected band sizes
(Figures 3 to 5).
<Figure num="3" caption="Colony PCR of pSB1C3-StayGold-iTag(T3C) plasmid
construct using 5' UTR-StayGold FWD and REV primers (left) and
pSB1C3-Mdh-Fls using T7pro-5UTR-inteinC FWD and 5UTR-inteinC-Fls REV
primers (right). Run against NEB 1 kb Plus DNA Ladder. Lanes 1 to 4
show DNA that was PCR-amplified out from pSB1C3-StayGold-iTag(T3C)
plasmids miniprepped from separate *E. coli* culture replicates (E1,
E2, E3, E4). Lanes 5 to 8 show DNA that was PCR-amplified out from
pSB1C3-Mdh-Fls plasmids miniprepped from separate *E. coli* culture
replicates (M1, M2, M3, M4). Expected band size of
pSB1C3-StayGold-iTag(T3C) was 1693 bp, and lanes 1 to 4 appear to be
correct. Expected band size of pSB1C3-Mdh-Fls was 3757 bp; lanes 5 to
8 contain a major band with the correct size, but also a lower and
upper band that may have resulted from nonspecific primer binding." src="https://static.igem.wiki/teams/4796/wiki/results/image12.png" />
<Figure src="https://static.igem.wiki/teams/4796/wiki/results/image10.png" />
<Figure num="4" caption="Diagnostic digest of pSB1C3-Mdh-Fls plasmid construct, run
against Froggabio 1kb+ plus ladder. Lanes 1 and 3 show undigested
pSB1C3-Mdh-Fls plasmids miniprepped from separate *E. coli* culture
replicates (M1, M3). Lanes 2 and 4 show pSB1C3-Mdh-Fls plasmids digested
with EcoRI-HF. Diagnostic digest appears to match virtual Benchling
digest." src="https://static.igem.wiki/teams/4796/wiki/results/image18.png" />
<Figure num="5" caption="Colony PCR of pSB1C3-PC-Dhak construct using F1 FWD and F13 REV primers. Run against Froggabio 1kb+ plus ladder. Lanes 1 to 8 show DNA that was PCR-amplified out from pSB1C3-PC-Dhak plasmids miniprepped from separate transformed *E. coli* culture replicates (M1 to M8). Expected band size was 756 bp. Lanes 1 and 2 appear to indicate correctly assembled pSB1C3-PC-Dhak plasmid constructs." src="https://static.igem.wiki/teams/4796/wiki/results/image1.png" />
<Figure
alternate
num="3"
caption={
<>
Colony PCR of pSB1C3-StayGold-iTag(T3C) plasmid construct using 5'
UTR-StayGold FWD and REV primers (left) and pSB1C3-Mdh-Fls using
T7pro-5UTR-inteinC FWD and 5UTR-inteinC-Fls REV primers (right). Run
against NEB 1 kb Plus DNA Ladder. Lanes 1 to 4 show DNA that was
PCR-amplified out from pSB1C3-StayGold-iTag(T3C) plasmids miniprepped from
separate <i>E. coli</i> culture replicates (E1, E2, E3, E4). Lanes 5 to 8
show DNA that was PCR-amplified out from pSB1C3-Mdh-Fls plasmids
miniprepped from separate <i>E. coli</i> culture replicates (M1, M2, M3,
M4). Expected band size of pSB1C3-StayGold-iTag(T3C) was 1693 bp, and
lanes 1 to 4 appear to be correct. Expected band size of pSB1C3-Mdh-Fls
was 3757 bp; lanes 5 to 8 contain a major band with the correct size, but
also a lower and upper band that may have resulted from nonspecific primer
binding.
</>
}
src="https://static.igem.wiki/teams/4796/wiki/results/image12.png"
/>
<Figure
alternate
num="4"
caption={
<>
Diagnostic digest of pSB1C3-Mdh-Fls plasmid construct, run against
Froggabio 1kb+ plus ladder. Lanes 1 and 3 show undigested pSB1C3-Mdh-Fls
plasmids miniprepped from separate <i>E. coli</i> culture replicates (M1,
M3). Lanes 2 and 4 show pSB1C3-Mdh-Fls plasmids digested with EcoRI-HF.
Diagnostic digest appears to match virtual Benchling digest.
</>
}
imgs={[
"https://static.igem.wiki/teams/4796/wiki/results/image10.png",
"https://static.igem.wiki/teams/4796/wiki/results/image18.png",
]}
/>
<Figure
alternate
num="5"
caption={
<>
Colony PCR of pSB1C3-PC-Dhak construct using F1 FWD and F13 REV primers.
Run against Froggabio 1kb+ plus ladder. Lanes 1 to 8 show DNA that was
PCR-amplified out from pSB1C3-PC-Dhak plasmids miniprepped from separate
transformed <i>E. coli</i> culture replicates (M1 to M8). Expected band
size was 756 bp. Lanes 1 and 2 appear to indicate correctly assembled
pSB1C3-PC-Dhak plasmid constructs.
</>
}
src="https://static.igem.wiki/teams/4796/wiki/results/image1.png"
/>
We used site-directed mutagenesis on the Staygold Expression Vector to
revert the T3C construct back to the original Control sequence. We
......@@ -75,9 +121,25 @@ followed up with a diagnostic digest to confirm the success of
site-directed mutagenesis, as it disrupts an engineered NdeI site at the
third amino acid on the intein (Figure 6).
<Figure src="https://static.igem.wiki/teams/4796/wiki/results/image3.png" />
<Figure num="6" caption="Diagnostic digest of mutated Staygold Expression Vector (Control) plasmid construct, run against Froggabio 1kb+ plus ladder. Lanes 1 and 2 show Staygold Expression Vector (Control) plasmids miniprepped from separate *E. coli* culture replicates and digested with NdeI and EcoRI. Diagnostic digest appears to indicate that the Ndel enzyme cut site was disrupted, as the bands match the virtual Benchling digest for pSB1C3-Staygold-iTag(Control)." src="https://static.igem.wiki/teams/4796/wiki/results/image11.png" />
<Figure
alternate
num="6"
caption={
<>
Diagnostic digest of mutated Staygold Expression Vector (Control) plasmid
construct, run against Froggabio 1kb+ plus ladder. Lanes 1 and 2 show
Staygold Expression Vector (Control) plasmids miniprepped from separate{" "}
<i>E. coli</i> culture replicates and digested with NdeI and EcoRI.
Diagnostic digest appears to indicate that the Ndel enzyme cut site was
disrupted, as the bands match the virtual Benchling digest for
pSB1C3-Staygold-iTag(Control).
</>
}
imgs={[
"https://static.igem.wiki/teams/4796/wiki/results/image3.png",
"https://static.igem.wiki/teams/4796/wiki/results/image11.png",
]}
/>
## Sequencing results
......@@ -88,28 +150,58 @@ confirm PC-Dhak despite multiple sequencing attempts so we used whole
plasmid sequencing. Through Plasmidsaurus, we were able to confirm that
the PC-Dhak was successfully cloned.
<Figure num="7" caption="Whole plasmid map of StayGold Expression Vector Construct, generated by Plasmidsaurus." src="https://static.igem.wiki/teams/4796/wiki/results/image16.png" />
<Figure num="8" caption="Whole plasmid map of PC-Dhak Construct, generated by Plasmidsaurus." src="https://static.igem.wiki/teams/4796/wiki/results/image13.png" />
## Chassis and Construct Characterization
**Growth Curve Comparison Between *V. natriegens* and *E.
coli***
<Figure num="9" caption="Growth curves of *E. coli* transformed with our constructs and grown in 150 uL of LB on a 96 well plate, normalized against LB only blank." src="https://static.igem.wiki/teams/4796/wiki/results/image2.png" />
<Figure num="10" caption="Growth curves of *V. natriegens* transformed with our constructs and grown in 150 uL of LB on a 96 well plate, normalized against LB only blank." src="https://static.igem.wiki/teams/4796/wiki/results/image14.png" />
Our growth curve comparisons between *V. natriegen*s and *E. coli*
(Figures 9 and 10) showed that *V. natriegens* had faster growth rates
(untransformed *V. nats* 0.0439h^-1^ compared to untransformed *E. coli*
0.0376h^-1^) and doubling times (untransformed *V. nats* 15.77h compared
to untransformed *E. coli* 18.41h). However, both bacteria cultures were
<Figure
alternate
num="7"
caption="Whole plasmid map of StayGold Expression Vector Construct, generated by Plasmidsaurus."
src="https://static.igem.wiki/teams/4796/wiki/results/image16.png"
/>
<Figure
alternate
num="8"
caption="Whole plasmid map of PC-Dhak Construct, generated by Plasmidsaurus."
src="https://static.igem.wiki/teams/4796/wiki/results/image13.png"
/>
### Chassis and Construct Characterization
#### Growth Curve Comparison Between _V. natriegens_ and _E.coli_
<Figure
alternate
num="9"
caption={
<>
Growth curves of <i>E. coli</i> transformed with our constructs and grown
in 150 uL of LB on a 96 well plate, normalized against LB only blank.
</>
}
src="https://static.igem.wiki/teams/4796/wiki/results/image2.png"
/>
<Figure
alternate
num="10"
caption={
<>
Growth curves of <i>V. natriegens</i> transformed with our constructs and
grown in 150 uL of LB on a 96 well plate, normalized against LB only
blank.
</>
}
src="https://static.igem.wiki/teams/4796/wiki/results/image14.png"
/>
Our growth curve comparisons between *V. natriegen*s and _E. coli_
(Figures 9 and 10) showed that _V. natriegens_ had faster growth rates
(untransformed _V. nats_ 0.0439h<sup>-1</sup> compared to untransformed _E. coli_
0.0376h<sup>-1</sup>) and doubling times (untransformed _V. nats_ 15.77h compared
to untransformed _E. coli_ 18.41h). However, both bacteria cultures were
only able to grow to an optical density of approximately 2 after 13
hours, which is far lower compared to literature values showing V. nats
could reach optical densities upwards of 15 after 8 hours[^1].
Furthermore, both *V. nats and E. coli* doubling times were
Furthermore, both _V. nats and E. coli_ doubling times were
significantly lower compared to literature values reporting 10 minutes
and 20 minutes respectively[^2][^3].
......@@ -117,27 +209,39 @@ Since our bacteria were grown in 96 well plates with limited LB media,
aeration, and agitation, these unfavorable conditions were likely the
largest limiting factor that prevented our cultures from reaching the
reported literature values. However, we were able to show that even
under poor growth conditions, both untransformed and transformed *V.
natriegens* with our plasmid constructs were able to grow faster than
*E. coli* counterparts. Our growth curve comparisons demonstrate that
under similar conditions, *V. natriegens* demonstrates accelerated
under poor growth conditions, both untransformed and transformed _V.
natriegens_ with our plasmid constructs were able to grow faster than
_E. coli_ counterparts. Our growth curve comparisons demonstrate that
under similar conditions, _V. natriegens_ demonstrates accelerated
growth rates thus confirming our choice in chassis for our protein
production pillar. We can confidently say that *V.natriegens* is both a
production pillar. We can confidently say that _V.natriegens_ is both a
suitable and effective choice in bacteria to use as cell lysate for CFPS
preparation.
### Methanol Death Curve
*V. natriegens*, is a halophilic and non methylotrophic bacterial
_V. natriegens_, is a halophilic and non methylotrophic bacterial
strain, thus the introduction of methanol would act to inhibit the
growth of the bacteria. To test the upper and lower bounds of growth
inhibition we performed two methanol tolerance experiments. Our first
methanol growth curve experiment tested untransformed V. *natriegens*
methanol growth curve experiment tested untransformed V. _natriegens_
tolerance in a range from 1-1000mM methanol, with a 2 factor dilution.
<Figure num="11" caption="Methanol death curve of untransformed *V. natriegens* Vmax X2, grown in 300 uL of LB at methanol concentrations ranging from 1-1000mM. OD600 measurements taken 135 minutes after inoculation, normalized against LB blanks." src="https://static.igem.wiki/teams/4796/wiki/results/image8.png" />
As seen in Figure 9, *V. nats* can survive in up to 500 mM of methanol.
<Figure
alternate
num="11"
caption={
<>
Methanol death curve of untransformed <i>V. natriegens</i> Vmax X2, grown
in 300 uL of LB at methanol concentrations ranging from 1-1000mM. OD600
measurements taken 135 minutes after inoculation, normalized against LB
blanks.
</>
}
src="https://static.igem.wiki/teams/4796/wiki/results/image8.png"
/>
As seen in Figure 9, _V. nats_ can survive in up to 500 mM of methanol.
This is useful as methanol is a strong carbon source due to its
widespread availability and 50% higher ATP yield per carbon relative to
glucose[^4]. Methanol is also a cheaper and more energy-dense fuel source
......@@ -153,9 +257,33 @@ Figure 9 also shows that at higher concentrations such as 1000 mM,
methanol can act as a kill switch, allowing for precise control over the
growth of our bacteria.
<Figure num="12" caption="Methanol death curve of *E. coli* transformed with our plasmid constructs, grown in 300 uL of LB at methanol concentrations ranging from 62.5-1000mM. OD600 measurements taken 135 minutes after inoculation and normalized against LB blanks at respective methanol concentrations." src="https://static.igem.wiki/teams/4796/wiki/results/image6.png" />
<Figure num="13" caption="Methanol death curve of *E. coli* transformed with our plasmid constructs, grown in 300 uL of LB at methanol concentrations ranging from 62.5-1000mM. OD600 measurements taken 255 minutes after inoculation and normalized against LB blanks at respective methanol concentrations." src="https://static.igem.wiki/teams/4796/wiki/results/image5.png" />
<Figure
alternate
num="12"
caption={
<>
Methanol death curve of <i>E. coli</i> transformed with our plasmid
constructs, grown in 300 uL of LB at methanol concentrations ranging from
62.5-1000mM. OD600 measurements taken 135 minutes after inoculation and
normalized against LB blanks at respective methanol concentrations.
</>
}
src="https://static.igem.wiki/teams/4796/wiki/results/image6.png"
/>
<Figure
alternate
num="13"
caption={
<>
Methanol death curve of <i>E. coli</i> transformed with our plasmid
constructs, grown in 300 uL of LB at methanol concentrations ranging from
62.5-1000mM. OD600 measurements taken 255 minutes after inoculation and
normalized against LB blanks at respective methanol concentrations.
</>
}
src="https://static.igem.wiki/teams/4796/wiki/results/image5.png"
/>
Our second experiment tested our assembled plasmid constructs in E. coli
to characterize potential metabolic burdens that the plasmids may have.
......@@ -167,13 +295,18 @@ construct caused the largest metabolic burden, likely due to the large
size of the recombinant protein product and the fact that it codes for
only the DhaK enzyme and not any prior enzymes in the methanol
metabolism pathway. However, when all three enzymes are together in the
*E. col*i coculture of both PC Dhak and Mdh Fls, the cell density was
*E. coli* coculture of both PC Dhak and Mdh Fls, the cell density was
restored back to similar levels as the control. This demonstrates the
success of the dual plasmid methanol utilization vector.
### CFPS Reporter Fluorescence Activity
### CFPS Reporter Fluorescence Activity
<Figure num="14" caption="Fluorescence measurements on CFPS run at 575/625 nm, at that wavelength, normalized against no plasmid control." src="https://static.igem.wiki/teams/4796/wiki/results/image9.png" />
<Figure
alternate
num="14"
caption="Fluorescence measurements on CFPS run at 575/625 nm, at that wavelength, normalized against no plasmid control."
src="https://static.igem.wiki/teams/4796/wiki/results/image9.png"
/>
As discussed in our project design page, we designed our methanol
utilization vector with a dual plasmid reporter system. Each of the
......@@ -195,23 +328,33 @@ system are greater than each of the plasmids individually, confirming
the restoration of mCherry-XL fluorescence and thus the success of our
split intein mechanism in CFPS.
<Figure num="15" caption="CFPS reaction fluorescence growth curve measured at 558/589 nm, normalized against no plasmid control." src="https://static.igem.wiki/teams/4796/wiki/results/image4.png" />
[^1]: Weinstock MT, Hesek ED, Wilson CM, Gibson DG. Vibrio natriegens as
a fast-growing host for molecular biology. Nature Methods. 2016 Oct
13;13(10):849-851. doi:10.1038/nmeth.3970
[^2]: Xu J, Dong F, Wu M, Tao R, Yang J, Wu M, Jiang Y, Yang S, & Yang
L. Vibrio natriegens as a pET-Compatible Expression Host Complementary
to Escherichia coli. Frontiers in microbiology. 2021;12, 627181. https://doi.org/10.3389/fmicb.2021.627181
[^3]: Gibson B, Wilson DJ, Feil E, Eyre-Walker A. The distribution of
bacterial doubling times in the wild. *Proc Biol Sci*.
2018;285(1880):20180789. doi:10.1098/rspb.2018.0789
<Figure
alternate
num="15"
caption="CFPS reaction fluorescence growth curve measured at 558/589 nm, normalized against no plasmid control."
src="https://static.igem.wiki/teams/4796/wiki/results/image4.png"
/>
[^1]:
Weinstock MT, Hesek ED, Wilson CM, Gibson DG. Vibrio natriegens as
a fast-growing host for molecular biology. Nature Methods. 2016 Oct
13;13(10):849-851. doi:10.1038/nmeth.3970
[^2]:
Xu J, Dong F, Wu M, Tao R, Yang J, Wu M, Jiang Y, Yang S, & Yang
L. Vibrio natriegens as a pET-Compatible Expression Host Complementary
to Escherichia coli. Frontiers in microbiology. 2021;12, 627181. https://doi.org/10.3389/fmicb.2021.627181
[^3]:
Gibson B, Wilson DJ, Feil E, Eyre-Walker A. The distribution of
bacterial doubling times in the wild. _Proc Biol Sci_.
2018;285(1880):20180789. doi:10.1098/rspb.2018.0789
[^4]:
Sarwar A., & Lee EY. Methanol-based biomanufacturing of fuels and
chemicals using native and synthetic methylotrophs. Synthetic and
systems biotechnology. 2023;8(3), 396--415. https://doi.org/10.1016/j.synbio.2023.06.001
[^4]: Sarwar A., & Lee EY. Methanol-based biomanufacturing of fuels and
chemicals using native and synthetic methylotrophs. Synthetic and
systems biotechnology. 2023;8(3), 396--415. https://doi.org/10.1016/j.synbio.2023.06.001
</div>
export default Page;
\ No newline at end of file
pages/usermanual.mdx
+ 2
− 2
View file @ 9fed04b8
......@@ -20,7 +20,7 @@ export const meta = {
Cell-free Protein Synthesis is the scalable, _in-vitro_ production of recombinant proteins. UBC iGEM’s CFPS kit, PILOT (Platformed Inteins: a Linked Orthogonal Toolkit), is a versatile intein-mediated tool designed to manufacture biologics by taking advantage of a *Vibrio natriegens* lysate alongside our 3 expression vectors. PILOT is a prokaryotic lysate bioproduction platform for simultaneous biosynthesis and purification of proteins. We have designed our primary expression plasmid to be modular, allowing for any researcher to easily synthesize and purify their genes of interest for a variety of research applications.
<Figure src="https://static.igem.wiki/teams/4796/wiki/usermanual/cfps.svg" num="1" caption="Graphic demonstrating a cell free protein synthesis reaction and its components."/>
<Figure src="https://static.igem.wiki/teams/4796/wiki/usermanual/cfps.svg" num="1" caption="Graphic demonstrating a cell free protein synthesis reaction and its components." alternate/>
## Project Pillars
......@@ -316,7 +316,7 @@ folding stability. This is highly pertinable to research groups trying
to evaluate and assess their engineered biologic/drug design. Knowing
how protein stability can be affected by systematic mutations along the
protein's primary sequence may be important for assessing binding
affinity to a particular drug target (See [modeling](/ubc-vancouver/proteinmodelling) page).
affinity to a particular drug target (See [modeling](/ubc-vancouver/model) page).
In another feature, we have modeled the MxyGyrA intein-protein fusion in
aqueous solvent. This provides users with a predictive model to
......
utils/SebneueRegular.otf deleted 100644 → 0
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utils/fonts.js
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import { Josefin_Sans } from "next/font/google";
// export const headerFont = localFont({ src: "./Besan.otf" });
export const headerFont = Josefin_Sans({
weight: "700",
subsets: ["latin"],
......
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