iGEM 2023 — Team cu-boulder

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{% block title %}Notebook{% endblock %}
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## Alpha Lactabulin notebook

2/2/23: designing Twist alpha-lactalbumin (a-lac) gene
2/14/23: First golden gate cloning
13.6 uL water
2 ul Ligase buffer
1 uL AmpBC backbone
1.4 uL a-lac gene
0.75 uL BsaI
1.25 uL ligase
2/20/23: performing E.Coli transformation and plating
2/21/23: plate did not grow overnight. Redoing the same golden gate procedure from the 14th but thermocycling for 18hrs instead of 1hr
2/22/23: transforming and plating
2/27/23: picked three white colonie for grow up
3/1/23: did mini preps to isolate the DNA
3/2/23: digest and gel
Digest:
13 uL water
2 uL buffer
3 uL DNA
0.5 uL Oligo
0.5 uL AarI
Gel:

![](https://static.igem.wiki/teams/4919/wiki/notebook1.png)
![](https://static.igem.wiki/teams/4919/wiki/notebook2.png)

##

3/9/23: DNA sent for sequencing
3/14/23: sequencing results aligned perfectly! Now adding promoter, Gmubi, and terminator, hstp.
3/20/23: Doing overnight grow up of glycerol stocks of the promoter and terminator
3/25/23: Doing a mini prep of the promoter and terminator
4/5/23: doing a 18hr golden gate
10.82uL water
2 uL ligase buffer
0.6 uL ChlorAD-B backbone
0.3 Gmubi
2 uL a-lac
1.75 uL hspt
0.75 PaqC1
0.5 uL restriction enzyme activator
1.25 uL ligase
4/6/23; transforming and plating
4/7/23: plate looks good grabbing white colonies for grow ups

![](https://static.igem.wiki/teams/4919/wiki/notebook3.png)

##

4/11/23: Minipreps of the three grow ups
4/12/23: digest and gel electrophoresis
Digest:
14 uL water
2 uL buffer
3 uL DNA
0.5 uL BsaI
0.5 uL BaeI
Thermocycle

![](https://static.igem.wiki/teams/4919/wiki/notebook4.png)
![](https://static.igem.wiki/teams/4919/wiki/notebook5.png)

##

Not seeing three separate bands we are looking for.
4/17/23: Redoing the golden gate but increasing concentrations
8.2 uL water
2 uL ligase buffer
1.3 uL ChlorA DB
3 uL a-lac
2.75 uL HSPt
0.75 uL PaqC1
0.75 uL activator
1.25 uL ligase
4/19/23: transfecting and plating
4/20/23: The plates have all blue conlines and no white colonies
5/15/23:Redoing golden gate but increasing inserts but not the backbone concentrations
6.25 uL water
2 uL ligase buffer
0.6 uL ChlorA DB
1 uL Gmubi
4 uL a-lac
3.5 uL HSPt
0.75 uL PaqC1
0.75 uL Activator
1.25 uL ligase
5/17/23: transforming and plating
5/18/13: the plates did not grow any colonies
5/26/23: Not sure why the plates are not growing. Performing digest of ChlorA DB, Gmubi, and HSPt to see if they have the right DNA

![](https://static.igem.wiki/teams/4919/wiki/notebook6.png)

lane (first and last lanes are 1kb ladder)
1; Gmubi high
2; " low
3; HSPt high
4; " low
5; Chlor P high
6; Chlor P low
7; Chlor B high
8; Chlor B low

![](https://static.igem.wiki/teams/4919/wiki/notebook7.png)

##

All bands look as expected so now will redo all the math for the golden gate procedure as the inserts do not seem to be the problem
5/29/23: new golden gate
7 uL water
2 uL ligase buffer
0.6 ChlorA DB
2.7 Gmubi
2 uL a-lac
3.2 uL HSPt
0.75 uL PaqC1
0.5 uL activator
1.25 ligase
Did a second golden gate where I doubled the volume of HSPt, Gmubi, and a-lac.
5/30/23: transformation and plating
5/31/23: first golden gate plate had two white colonies I picked for grow ups

![](https://static.igem.wiki/teams/4919/wiki/notebook8.png)

##

6/2/23: mini preps, digest, and gel electrophoresis
Did a double restriction digest because BsaI cuts two fragments of very similar lengths
Digest:
14 uL water
2 uL buffer
3 uL DNA
0.5 uL BsaI
0,5 uL BaeI
Thermocycle
Gel electrophoresis:
lane 1 ladder
lane 2 sample 1 just BsaI
Lane 3 sample 1 just BsaI
Lane 4 sample 2 just BsaI
Lane 5 sample 2 just Bsal
Lane 6 sample 1 just BaeI
Lane 7 sample 1 just BaeI
Lane 8 uncut sample 1
Lane 9 uncut sample 2
lane 10 ladder
bottom lanes:
lane 1 ladder
lane 2 sample 2 just BaeI
Lane 3 sample 2 just BaeI
Lane 4 sample 1 uncut
Lane 5 sample 2 uncut

![](https://static.igem.wiki/teams/4919/wiki/notebook9.png)
![](https://static.igem.wiki/teams/4919/wiki/notebook10.png)

##

Picked colonies for an overnight grow up
7/18/23:
Digest and gel electrophoresis
Digest:
14 uL water
2 uL buffer
3 uL DNA
1 uL PSTI
Thermocycle
gel key:
Expected

![](https://static.igem.wiki/teams/4919/wiki/notebook12.png)
![](https://static.igem.wiki/teams/4919/wiki/notebook13.png)

Actual
1kb ladder, 1.1, 1.2, 1.3, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7

##

First three lanes are showing the correct band lengths
2 first lanes sent off for sequencing
8/8/23: all sequences aligned correctly!
8/25/23: Found out from other experiments the RUBY marker is not working as desired so will be now using a GUS marker and redoing the last plasmid golden gate with GUS in place of RUBY
10/5/23: GUS plasmid was constructed by a lab Technician and is now ready to go into the last backbone
Golden gate
11.3 uL water
2 uL ligase buffer
1.2 uL pBEHA2 backbone
1.2 uL GUS
2.3 uL Chlor A DB/a-lax/gmubi/HSPt
0.75 uL BsaI
1.25 uL ligase
10/6/23: transformation and mini prep
10/10/23: digest and gel electrophoresis
Digest:
14.5 uL water
2 uL buffer
3 uL DNA
0.5 uL BsaI
Thermocycle
Gel electrophoresis

![](https://static.igem.wiki/teams/4919/wiki/notebook14.png)
![](https://static.igem.wiki/teams/4919/wiki/notebook15.png)

##

Can’t see the lower band so will do a digest with a different restriction enzyme to get cut sites that are easier to visualize on a gel

## Beta Lactoglobulin notebook

DNA shipment and processing:
We received the TWIST order this weekend. Planning on transfecting the Bglobin DNA into NEB chemically competent cells.
DNA is stored in box label: B-Lactoglobulin
1000ng DNA was diluted with 20ul H20 for a concentration of 20ng/ul
Initial GoldenGate:
2/13/2023
Bglobulin insert (30ng goal @ 50ng/ul)- 1ul
Amp B BC Plasmid(70ng goal at 140ng/ul)- .5ul
T4 Ligase buffer (10x)- 2ul
T4 Ligase- 1.25ul
BsaI- .75ul
H20- 14.5ul
Total Volume- 20 ul
60 minute Golden Gate ran, store in Bglobulin box.
Cell transformation:
Date performed: 02/15
Changes in yellow
Colony selection:
Plates grew well from 2/15-2/16 with many white colonies and no blue colonies.
4 colonis were picked (2 from each plate) and grown out overnight (2/16-2/17)
On 2/17 grow outs were drained and placed in freezer.
Initial Miniprep:
2/20: Dna was miniprepped
Dna Digestion for insert conformation:
02/27/2023
Dna was sigested with PAQC1
13ul H20
2ul Cut Smart
3ul DNA
.5ul PAQC1
.5ul Activator
---------------
20 ul total
60 min GG
Gel For Confirmation:
Confirmed..
DNA Sequencing Results:
Perfect, both BG 2,1 and BG 1,1 contain no errors
Promoter/ Terminator Golden Gate Planning:
A- Glycine Max Ubiquitin promoter - B - Beta Lactoglobulin - C - Arabadopsis therm HSP Terminator- D
into
A- XX -D-B Plasmid
PAQC1 is used to open primary assembly
Golden Gate- Preformed on 3/13/23 :
Chlor A-DB- 55ng/ul stock- .75ul
GMubi- 104ng/ul stock- .5 ul
Beta Lactoglobulin (BG 2,1)- 60 ng/ul- 1ul
Arabdopsis Term- 85 ng/ul
Cell Transformation Chlor ADB with B globulin and promoter/term:
Date performed: 03/14/2023
Notes:
Used 2 5ul aloquats of the same cells, i am planning on making 4 plates 2 from each.
Pro- Bg- Term grow outs:
Went pretty well, not a lot of colonies but there is atleast 1 left on each plate.
Mini Prep concentrations:
1.1
100 ng/ul
1.2
100 ng/ul
1.3
150 ng/ul
2.1
150 ng/ul
Pro- Beta globulin- Term confirmatory gel:
Virtual Digest:
Real BsaI Digest:
BG assembly + Chlor BD Ruby Planning
plSUK backbone- .05 picomolar = 150 nanograms - 1.75 ul
Betaglobulin assembly - 1,500 Bp- .1 picomolar= 97.5 nanograms- .75 ul
\*\* used Betaglobulin - promoter/ Term assembly 2.1 from plate (A1-2) colony #1 \*\*
Ruby - 6000 Bp- .1 picomolar = 390 nanograms- 1.75 ul
T4 Ligase buffer (10x)- 2ul
T4 Ligase- 1.25ul
BsaI- .75ul
H20- 11.75 ul
Total Volume- 20 ul
T4 Ligase buffer (10x)- 2ul
T4 Ligase- 1.25ul
BsaI- .75ul
PaqcI - .5 ul
PaqcI activator - .5 ul
H20- 12.4
Total Volume- 20 ul
7/12/23 Golden Gate Planning for Recovered Lactoferrin to go into simple ( AmpB A-B)
AmpB A-B (@97ng/ul) - 85ng required = .85ul
Lactoferrin Recovered 2 (@80 ng/ul) - 200 ul required = 2.75 ul
BsaI- .75ul
T4 Ligase Buffer (10x) - 2ul
T4 Ligase - 1.25ul
H20- 12.4 ul
Total Volume- 20ul
7/26/2023
Betaglobulin-PBEHA and Lactoferrin-Chlor-Adb transformation
LB Agar Plate recipe: Kanamycin
(Per 20ml agar plate)
Xgal- 40ul
IPTG- 100x- 200ul Iptg
Kanna - 20ul
LB Agar Plate recipe: Chlor
(Per 20ml agar plate)
Xgal- 40ul
IPTG- 100x- 200ul Iptg
Chlor- 20ul
10/6/2023
Betaglobulin-PBEHA with GUS marker Golden Gate
Golden Gate Recipe
pBEHAbackbone- .05 picomolar = 220nanograms (@ 250 ng/ul)= - .75 ul
Betaglobulin assembly - 1,500 Bp- .1 picomolar= 82.5 ng @ ( 150ng/ul)= .75
\*\* used Betaglobulin - promoter/ Term assembly 2.1 from plate (A1-2) colony #1 \*\*
GUS marker- .75 ul
T4 Ligase buffer (10x)- 2ul
T4 Ligase- 1.25ul
BsaI- .75ul
PaqcI - .5 ul
PaqcI activator - .5 ul
H20- 12.4
Total Volume- 20 ul
Mini Prep 10/8/23
Prepped with Quiagen Mini-prep. Miniprep was 50ul at 200ng/ul.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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The CU-Boulder iGEM team has demonstrated notable success in engineering plants. Our team focuses on utilizing soybeans to produce our proteins as it is a sustainable and inexpensive crop to mass produce. With that, our genetic modifications must be optimized for expression within the soybean. The first modifications we made was codon optimization of our bovine genes to be expressed in soybeans. The codons that code for a protein in one organism are not favored by another organism and this is the case for cow milk proteins and soybeans. The codons have been changed for the soybean optimized versions to produce the same amino acid but using a codon that the soybean is more familiar with. Another thing we had to engineer to utilize the soybean as a chassis is the export signals. The export signals for mammals and plants are significantly different, so we replaced the bovine export signal with a plant export signal for proper function in the soybean.

Further engineering techniques the CU-Boulder iGEM team has utilized and fulfilled is the use of Golden Gate cloning. The idea is when we are making the vector with all our parts such as the promoter, desired sequence, terminators, ect., that the insertion of the part cannot be reversed. This ensures that every vector going through the golden gate has the added sequence permanently attached. We also utilize very specific cut sites to ensure the desired sequence is inserted at a specific site as well as preventing the sequence from being reversed in order.

The CU-Boulder iGEM team plans to test our soybean protein sequence via western blot. This page will be updated with those results and what potential design changes we can utilize in the future.

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### Constructing, Introducing, Screening, Replicating, and Isolating the Newly Constructed Plasmid

**Newly constructed plasmids are integrated into E. coli as a means to screen correctly
assembled colonies, replicate it into quantifiable amounts, and then isolate it back out to be used for the next
assembly.**

* [Constructing: Golden Gate Assembly](https://static.igem.wiki/teams/4201/wiki/experiments/golden-gate-2-protocol.pdf)
* [Introducing: Electroporation](https://static.igem.wiki/teams/4201/wiki/experiments/electroporation-protocol.pdf)
* [Screening: Plating](https://static.igem.wiki/teams/4201/wiki/experiments/plating-protocol.pdf)
* [Replication: Colony Selection and Overnight Broth](https://static.igem.wiki/teams/4201/wiki/experiments/broth-protocol.pdf)
* [Isolation: Qiagen QIAprep Miniprep](https://static.igem.wiki/teams/4201/wiki/experiments/miniprep-protocol.pdf)
* [Plant Transformation](https://static.igem.wiki/teams/4201/wiki/experiments/plant-transformation-protocol.pdf)

---

### Restriction Enzyme Sequence Verification

**While the antibiotic resistance and blue/white coloration suggest a successful goldengate
assembly, this can be further verified through a restriction enzyme digest and gel electrophoresis.**

* [Restriction Enzyme Digest](https://static.igem.wiki/teams/4201/wiki/experiments/digest-protocol.pdf)
* [Gel
  Electrophoresis](https://static.igem.wiki/teams/4201/wiki/experiments/gel-protocol.pdf)

---

### Competent Cell Preparation

**It is much cheaper to reproduce your own stocks of electrocompetent cells than to be
consistently buying more. Competent cell preparation consists of multiplying a strain of cells in a nutrient rich
broth, spinning them down, and rinsing them several times to remove any salts present. These new stocks of cells
are then aliquoted into eppendorfs and stored at -80\*C.**

* [*E. coli* Competent Cell Preparation](https://static.igem.wiki/teams/4201/wiki/experiments/e-coli-prep-protocol.pdf)
* [*A. tumefaciens* (Agrobacterium) Competent Cell Preparation](https://static.igem.wiki/teams/4201/wiki/experiments/agrobacterium-prep-protocol.pdf)

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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