iGEM 2022 — Team ndsu

# **Notebook**

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## June

**6/28, MRH:** Liquid broth (LBB) and agar plates (LBA+Km and LBA+Amp) were made.

**6/29, MRH:** Samples B0015\_DE, BCD12\_BC, DVK\_EF, and J23100\_AB were plated on the appropriate antibiotic
agar plate and incubated at 37°C.

**6/30, MRH:** Liquid cultures were made for all of the strains, they were grown in the appropriate antibiotic
media. Incubated at 37°C.

## July

**7/1, MRH:** A plasmid prep was done on all of the cultures following the protocol found in the qiagen spin
miniprep kit (used the altered protocol written by Jake and Barney).

**7/6, MRH:** The plasmid prep samples were nanodropped with the following concentrations:

* B0015: 286.7 ng/ul
* BCD12: 215.6 ng/ul
* DVK: 270.0 ng/ul
* J23100: 62.9 ng/ul

**7/12, DTJ:** The genes from IDT were used along with parts B0015, BCD12, DVK, and J23100 in a golden gate
reaction using restriction enzyme BSA1. Of the 10 plasmids created, 5 plasmids have constitutive promoters and 5
have inducible promoters.

**7/19, DTJ:** A liquid culture of DH5a E. coli were made competent, flash frozen in liquid nitrogen, and
stored at -80 C.

**7/20, DTJ:** Genes from IDT along with parts B0015, BCD12, DVK, and J23100 were used to create 2 of the 5
plasmids with constitutive promoters and 2 of the 5 plasmids with inducible promoters. Once representative
plasmids from each group are confirmed working, the rest will be created. The resulting plasmids are stored at -20
C overnight for the transformation the next day. Reaction tubes are labeled 21, 23, 26, and 28 recipes for which
are stored in an Excel file.

**7/21, TRP:** Chemical transformation with GG reactions 21, 23, 26, 28, and DVK (as a positive control) were
completed using competent E. coli transformation protocol. 3uL of DNA was added to 30uL of DH5α that were made on
7/19. 967uL of SOC was added to the mixture instead of 950uL.

**7/26, DTJ:** As previous transformation/culture attempts were unsuccessful, the transformation protocol was
repeated using the 3uL of the DVK plasmid, 30uL DH5α competent cells created in the lab, and 30uL purchased DH5α
competent cells as a positive control. The sample with purchased DH5α cells is labeled “+ ctrl” and the sample
with cells created in the lab is labeled “iGEM made”

**7/27, DTJ & CLO:** As the transformation and culture of DVK worked properly in both purchased cells and the
competent cells created in our lab, an additional 10 transformations were performed. Golden gate constructed
plasmids 21, 23, 26, and 28 and DVK were transformed into both purchased cells and our competent cells. Samples
labeled with “+” indicate that they are made with purchased cells. As there was not a sufficient amount of
competent cells to be used for all the transformations, DVK+ was made with purchased competent cells from the
previous day that was stored at 4 C. 28 and DVK were also made with our competent cells from the previous day that
were stored at 4 C. The rest of the samples were made with cells that were stored at -80 C.

After shaking at 37°C for 3 hours samples 21, 23, 26, 28, DVK, 21+, 23+, 26+, 28+, and DVK+ were plated. Samples
26, 28, 26+, and 28+ fell off the shaker, so growth was expected to be less for those samples. Each sample was
plated at 10^0, 10^-1, and 10^-2 (Dilution protocol). They were put in the incubator at 37°C.

**7/28, CLO:** Plates were taken out of the incubator in the AM and put in the fridge. All samples grew except
for sample 28.

**7/29, MRH and CLO:** Made media and poured plates. Streaks were made of 4 isolated colonies for 21, 23, 26,
and 28+ (28 did not grow).

## August

**8/01, CLO:** Streaks were in the incubator over the weekend so they were overgrown and thrown away. Single
colonies were taken from 21, 23, 26, and 28+ and streaked onto new plates to grow at 37°C overnight.

**8/02, JDS & CLO:** Plate 23 did not grow and only 1 of the 4 streaks on plate 26 grew. Because of this, we
used the original transformation plates of 23+ and 26+ for the liquid cultures/isolation streaks. The original
plates for 23 and 26 had little growth. The isolation streaks of 28+ and 21 grew and were used for their liquid
cultures. 4 different colonies from each strain (21, 23+, 26+, 28+) were inoculated in liquid culture, then
streaked on new plates using (2 colonies per plate). We ended the day with 16 liquid cultures and 8 plates.

**8/03, DTJ & JDS & CLO:** DTJ: Plasmid prepped of all 16 cultures from 8/02 and eluted with 50ul of water.
JDS: Using Geneious software, the combination of a Cla1 and EcoR1 enzyme was found to digest pGEC 021-030 with a
unique fragment pattern on a digital electrophoresis gel. CLO: The plasmid prep samples were nanodropped with the
following concentrations:

* 21-1: 313.4 ng/ul
* 23+-2: 156.0 ng/ul
* 26+-3: 41.8 ng/ul
* 28+-4: 246.2 ng/ul

All 16 plasmid preps were digested with both the EcoR1 and Cla1 enzymes in a single pot reaction. Each tube
contained 2.5ul rCutSmart Buffer, 0.5ul EcoR1, 0.5ul Cla1, and 21.5ul of the respective plasmid prep. The 16 tubes
were put into the thermocycler and incubated at 37°C for 40 minutes then 65°C for 20 minutes.

**8/04, TRP & DTJ:** The gel was run following the 1.5% Gel protocol (Larger gel). The samples were loaded in
numerical order as listed on 8/2 and below. When imaging, we initially saw no bands at all. Barney suggested
soaking the gel in a Midori Green solution before imaging again. After letting it sit for 30ish minutes, we imaged
again and saw the bands below. One thing to note is the top left corner is a bit wavy. The gel looked completely
set when pulling the comb out but was not in that area. Barney said it would be okay to run and would turn out
still.

* Wells 1, 10, and 19: 1kb Ladder
* Well 2: 21-1
* Well 3: 21-2
* Well 4: 21-3
* Well 5: 21-4
* Well 6: 23+-1
* Well 7: 23+-2
* Well 8: 23+-3
* Well 9: 23+-4
* Well 11: 26+-1
* Well 12: 26+-2
* Well 13: 26+-3
* Well 14: 26+-4
* Well 15: 28+-1
* Well 16: 28+-2
* Well 17: 28+-3
* Well 18: 28+-4

![Gel Image](https://static.igem.wiki/teams/4258/wiki/images/notebook/gel8-4.png)
![Expected Gel](https://static.igem.wiki/teams/4258/wiki/images/notebook/gel21-28.png)

**8/09, MRH & JDS:** Poured ~15 LBA+Amp plates and struck 2 strains from the -80C freezer.

**8/10, TRP:** Made liquid cultures of RBS-HE and mScarlet with a negative control using LB media. Grown
overnight in 37C shaker.

**8/11, CLO:** Plasmid prepped RBS-HE and mScarlet.
Concentrations: RBS-HE= 140.1 ng/ul, mScarlet= 198.8 ng/ul.
A restriction digest of the plasmid preps was done with Bsa1.

**8/12, CLO:** A small 1.5% gel was made (60 mL 0.5x TAE, 0.9G agarose, 5 uL midori green).

* Well 1: 1kb ladder
* Well 2: mScarlet (C99)
* Well 3: RBS-HE (BCD2)
![Gel Image](https://static.igem.wiki/teams/4258/wiki/images/notebook/gel8-12.png)
![Expected Gel](https://static.igem.wiki/teams/4258/wiki/images/notebook/gel8-12exp.png)

**8/15, JDS & CLO:** Diluted DNA parts to standard concentrations for Golden Gate cloning. All parts will
now be at the correct concentration to add 1.0ul to Golden Gate cloning reactions.

| Parts | Current conc. (ng/ul) | Target conc. (ng/ul) | DNA (ul) | H2O (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- |
| (1) 100 | 26 | 13.0 | 37.0 | 50 | |
| (2) 100 | 26 | 13.0 | 37.0 | 50 | |
| (3) 100 | 26 | 13.0 | 37.0 | 50 | |
| (4) 100 | 26 | 13.0 | 37.0 | 50 | |
| (5) 100 | 19.5 | 9.8 | 40.2 | 50 | |
| (6) 100 | 33.8 | 11 | 22 | 33 | |
| J23100\_AB 62.9 | 55.9 | \* | \* | 20 | |
| BCD2\_BC 140.1 | 57.2 | 40.8 | 59.2 | 100 | |
| B0015\_DE 286.7 | 57.2 | 20.0 | 80.0 | 100 | |
| DVK\_AE 270 | 70.2 | 20.0 | 57.0 | 77 | |
| mScarlet-I 198.8 | 72.8 | 36.6 | 63.4 | 100 | |

Used the “GG Cloning Calculator” Excel sheet to determine the amount of each component for each Golden Gate
reaction.

Thermocycler settings:

30x cycles:

+ 37°C for 1 minute
+ 16°C for 1 minute

1x cycle:

+ 60°C for 5 minutes

4C hold

**8/16, DTJ & MRH & CLO:** SOC media was made by autoclaving 400mL SOB media, adding 8mL autoclaved 1M
MgSO4, and adding 8mL 1M glucose. Transformations were done for samples 25, 31, 35, 36, 37, and DVK\_AE (plasmid
prep).

pGEC021 was struck from wedge 1 of the 8/1 plate, and pGEC023+ was struck from colony 3 on the 7/26
transformation plate.25, 31, 35, 36, 37, and a DVK control were also plated. All plates were incubated at 37°C
overnight.

**8/17, CLO:** 25, 31, 35, and 37 had very little growth, so they were left in the incubator for an
additional day. Liquid cultures of 21, 23, and 36 were made– two of each, one had arabinose added at the start,
the other will have arabinose added tomorrow.

**8/18, CLO & MRH:** All liquid cultures grew, and 36+ arabinose was the only pink one. Made more LBB.

**8/22, JDS:** Designed new CD sequences (11) through (21). All sequences were designed with Bsa1 sites
flanking the CD region. Then flanked by Bpi1 sites for the option to clone into the pOGG006 vector for
immortalization in a plasmid form.Then an additional AAA at the 5’ end and a TTT at the 3’ end. (21) has the
AmilCP gene which is another chromoprotein (blue/purple) from the 2013 iGEM team. TwistBioscience website
identified hard to synthesize regions in the GS linker and changed the codons to make synthesis possible. The
sequences are as follows:

+ (11)His-meffRed-Linker-CBDfimi
+ (12)His-meffRed-Linker-CBDclos
+ (13)CBDfimi-Linker-meffRed-His
+ (14)CBDclos-Linker-meffRed-His
+ (15)meffRed-His\_NegControl
+ (16)His-mScarlet-Linker-CBDfimi
+ (17)His-mScarlet-Linker-CBDclos
+ (18)CBDfimi-Linker-mScarlet-His
+ (19)CBDclos-Linker-mScarlet-His
+ (20)mScarlet-His\_NegControl
+ (21)AmilCP-His\_NegControl

Additionally, pGEC036 and pGEC037 were restruck from their 8/16 transformation plate. Three colonies from each
plate were struck onto a new LBA+Km plate divided into 3 wedges.

**8/23, DTJ & CLO & JDS:** Blunt ligation and transformation. Parts used in the blunt ligation were 1, 5,
and +( the positive control in the kit) and tubes were labeled as such.

Diluted 21-3, 23-3, 26-2, 28-1 to 30 ng/uL
(were 414.8 ng/ul, 258.5 ng/ul, 262.6 ng/ul, and 189.3 ng/ul respectively) and sent to Plasmidsaurus.

Liquid culture of 36 and 37 (3 each from the plates streaked 8/22). Added arabinose to the 36 cultures (1%, so
500 uL). Plated transformations of 1, 5, and positive control at 10^0 and 10^-1 on amp plates.

**8/24, CLO:** Plasmid prepped 37A, 37B, 37C, and 36A. The concentrations were:

+ 37A: 162.0 ng/ul
+ 37B: 231.5 ng/ul
+ 37C: 221.9 ng/ul
+ 36A: Contaminated

**8/29, JDS:** PlasmidSaurus results arrived. Used Geneious software to visualize the sequences
Plasmidsaurus sent back. P21 and p28 looked identical to the expected sequences barring 2-3 SNPs. p23 and p26
showed sequences that appeared to be undigested DH5a competent cells. This tells us that at least 2 of our GG
constructs assembled correctly and should have functioned properly. The fact that we did not see any red on the
meffRed constructs and we did see color on the mScarlet controls suggest that there is something wrong with the
meffRed sequence.

On second review p21 and p28 are also not perfect. They have a section of IS1 that should have been excised
during GG. In Conclusion - The problem is likely the GG Cloning, either it did not digest correctly or the
cloning did not assemble correctly. The IS1 sequence could for some reason be interfering with the color for
some reason too.

## September

**9/6, DTJ:** Golden gate reactions were done for samples 37, 55, 60, and 62. After the reaction was completed
on the thermocycler all the samples along with DVK were transformed into DH5Alpha competent E.Coli created in the
lab. Samples were then shaken at 37°C for 2.5 hours before being plated at 10^0, 10^-1, and 10^-2.
All 15 plates were incubated overnight at 37°C.

**9/7, JDS:**

Transformation results:
![Plate 37](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-7-1.png)
![Plate 55](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-7-2.png)
![Plate 60](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-7-3.png)
![Plate 62](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-7-4.png)

All of the plates had a small number (5-10) of big white colonies that finished in frequency with the dilution;
these are likely KmR mutants of the competent *E. coli*. p37 worked great as a positive control. There were
hundreds of small pink colonies on the 10^0 plate. he only other transformation with any colored colonies was p60
(the mScarlet test); it only had ~50 small pink colonies on the 10^0 plate however, so the efficiency of the
cloning was lower. p55 also had the same colony types as the successful transformations, but no colored colonies.
MeffRed may take more time to develop. p62 only had the large colony types. The cloning may have went wrong here
and that is why no small potentially blue colonies are present. The 10^-1 dilution plate of each transformation
was placed in the fridge, the other plates were all left in the 37°C incubator to determine if color would
eventually develop.

Lab work:

Three each colonies from p55 and p60 were selected to be streak purified. Three pink colonies were selected
from the p60 plate. Three small white colonies were selected from the p55 plate (hoping they turn red/pink
eventually). Each strain was struck onto LBA+Km plates divided into thirds.

**9/13, DTJ & JDS:** Diluted CD parts (16), (17), (18), and (19) from dry pellet in tube to 20 ng/ul. Golden
Gate cloned (16), (17), (18), and (19).

Transformation:

Followed the transformation protocol on the back of the NEB 5-alpha Competent E. coli (High Efficiency) with a
few differences: Used our own competent cells (made 7/19/22), added 2ul of GG reaction to the competent cells, and
shake at 37C for 3 hours. Plated 10^0 and 10^-1 dilutions of the 5 GG products and 1 DVK\_AE positive control.
Plates were left to incubate at 37°C overnight.

**9/14, JDS & CLO:** Poured 13x plates of LBA+Km^25. Melted the LBA made on 8/9/22 and added 175ul of Kanamycin
stock (50mg/ml) to the 350ml of LBA.

![Plate 46](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-14-1.png)
![Plate 47](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-14-2.png)
![Plate 48](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-14-3.png)
![Plate 49](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-14-4.png)
![Plate 50](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-14-5.png)
![DVK Plate](https://static.igem.wiki/teams/4258/wiki/images/notebook/plate9-14-6.png)

All transformation plates appear successful. Each 10^-1 plate had ~100 colonies except for the DVK\_AE control
which was much more dense (~1000 on the 10^-1 plate). This is likely due to a higher amount of plasmids in the
DVK\_AE plasmid prep than the GG reactions. No color yet as these are arabinose inducible.

Three large colonies were selected from each of the five 10^-1 GG transformation plates (pGEC046-pGEC050).
These colonies were wedge struck onto LBA+Km25 plates divided into thirds then left to incubate at 37°C overnight.

**9/15, MJV:**

Started liquid cultures of the new GoldenGate Products (pGEC046-050) from 9/14. Left to incubate at 37°C
overnight.

**9/16, CLO:** Only pGEC046 and pGEC050 were pink. Plasmid prepped 46C and 50A.

**9/20, MRH & DTJ & JDS:** 5mL cultures of samples 46, 47, 48, 49, 50, and 60 were made as well as a negative
control with no culture. 1.8L LB media was autoclaved and kanamycin was added. 50mL of 10% w/v arabinose was made,
filtered, and placed in the freezer.

**9/21, DTJ & CLO & JDS:** Sample 60, his-tagged mScarlet with no CBD, was pink after overnight growth as it
has a constitutive promoter. The negative control had no growth and the other samples were cloudy. 1mL of sample
60 along with 46, 47, 48, and 49 were then added to 250mL LB with kanamycin. 10ml of Arabinose was added to the
cultures once they reached a DC600 between 0.6 and 0.8 (recorded below).

* 46: 0.766
* 47: 0.720
* 48: 0.829
* 49: 0.844

Three hours later none of the cultures were pink. 50ml of 10% w/v arabinose solution was made (5g of arabinose in
water) and filter sterilized. Another 10ml of Arabinose was added to each 250ml culture and allowed to shake at
37°C overnight.

**9/22, CLO:** None of the 250ml cultures from yesterday were pink in the morning. Not even the constitutive.
Dr. Geddes thinks there might have been contamination.

**9/26, CLO & DTJ & JDS:** Sent the Plasmid Preps of pGEC046 and pGEC050 from 9/16/22 to PlasmidSaurus. 1.8L LB
media was made and autoclaved, 100mL 10% w/v arabinose solution was made and filter sterilized, and used 250mL
flasks were autoclaved for the first time.

**9/28, MRH:**
Acid washed the 250mL flasks.

**9/29, DTJ & CLO & JDS:** 100 mL cultures of samples 46, 48, 49, 50, and 60 were started from the overnight
liquid cultures. After excess growth, the cells were diluted enough to have an optical density of 0.7 and
arabinose was added. After 3 hours of incubation with arabinose, only the constitutively expressed sample (60) was
pink. To address this, 50mL of each culture was removed and centrifuged to form pellets while the cultures were
once again diluted to 150mL with additional arabinose and left to grow overnight.

**9/30, DTJ:** After growing overnight, inducible sample 50 was visibly more pink than constitutive sample 60
even though both express the same protein. Sample 48 was not pink at all, Sample 49 was slightly pink, and sample
46 was visibly very close in vibrance to sample 50. All cultures were centrifuged to form pellets and stored at
4°C.

![Pellets](https://static.igem.wiki/teams/4258/wiki/images/notebook/pellets9-30.png)

## October

**10/3, JDS:** Liquid cultures of pGEC047A, pGEC048C, and pGEC049C were started in LBB+Km25 and shook at 37C
overnight.
Fresh plates of pGEC046C, pGEC047A, pGEC048C, pGEC049C, and pGEC050A were streaked on LBA+Km25. Left in the 37°C
incubator overnight.

**10/4, DTJ:** Plasmid prepped pGEC047A, pGEC048C, and pGEC049C.

**10/5, CLO:** Sent plasmid preps from yesterday off to plasmidsaurus.

**10/7, DTJ & WW:**
Cell pellets 46, 47, and 50 were lysed using the ThermoFisher Scientific B-PER lysis kit. Despite most of the
protein being insoluble, the supernatant was removed from each sample for testing. Sample 50 had the most soluble
protein. In order to have direct comparisons between the different constructs, serial dilutions of sample 50 were
performed and fluorescence was measured. A 1:16 dilution of sample 50 was found to be roughly equivalent to 46 and
47. Dyeing a t-shirt resulted in very weak visual results. Since 46 was the most promising sample, it and the 1:16
dilution of 50 were compared by creating 3 replicates of cotton t-shirt pieces that were dyed with 1mL of each
solution. They were incubated at room temperature overnight in falcon tubes.

**10/8, DTJ & WW:**
Samples from the previous night were imaged using a trans-illuminator. Then 40mL water was added to each sample
and shaken at 250rpm for 30 minutes as a standardized wash procedure. Finally, the samples were re-imaged on the
trans-illuminator.

**10/9, JDS:**
PlasmidSaurus Results:

* pGEC046 = as expected - AraC+6xHis+mScarlet+Linker+CBDfimi+B0015+DVK
* pGEC047 = as expected - AraC+6xHis+mScarlet+Linker+CBDclos+B0015+DVK
* pGEC048 = Incorrect - - AraC+CBDfimi+Linker+meffRed+6xHis+B0015+DVK
* pGEC049 = as expected - AraC+CBDclosr+mScarlet+6xHis+B0015+DVK
* pGEC050 = as expected - AraC+mScarlet+6xHis+B0015+DVK

When designing the synthesized (16) CD gene fragment, the meffRed sequence must not have been changed to mScarlet
as was intended.
pGEC048’s incorrect sequence explains why it had almost no pink color in culture.

**10/10, JDS:**
Liquid Cultures of the following strains were started in LBB+Km^25:

pGEC046, pGEC047, pGEC048, pGEC049, pGEC050, and pGEC060.

**10/11, JDS:**
The following strains were stocked in the -80C freezer in an 8% DMSO solution:

pGEC046, pGEC047, pGEC048, pGEC049, pGEC050, and pGEC060.

{% endblock %}

# **Engineering Success**

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![Constructs](https://static.igem.wiki/teams/4258/wiki/images/engineering/constructs.png)

*J23100\_AB is a high expression Anderson series promoter from the CIDAR MoClo Parts Kit. BCD12\_BC is a medium
expression bicistronic ribosome binding site from the CIDAR MoClo Parts Kit. BCD2\_BC is a high expression
bicistronic ribosome binding site from the CIDAR MoClo Parts Kit. B0015\_DE is a double terminator from the CIDAR
MoClo Parts Kit. DVK\_AE is a kanamycin resistant destination vector from the CIDAR MoClo Parts Kit. For more
detailed information on parts (6) and (11) through (21), please reference our [parts page](https://2022.igem.wiki/ndsu/parts).*

## Engineering Design Cycle

---

An important aspect of iGEM is successfully following the engineering design cycle. This process starts with
design, building, testing, learning, and then starts again at design. This is a continuous process that doesn’t
need to be ‘completed’ but should have gone through in entirety at least once. Following this cycle is essential
in project creation, development, and improvement. Our team has followed this cycle through multiple iterations
due to difficulties in our original design. The design cycle is reflected in our team's selection of segments for
construction, and successful expression of vibrant cellulose binding dye.

## Cycle 1 - Formation of Alternative to Synthetic Dye

#### 1. Design

---

The problem of synthetic dyes was identified with water quality being a predominant factor. A protein based dye
that had an improved ability to cotton would provide an alternative to hazardous synthetic dyes.

There are a variety of naturally produced vibrant protein chromophores that are non-toxic to humans. Chromoproteins
will be used as a chromophore due to their visibility under normal light and well established pathways in bacteria,
specifically meffRed. A protein domain for cellulose binding has already been identified allowing for a potentially
easy way to improve the cellulose binding of a protein based dye.

Our alternative dye would have to be produced in large quantities so a rapidly reproducing chassis is essential. The
CIDAR MoClo Parts Kit has a wide array of established modular components designed to create tailor made expression
vectors through Golden Gate cloning. Designed gene blocks need to be compatible with Golden Gate cloning in the
MoClo scheme to fit the parts available in the CIDAR MoClo Parts Kit.

Our bacterial strain is easily manipulated for many iterative genetic experiments and is low maintenance. DH5-alpha
*E. coli* will suffice for early experimentation.

Software for building gene blocks and testing their cloning digitally would be necessary. We will use Geneious
software.

#### 2. Build

---

The construction of our build will revolve around Golden Gate cloning in the MoClo scheme.

* Fast and efficient method of cloning that relies on type two restriction sites that are excised after ligation
  which prevents backwards reactions (Golden Gate cloning).
* Genetic segments for all parts can be obtained from online databases and sequences can be edited to contain
  Golden Gate cloning restriction and recognition sites.

The combination of meffRed and cellulose binding domain could cause misfolding during translation. Therefore
multiple orientations were designed with two different types of cellulose binding domains to try and maintain the
efficacy of the recombinant protein.

A histidine tag was included in the build to ensure easier separation of protein during purification. The position
of the tag varied with the different orientations of each construct, on either the amino or carboxyl terminus.

#### 3. Test

---

The construction of our build will revolve around Golden Gate cloning in the MoClo scheme.

The constitutive conjugants were observed for visible dye production. There was no visible color, even in the
positive control with no cellulose binding domain. The inducible conjugants were grown on media with arabinose and
also screened for visible dye production. There was no visible color.

Colonies were selected to be plasmid prepped, undergo restriction digest, and run on an agarose gel to verify
correct construction.

#### 4. Learn

---

After verification via plasmid digest we saw that none of the constructs had assembled correctly. To solve this issue we first decided to switch out some of our modular parts from the MoClo kit to see if that was the problem and had an easier time assembling.

## Cycle 2 - Increased Expression

#### 1. Design

---

The new construction switched RBS from BCD12\_BC to BCD2\_BC as seen in pGEC031 to pGEC035. This new RBS was higher strength, which would also increase expression of protein.

In order to see if our ordered coding sequence was the problem we used mScarlet-1 (c99) from the CIDAR MoClo Extension kit as a positive control to see if the cloning process wasn’t working or if our sequences were flawed. This is the pGEC037 construct. All parts in pGEC037 come from the established CIDAR MoClo kit.

Additionally we used the c99 mScarlet-1 control to test our arabinose inducible promoter sequence ((6)). This is the pGEC036 construct. All parts in pGEC036 come from the established CIDAR MoClo kit except the (6) arabinose inducible promoter sequence.

#### 2. Build

---

The mScarlet-1 was already available and constructed to fit in our modular cloning system, so no new sequences had to be synthesized and ordered. Golden Gate cloning was used again to make the process faster.

#### 3. Test

---

None of the colonies with meffRed expressed color. Both pGEC037 and pGEC036 (after adding arabinose) produced visibly pink cultures under normal light.

Colonies were again plasmid prepped, underwent restriction digest, and were ran on an agarose gel for assembly verification.

#### 4. Learn

---

The agarose gel verification isn’t the most reliable method, so we also sent these constructs to Plasmidsaurus for next-generation sequencing. The results showed that the constructs pGEC031-pGEC035 did not assembled correctly, but the sequence of pGEC037 showed the expected components and layout. This means that we are correctly performing the Golden Gate reactions correctly, and that there is an issue with our designed coding sequences. The sequence of pGEC036 also proved to be a correct assembly. This means that the (6) arabinose sequence is correct. This leaves our CD sequences (1) through (5) as the reason for the unsuccessful cloning.

## Cycle 3 - Redesigning CD Sequences and Substitution of meffRed with mScarlet-1

#### 1. Design

---

Due to the issues with the CD modules coding sequences (1) through (5), they were redesigned as (11) through (15). Golden Gate compatible Bpi1 sites were added on the outsides of (1) through (5) for the option to immortalize the parts as plasmids. Additionally, the extra nucleotides added to the ends of the DNA sequences should allow the restriction enzyme to work more effectively during cloning. The new designs were made with meffRed as well as mScarlet-1 and AmilCP as the chromophore. These designs include (16)-(21) in the constructs table. mScarlet was chosen because it was very vibrant when expressed as our positive control, and would be easier to use to test the binding domain. AmilCP was also included, which is a blue chromoprotein. This was just done to determine if meffRed specifically is a poor chromoprotein or if chromoproteins in general will be hard to express.

#### 2. Build

---

The same building technique was implemented, using Golden Gate cloning for construct assembly. However, only a few constructs of each orientation were constructed to save time and streamline the testing process. Initially pGEC037, pGEC055, pGEC060, and pGEC62 were the only four assembled for the first round of testing. pGEC037 grew well which indicated that the cloning and transformation were successful. pGEC055 had small colonies that were very light pink, while pGEC060 also had small colonies but were vibrantly pink. pGEC062 had no color. This led us to move forward with mScarlet-1 as our protein based chromophore due to the success of pGEC060.

Next all of the inducible constructs using mScarlet-1 were cloned, which is pGEC046, pGEC047, pGEC048, pGEC049, and pGEC050. The inducible promoter was used over the constitutive because of the long growth period of the constitutive colonies due to constant expression of protein. The inducible colonies were originally grown without the inducer to encourage strong growth before protein expression. When grown with the inducer, constructs pGEC050 and pGEC046 were the most pink, pGEC049 was slightly pink, and pGEC047 and pGEC048 were not pink. This result led us to choose pGEC050, pGEC049, and pGEC046 for further testing.

All of the assembled inducible constructs were sent to Plasmidsaurus for next-generation sequencing. The constructs sent for sequencing all had correct assemblies except for pGEC048. pGEC048’s sequence has a meffRed sequence where a mScarlet sequence was expected. The lack of a vibrant pink color pGEC048 is then easily explained by the significantly worse meffRed sequence as seen in previous experiments. The remaining constructs also have large differences in vibrance of color. Since we know that they are assembled correctly from the Plasmidsaurus sequences, we attribute these changes in phenotype to the differences in orientation and cellulose binding domain.

#### 3. Test

---

Since visibly red colonies were produced and the plasmid constructs were verified. The cellulose binding domain needed to be tested. Due to time constraints, we were unable to set up an assay to determine if the dissociation coefficient was the same as the 0.6 μM coefficient reported in the literature but we still wanted to find preliminary results to support or reject the idea that the binding domain was functional. Our initial method of testing involved creating handmade cotton filters to run cell lysate through and measure what was unable to bind to the cotton. Our crude handmade filters were not sealed properly, causing the solvent to leak everywhere but the collection tube. As a result, a new assay was created using t-shirt swatches to visually distinguish between mScarlet with and without a cellulose binding domain. While this experiment was only qualitative by nature, it provides preliminary information on whether or not the cellulose binding domain was functional until more thorough assays can be performed.

In order to have direct visual comparisons between the constructs with and without cellulose binding domains, serial dilutions of sample pGEC050 were performed and fluorescence was measured using a Cytation 5 plate reader. A 1:16 dilution of sample pGEC050 was found to be roughly equivalent to pGEC046. Sample pGEC046 and the 1:16 dilution of pGEC050 were compared by creating 3 replicates of cotton t-shirt pieces that were dyed with 1mL of each solution. They were incubated at room temperature overnight in falcon tubes. Samples from the previous night were imaged using a trans-illuminator. Then 40mL water was added to each sample and shaken at 250rpm for 30 minutes as a standardized wash procedure. Finally, the samples were re-imaged on the trans-illuminator and the images were compared.

#### 4. Learn

---

This assay, while somewhat functional, could be vastly improved upon. First, since most of our protein was expressed in inclusion bodies, we had very low protein concentrations to test. This resulted in the need to adjust the contrast of photos after taking them to visually compare differences between the two test groups (adjusting the brightness of images was noted in the relevant results page). Re-solubilizing and then refolding inclusion bodies would result in higher concentrations of produced dye making evaluation easier. Furthermore, the experiment did not use purified protein, instead the cell lysate was used. The detergent in the B-PER lysing agent could interfere with the cellulose binding domain and as a result could skew results. While testing of protein binding under different conditions is necessary to create a fully characterized product, the test should be performed with purified protein first as a baseline. Future testing will take advantage of the 6x-His tag present on the constructs for IMAC and then desalting through dialysis. This will ensure that the charged residues responsible for specific binding to cellulose are not interfered with during testing. Finally, quantitative testing should be prioritized in the future over the qualitative method we used for these preliminary results.

We also learned that our constructs were able to express natural protein dyes with preliminary results indicating functionality and promise with further optimization.

{% endblock %}

# **Experiments**

{% extends "teamlayout.html" %}
{% block page\_content %}

## Plasmid Prep

1. Pool 1 mL culture in 1.5 mL Eppendorf tube by spinning down 1.5 mL at a time and removing
   supernatant (3-4 times).
2. Resuspend cells in 250 µL of buffer P1 (with RNAse added) in an Eppendorf tube.
3. Add 250 µL Buffer P2 and mix by gently inverting 5 times, leave sit maximum 5 minutes.
4. Add 350 µL Buffer N3 and mix by inverting 5-10 times. Do not vortex.
5. Spin for 10 minutes at 13000 rpm in centrifuge.
6. Apply 800 µL of supernatant to a QIAgen spin column.
7. Centrifuge for 60s, discard flow through.
8. Wash by adding 750 µL Buffer PE (with ethanol added) and spinning for 60s. Remove flowthrough.
9. Spin for an additional 2 min to remove residual PE buffer and discard flowthrough.
10. Transfer to a new Eppendorf tube. Add 50 µL ddH2O to the center of the column, let sit 3 min, then
    centrifuge 60s. Discard the spin column.

## Competent *E. coli* transformation

1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix
   gently and
   carefully pipette 50 µl of cells into a transformation tube on ice.
2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5
   times to mix
   cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm).
8. Warm selection plates to 37°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial
   dilutions in SOC.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.

## Dilutions

* 10^-1 plate: 100 µL of original
* 10^-2 plate: 10 µL of original + 90 µL of S.O.C.
* 10^0 plate: Spin down remainder @ 13,000 rpm for 1 min, pour off supernatant, resuspend pellet in
  100 µL
  of S.O.C., then plate all 100 µL

## Gel Electrophoresis

1. Larger: 120ml 0.5x TAE buffer + 1.8g agarose, Smaller: 60mL 0.5x TAE buffer + 0.9g agarose.
   Microwave
   40-80s, then 5 µL Midori Green
2. Cast the gel in the gel rig if there is the rubber seal, or tape it if you can't find a rubber
   sealed one.
   Put in the comb.
3. Ladder = 15µl “1Kb+ GeneRuler” Ladder + 3µl “Purple Loading Dye” dye.
4. Samples (16x) = 25µl of Digest reaction + 5µl “Purple Loading Dye” dye. You can mix dye into the
   same tube
   we did the digest in.
5. Once the gel is cooled, take out the comb and orient the gel so that the wells are on the black
   side. The
   DNA will “Run to Red”. Fill the gel rig with more 0.5x TAE until it fills and covers the wells.
6. Add ladder + samples to wells.
7. Run at 100V for 60 minutes.
8. Take gel out and image with “UV365”.

## Restriction Digest

EcoR1 and Cla1:

* 2.5ul rCutSmart Buffer, 0.5µl EcoR, 0.5µl Cla1, 21.5µl DNA.
* Thermocycler: 37C for 40min then 65C for 20 min.

Bsa1

* 5 µL rCutSmart Buffer, 5µL Bsa1, 1 µg DNA (1000/concentration), water to 50 µL
* Thermocycler: 37°C for 5–15 minutes, 80°C for 40 min

## Golden Gate Cloning

| Reagent | Assembly Reaction(ng/ul) |
| --- | --- |
| DNA parts  * Synthesized gene fragments * Plasmids designed for BsaI Golden Gate  40 fmol | |
| T4 DNA Ligase Buffer (10X) 2 µl | |
| NEB Golden Gate Enzyme Mix (BsaI-HFv2) 1.5 µl | |
| Nuclease-free H2O to 20 µl | |

Run the Golden Gate mixture in a thermocycler with following program:

30 cycles of:

* 37°C for 1 minute
* 16°C for 1 minute
* 60°C for 5 minutes

Hold at 4°C

{% endblock %}

# **Results**

{% extends "teamlayout.html" %}
{% block page\_content %}

## Initial Results

---

After working through many different constructs, we were able to successfully produce several cultures that were
both red and had the cellulose binding domain present within the open reading frame. There were noticeable
differences in vibrance across different parts after pelleting overnight cell cultures.

Constructs 50 and 60 both contained only mScarlet with a 6x-His tag in the open reading frame. The visual differences in vibrance show that the arabinose operon ((6)) resulted in significantly higher expression than the high strength constitutive promoter (J23102).

Constructs 46 and 49 both contained a cellulose binding domain in their open reading frame. 46 has the CBDfimi domain and 49 has the CBDclos domain. The differences in vibrance between these two strains shows that the choice of cellulose binding domain either affects overall expression or the vibrance of an individual protein.

Construct 48 contained the chromoprotein meffRed and CBDfimi , which similar to previous attempts, resulted in no color.

These initial results are confirmed with verified plasmids sequences from PlasmidSaurus seen below.

![Pelleted Samples](https://static.igem.wiki/teams/4258/wiki/images/results/tubes.jpg)

*Cell pellets of various* E. coli *cultures containing our recombinant protein constructs. Each part
corresponds to the open reading frame shown above.*

![pGEC046](https://static.igem.wiki/teams/4258/wiki/images/results/circle46.png)
![pGEC048](https://static.igem.wiki/teams/4258/wiki/images/results/circle48.png)
![pGEC049](https://static.igem.wiki/teams/4258/wiki/images/results/circle49.png)

![pGEC050](https://static.igem.wiki/teams/4258/wiki/images/results/circle50.png)
![pGEC060](https://static.igem.wiki/teams/4258/wiki/images/results/circle60.png)

![All of the sequences](https://static.igem.wiki/teams/4258/wiki/images/results/allpgec.png)

*Plasmid constructs pGEC046, pGEC048, pGEC049, pGEC050, and pGEC060 visualized in Geneious software for ease of comparison.*

## Cell Lysis

---

![Lysed Samples](https://static.igem.wiki/teams/4258/wiki/images/results/epin-tubes.jpg)

*Samples 46 (left), 49 (middle), and 50 (right) after lysing cells and centrifuging insoluble material.*

After lying our cell cultures with B-PER, lysozyme, and DNASE I, and centrifuging the remaining insoluble material,
it was clear that a vast majority of our protein was sequestered into insoluble inclusion bodies. This makes sense
as in order to visually see color in the colonies, even cell cultures with inducible promoters needed to be grown
overnight.

## Visual Results

---

Due to time constraints, we were unable to purify our cell lysate, determine concentration, or determine the
dissociation constant of our fusion protein. However, we wanted to qualitatively show that our constructs were
better at binding to cotton than the mScarlet negative control as a preliminary result. To make a direct visual
comparison, the mScarlet negative control was diluted 1:16 to give the same fluorescence as the fusion protein.
Applying the samples to a t-shirt produced results too transparent to see, showing that a much higher concentration
is needed for the dye to be commercially viable.

![Samples on Shirt](https://static.igem.wiki/teams/4258/wiki/images/results/shirt.jpg)

*Cotton t-shirt after directly applying 1mL of each sample.*

![Pre-wash](https://static.igem.wiki/teams/4258/wiki/images/results/results1.jpg)
![Post-wash](https://static.igem.wiki/teams/4258/wiki/images/results/results2.jpg)

Despite this shortcoming, we still wanted to determine if the cellulose binding domain was functional. Since our
proteins exhibit fluorescence, we could image dyed t-shirt swatches with a trans-illuminator. Three replicates of
both sample 46 and 50 were created by dyeing cotton t-shirt swatches with 1mL of the respective solution and
placing them in falcon tubes at room temperature overnight. The following day, the samples were imaged on a
trans-illuminator before washing. Washing took place by adding 40mL water to each tube and shaking at 250rpm for
30 minutes. Images were then taken after washing to show if the cellulose binding domain helped mScarlet stay
attached to the cotton.

![Pre-wash with reduced brightness](https://static.igem.wiki/teams/4258/wiki/images/results/results3.png)
![Post-wash with reduced brightness](https://static.igem.wiki/teams/4258/wiki/images/results/results4.jpg)

*Experimental results of the wash experiment. Pre-wash (top left), post-wash (top right), pre-wash with reduced
brightness (bottom left), and post-wash with reduced brightness (bottom right).*

Quantitative testing after purification of our protein is required to definitively show that the cellulose binding
domain is functional. However, the results from this experiment suggest that it is functional, even in the presence
of
the B-PER detergent used for cell lysis.

## Conclusions

---

* A successful fusion protein between mScarlet and a cellulose binding domain was created and verified with
  sequencing.
* The choice of cellulose binding domain and chromoprotein is important in overall function because pGEC049 was
  visibly less vibrant than pGEC046.
* Due to the necessity of producing large amounts of protein to be visible as a dye, inclusion bodies will likely be
  present. As a result, inclusion body purification must be taken into account in large scale production.
* All three of the pGEC046 replicates showed a successful initial staining before the wash, but the pGEC050
  replicates
  struggled even with initial staining. This implies that the cellulose binding domain present in pGEC046 is
  important
  in the initial binding to cotton.
* The pGEC046 replicates retained more color after the wash. The third replicate of pGEC050 showed a clear loss of
  color after its wash, while the 3 replicates of pGEC046 showed retention of color. This indicates that pGEC046 is
  more resistant to washing and that the cellulose binding domain is important for permanent staining.

## Future directions

---

There are many more areas to work on in order to make Dyenamix a reality. A few of these objectives include:

* Solubilizing inclusion bodies and then purifying each construct through IMAC chromatography for quantitative
  testing
* Determining the dissociation constant between each construct and its cellulose substrate
* Quantifying a necessary protein concentration for commercial dyeing
* Performing directed evolution assays to increase vibrance of successful constructs
* Determining persistence of dyed fabric under various washing procedures
* Creating new constructs with different colors

{% endblock %}