iGEM 2022 — Team unicamp-brazil

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}The step by step of our lab processes{% endblock %}
{% block page\_content %}

# March to June 2021

Eduardo Menoti Silva, Gustavo Seguchi, Julia Ferreira Oliveira, Larissa Monteiro, Luiza Hesketh Gomes, and Michele De Vuono Geismar Petineli decided to participate in iGEM 2022

# July to December 2021

Recruit members outside the Institute of Biology

Vistis to waste management facilities, meeting, meetings and more meetings

First Styropolis project draft: Engineer bacteria to convert polystyrene into PHA ot PHB

Discussions with past iGEM teams

Design visual identity for the team

Design documentation for sponsors

Contact sponsors

# January to March 2022

Preliminary trials with different solvents to shrink polystyrene

Acquire *Streptomyces*, *Bacillus* and *Lactobacillus* as candidate chassis

Request Freegenes toolkit

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/limoneno.gif)

Cultivation of mealworms fed on polystyrene as a source of gut bacteria expressing polystyrene degrading enzymes

![](https://static.igem.wiki/teams/4435/wiki/pages/human-practices/larvas.png)

# April 2022

Pay iGEM

Order plastic ware, enzymes, kits and chemicals

# May 2022

New interviews and calculations revealed that Styropolis would not be beneficial to society.

PANIC!!!! Back to the drawing board.

Half of the team left

Why not work with a different bacterium that can convert other waste product into a high value product?

Open selection process for Unicamp\_brazil team and Synthetic Biology club

# June 2022

Recruit new team members (Unicamp\_Brazil and Synbio club)

Meeting with Dr. Hernane Barud to learn about BC production.

Definition of the Chassis: *Komagataiebacter*

Product: bacterial cellulose

Challenge: how can we engineer bacteria to produce more cellulose at lower cost?

iGEM distribution kit stuck at customs

Consumables arriving at the lab

# July 2022 : first week

iGEM distribution still stuck at customs (daily calls to UPS, customs, ministry of science, and ministry of agriculture to try and free the samples).

design, create Gcode and print the first mold for BC production in defined shapes.

Consumables arriving at the lab

# July 2022: second week

Michele and Larissa travel to Araraquara for hands-on training in *Komagataeibacter* cultivation and BC production

*Komagataeibacter rhaeticus*AF1 and *K. Medellinensis* ID13488 arrive at Unicamp.

iGEM distribution still stuck at customs (daily calls to UPS, customs, ministry of science and ministry of agriculture to try and free the samples)

Design engineering strategy (select relevant plasmids from toolkit and begin custom primer and gene design)

Preparation of the media

Test different protocols for preparation of ultracompetent *E. coli* DH5alpha cells (best competence achieved with cells prepared with Inoue buffer (stored at -80oC) (protocol by Sambrook and Russel).

DH5aplha transformation:

Slowly thaw competent cells on ice

Add 2-5 uL of DNA to 40-60 uL of competent cells in a fresh sterile tube

Incubate for 30 min

Heat shock at 42oC for 45 sec

Place on ice

Add 500 uL of SOC

Recover by incubation at 37oC for 1h

Spin for 2 min at 10000 rpm

Discard supernatant by inversion

Ressuspend cell pellet in the residual liquid and plate onto selective LB media

Calculate transformation efficiency: 2,8 x 106 colonies/ug DNA

# July 2022: third week

Distribution kit arrived!!!

Prepare LB (10 g tryptone; 10 g NaCl; 5 g yeast extract; add dH2O; adjust pH to 7; 15 g agar; complete to 1 L) and HS (50 g glucose; 0,75 g MgSO4; 2 g KH2PO4; 4g yeast extract; 20 ml ethanol; complete to 1 L with dH2O; sterile filter)

Transform DH5alpha with plasmids from distribution plate 1 positions:

1c, 1e, 1g, 1i, 1k, 1m (chloramphenicol)

2k, 2m (kanamycin)

3l, 3n, 3p (chloramphenicol)

4a (kanamycin)

5b, 5d, 5f, 5h, 5j, 5l, 5n, 5p (chloramphenicol)

10i, 10k, 10m, 10o (kanamycin)

12e, 12g, 12i, 12k, 12m, 12o, 14a, 14c, 14e (chloramphenicol)

Ressuspend DNA from iGEM plates in 10 uL ddH2O and use 2 uL to transform 40 uL of competent DH5alpha

Inoculate K. rhaeticus AF1 and K. medellinensis in HS with cellulase for transformation - cellulase not working - repeat

# July 2022: fourth week

Make glycerol stocks of iGEM plasmids in DH5aplha (750 uL culture + 250 uL of 60% glycerol)

Miniprep (Bioflux kit) all relevant iGEM plasmids

Inoculate transformantes for Interlabs experiments 1, 2 and 3 (50 mL tubes; 2 independent cultures each)

Perform all 3 Interlabs experiments according to protocols provided

Finalize strain, primers and gene designs

Inoculate K. rhaeticus AF1 and K. medellinensis in HS with new cellulase for transformation; measure ODs:
1. 0,548
2. 0,673
3. 0,687
4. 0,523
5. 0,438

Adjust OD to 0,04 and incubate for 24 h; measure ODs:
1. 0,334
2. 0,335
3. 0,337
4. 0,255
5. 0,320

Measure OD and incubate for another 24 h;
1. 0,831
2. 0,492
3. 0,493
4. 0,5
5. 0,490

Repeat due to contamination and after 3 days, prepare electrocompetent according to protocol developed by iGEM Imperial team 2014. Transform plasmids BBa\_J428346, BBa\_J428347 and BBa\_J428349.

# August 2022: first week

Evaluate *Komagataeibacter* transformations. No difference between bacteria transformed with plasmids or negative control. Conclusion: contamination. Repeat.

Place gene and primer synthesis order with IDT

Grow membranes in shapes

Repeat Interlabs experiments

# August 2022: second week

gene and primer synthesis order with IDT finally accepted

# August 2022: fourth week

Transform DH5alpha with part BBa\_J04450 for shipment to iGEM UFMG team (LB chloramphenicol)

Transform DH5alpha with FLP (iGEM distribution kit plate 2 position E6) (LB Kan)

*K. rhaeticus* AF1 transformation……FAIL

# September 2022: second week

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura1.png)

As IDT parts are not arriving on time, design new strategy and transform DH5alpha with new plasmids from the iGEM distribution plates:

Plate 1 positions:
15i, 15c, 15g, 13o, 15a, 15e, 23g, 9p, 13m, 23a, 13a, 16g, 13g, 13c, 13e (CmR)
2k, 2m, 4a (KanR)
6k, 6m, 8a (SpecR)
13p, 1o (CmR)
10k, 10m, 10o, 12a, 12c, 10g (CmR)

Plate 2 positions:
D4, F6, D8 (AmpR)
11i (CmR)
11k (KanR)
11m (tetracycline)
6E (AmpR)

Design and order primers for synthesis with Exxtend. Primers for marker swap of JUMP plasmids (with broad spectrum ori).

PCR products of AmpR (2x) and CmR (2x) with primers with adaptors for ligation with Jump backbones (Purify PCR products with BioFlux kit)

Transform K. rhaeticus AF1……FAIL

Grow K. rhaeticus AF1 and K. medellinensis in different media to quantify BC production

# September 2022: third week

Minipreps of toolkit parts for new construct design (low yield; shaker kept at 30oC due to *Komagataeibacter* cultures; probably low yield due to poor E.coli growth; solution: incubate for longer and with larger volume in larger tubes)

Weight BC wet sheets:
AF1- wet sheet 1,113 g
AF1 – wet sheet 1,1949 g
Medel – wet sheet 1,1754 g

Dry sheets:
AF1- 0,0163 g
AF1 – lost
Medel – 0,0170 g

Repeat PCR of backbones for marker swap: fail. In the same gel the last 4 lanes show the PCR products of AmpR (2x) and CmR (2x) with primers with adaptors for ligation with Jump backbones.

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura2.png)

New PCR of Jump plasmids. Mix per reaction (prepare on ice; prepare x8 and distribute into 0,2 ml strips for gradient)
5 ul of 5x Q5 reaction buffer
2 ul of 2,5 mM dNTPs
0,125 ul of 100 uM primer F
0,125 ul of 100 uM primer R
0,1 ul template plasmid (10 ng/ul)
0,25 ul Q5 DNA polymerase
5 ul Q5 high GC enhancer
12,5 ul ddH2O

Reaction conditions:
98oC for 1 min
98oC for 15 sec
50-60oC for 30 sec
72oC for 1,5 min
back to step to 30x
72oC for 5 min
hold at 4oC

All reactions worked!!! (Purify PCR products with BioFlux kit). Expected band sizes:

2k (BBa\_J428346)– 2,7 kb

2m – 2 kb

6k (BBa\_J428366)– 2,7 kb

6m – 2 kb
2 lanes of failed site directed mutagenesis

4a – 4,2 kb

8a – 4,2 kb

Quantification after purification:
6k – 29,15 ng/ul
6m – 35 ng/ul
8a – 36,15 ng/ul

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura3.png)

Attempt Goldengate – fail

Transform K. rhaeticus AF1……FAIL

1% agarose gel of miniprep of new parts

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura4.png)

Agarose gel of PCRs of 5´homology arms (K. rhaeticus AF1)

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura5.png)

# September 2022: fourth week

GoldenGate to combine JUMP plasmids 2k, 2m, 4a, 6k, 6m, 8a with AmpR or CmR

0, 5 ul T4 DNA ligase
2 ul 10 x T4 ligase buffer
0,5 ul *Sap*I
3 ul plasmid backbone
1 ul AmpR or CmR

Incubate at 37oC for 1h, 16oC 5 min, 55oC 5min, hold at 4oC

Transform 5 ul of product into DH5alpha. Select on LB amp or LB chloramphenicol (it worked!!!)

Inoculate for minipreps

Minipreps on 26/09

Transform DH5alpha with iGEM plasmids 2c, 2e and 2g (distribution plate 1) – plate onto LB kan

26/09. Synthetic LexRO and FLPe arrived!!!!!!

Golden Gate LexRO and FLPe into plasmid 13p (iGEM backbone) – fail

(26/09)

Measure OD of *Komagataeibacter* cultures to adjust OD:

*K. medellinensis*: 0,354
*K. rhaeticus* AF1: 0,605

BRICS measurement:

5,2

5,2

3,8
3,7
3,7
3,5

(27/09)

OD for preparation of competent *K. rhaeticus*:

1. 0,282 0,346 0,412

2.

0,260 0,382 0,625

3.

0,283 0,384 0,637

4.

0,263 0,542 0,709

5.

0,263 0,364 0,797

BRICS measurement:

5,1

5,2

3,7

3,2

3,3

3,2

(28/09)

BRICS measurement:

5,2

5,1

3,2

3,0

3,0

3,0

(29/09)

BRICS measurement:

5,1

5,1

3,2

3,2

3,1

3,1

Transform K. rhaeticus AF1 and K. medellinensis……maybe….. no…. it's a FAIL

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura6.png)

(28/09) IDT primers and short parts arrived!!!!!! Ressuspend plasmid DNA with 40 uL of H2O for 100 ng/ul stocks

Golden Gate of new parts into iGEM backbone

TU4p\_RBS

TU\_term

TU3p\_c

TU3p\_a

Dgc1

TU3p\_b

TU\_RBS

(Did not work)

PCR of *Komagataeibacter* genes with new IDT primers

galU

pgm (1,7 kb)

ndp (0,5 kb)

GK

TU1a (homology arm) (1,4 kb)

TU1b (homology arm) (1,4 kb)

TU4a cds (homology arm)

TU4b cds (homology arm) (1,4 kb)

PCR mix:

0,25 ul primer F

0,25 ul primer R

4 ul dNTP 2,5 mM

10 uL Q5 buffer

0,1 ul genomic DNA

0,5 ul Q5 DNA polymerase

10 uL PCR enhancer

complete to 50 uL with H2O

PCR conditions:

98oC for 5

98oC for 30 sec

55oC for 20 sec

72oC for 45 sec

back to 2 30x

72oC 5min

hold at 4oC

Gel of PCR from genomic DNA of K. rhaeticus AF1:

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura7.png)

Gel of PCR from genomic DNA of K. medellinensis:

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura8.png)

Purify successful PCRs and optimize other with gradient

# October 2022: first week

(01/10)

BRICS measurement:

5,1

5,05

3,2

3,1

3,05

3

(04/10)

BRICS measurement:

5,1

5,1

3,2

3,1

3,1

3,1

Gradient PCR (50-60oC annealing temperature) of genomic DNA AF1 for amplification of galU, GK and homology arms TU1b and TU4a; and genomic DNA medellinensis for amplification of galU, GK and homology arms TU1a , TU1b and TU4a.

Gel of PCR from genomic DNA of K. medellinensis:

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura9.png)

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura10.png)

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura11.png)

Conclusion: optimum annealing temperature for galU F and R is from 60-65oC; GK F andR is from 60-65oC; Homology arm primers- ideal annealing temperature between 67.9 and 70.1oC.

Transform DH5alpha with iGEM plate 1 plasmids 18m and 18o (type CDS); 16c and 16g (type promotor); 16e and 16o (type RBS); 18c and 20c (type terminator).

Construct parts parts BBa\_K4435315, BBa\_K4435316, BBa\_K4435317:

PCR mix:

0,25 ul primer AdapterTUred\_F

0,25 ul primer AdapterTUred\_R

4 ul dNTP 2,5 mM

10 uL Q5 buffer

0,1 ul mniprep 13p

0,5 ul Q5 DNA polymerase

complete to 50 uL with H2O

PCR conditions:

98oC for 5´

98oC for 20 sec

54 to 66oC for 20 sec

72oC for 45 sec

back to 2 30x

72oC 5min

hold at 4oC

PCR worked well! Purify with Bioflux (elute in 30 uL) and proceed to Goldengate

GoldenGate of red adapters:

2ul 10x T4 ligase buffer

3 ul backbone miniprep (6K, 6M and 8A)

0,5 ul T4 ligase

0,5 ul *Bsa*I

1 uL purified PCR

13 ul H20

incubate at 37oC for 1h, 16oC 5 min, 55oC 5 min, hold at 4oC

Transform DH5aplha

It worked! The MCS with marker was swapped! Successful exchange between MCS of level 2 plasmids 6K, 6M and 8A for MCS pOdd compatible, also exchanging the GFP reporter for the red reporter.

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura12.png)

Golden gate to insert TU1 and TU2 into pOdd1 and pOdd2 (using *Sap*I)

Dry and weigh BC sheets – before drying 14,58 g. After 24 hours at 65oC – 0,157 g

Optimize antibiotic concentrations in HS plates.

Amp 200/300/400 mg/L

Kan 100/150/200/300 mg/L

Chlo 50/100/150/200 mg/L

Spec 50/100/150/200/250 mg/L

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura13.png)
![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura14.png)
![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura15.png)
![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura16.png)
![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura17.png)

Conclusion: the antibiotic concentrations recommended in the literature are excessive for both AF1 and medellinensis. This might explain why are transformations are not working. Even with the plasmids the antibiotic concentration on the plates might still be too high.

test new parameters for electroporation and selection of K. rhaeticus AF1 transformants on plates with lower antibiotic concentration. It WORKS!!!!!!!

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura18.png)

culture AF1 for HPLC

typical task list for the day:

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura19.png)

# October 2022: second week

Transform K. rhaeticus AF1 with the Unicamp\_brazil plasmid BBa\_K4435305.

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura20.png)

Goldengate for assembly of CDS, promotor, RBS and terminator into iGEM backbones. Transformations OK. Innoculate 1 colony each for miniprep. Run gel of minipreps (not digested – supercoiled band evident). Candidates for parts BBa\_K4435001 to BBa\_K4435017:

LexRO

Flpe

dgc1

galU Kr

pgm Kr

ndp Kr

GK Kr

galU Km

pgm Km

ndp Km

Gk Km

18m (original plasmid)

TUp\_a

TUp\_b

TUp\_c

16c

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura21.png)

Conclusion: some promising candidates. Must sequence and inoculate more for minipreps.

Construct parts BBa\_K4435306, BBa\_K4435307 and BBa\_K4435314 (select red colonies).

![](https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura22.png)

Conclusion: red and green colonies in all plates. Pick 2 red colonies from each plate for miniprep and sequencing. Phenotypically successful.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

The Cellulopolis project involves multiple stages in the development of a Synthetic biology-based approach to solving real-life problems. In the course of our work, we dedicated ourselves engineer solutions at multiple levels, from problem identification to the final product. In the table below we enumerate key steps in our journey as examples of our engineering success.

# Key engineering success

| Task | Intermediate steps | Current status |
| --- | --- | --- |
| [Define project that can have a positive outcome for our environment and society](#define_project_that_can_have_a_positive_outcome_for_our_environment_and_society) | Design of the Styropolis project. Abandoned in May as would have a negative environmental impact when pollution due to transport was accounted for. Switch efforts to Cellulopolis. | All results obtained thus far are consistent with the potential of positive impact of Cellulopolis. |
| Acquire suitable chassis and learn how to cultivate it (growth conditions and BC production) | Invited to UNESP in Araraquara for hands on training | We established good cultivation conditions for *K. rhaeticus* AF1 and *K. medellinensis* and produce BC in the lab. |
| [Define antibiotic concentrations and establish electroporation protocols](#define_antibiotic_concentrations_and_establish_electroporation_protocols) | Standard protocols for closely related *K. rhaeticus* iGEM strain was not applicable to AF1 | We established new growth conditions and electroporation protocol, achieving good transformation efficiency. We confirmed that AmpR and KanR can be used for selection in AF1, but have no conclusive results on CmR or SpecR. |
| Identify candidate plasmid backbones | Literature searches point towards the use of RK2, pBBR1 and RSF1010 replication origins | Thus far we validated the efficiency of pBBR1 and RSF1010 as ori in AF1, but have no conclusive results on RK2. |
| Identify candidate promotor, RBS, reporter, selection marker and terminator | Order synthesis or employ parts from iGEM toolkit | Subcloning into level 0 plasmids apparently successful (colony numbers and gel profile). Minipreps awaiting sequencing results. |
| Model metabolism to establish targets for engineering | Suggestions of major changes (bimodal control of bcs) and of individual genes which overexpression might favor BC production yields | Awaiting cloning into level 1 and 2 plasmids, as well as genomic integration, for testing |
| Design primers for cloning | All PCRs successful | Transformations into level 0 plasmids successful. Minipreps awaiting sequencing results. |
| Design genes for synthesis | Genes recently arrived | Transformations into level 0 plasmids successful. Minipreps awaiting sequencing results. |
| Design new level 1 and level 2 backbones with selection markers compatible with *Komagataeibacter* | Level 2 plasmids with selected ori encoded SpecR, not used for *Komagataeibacter* | PCRs successful  Validation in *E. coli* successful  Transformation into *Komagataeibacter* successful |
| Design constructs for genomic integration | PCRs from genomic DNAs from *K. rhaeticus* AF1 and *K. medellinensis* ID13488 successful | Integration cassette under construction |
| [Design new level 2 backbones with adapters compatible with assembly of 4 TUs derived from pOdd1-4](#design_new_level_2_backbones_with_adapters_compatible_with_assembly_of_4_tus_derived_from_podd1-4) | Adapters PCRs successful | *E. coli* colony phenotype consistent with successful level 2 adaptor constructs! |
| Design of level 1 composite parts | Most level 0 parts required are ready | Under construction |
| Design of level 2 composite parts | Under construction | Under construction |
| Design of a low cost bioreactor | Constructed | Functional but requires optimization (current tubing not resistant to autoclaving) |
| Design of a container for light induction | Constructed | Awaiting light responsive candidate strains for testing |
| [Design of molds for custom BC production](#design_of_a_container_for_light_induction_and_design_of_molds_for_custom_bc_production) | GCode generated and 3D printing done | Molds efficient in limiting BC shapes allowing the formation of membranes suitable for coating spheres or tests in 24 well plates |
| [Cultivate of human cells using BC as substrate](#design_of_a_container_for_light_induction_and_design_of_molds_for_custom_bc_production) | Fibroblasts and melanoma cell lines | Cells adhere and grow well on BC |

---

# Define project that can have a positive outcome for our environment and society

Our engineering started with the conception of the project, which focused on dealing with environmental problems and providing a positive outcome for society. We began constructing it from the information we collected with research on waste management and visits to recycling centers. This culminated with the idea of working with an enzyme capable of degrading polystyrene, the main component of styrofoam, which has almost no destination other than landfills, because of the lack of profitability in its recycling process. Thus, we began to run the tests for pre-treatment of the material and encountered some barriers due to the approach’s inefficiency, such as a negative environmental impact when pollution due to transport was accounted for. With this scenario, we learned that our first idea could be unfeasible and re-set the design project design for the production of BC from agroindustrial residues, a project that actually shows consistent results regarding positive social and environmental impact.

# Define antibiotic concentrations and establish electroporation protocols

An important step in engineering our chassis is the transformation of *Komagataeibacter* with the plasmids of interest. We planned this step from extensive literature research and protocol analysis in order to tailor efficient instructions for the strain of interest. We tested the original transformation protocols published for *K. rhaeticus* iGEM, using our *Komagataeibacter* rhaeticus AF1 strain. Unfortunately, our first attempts were not successful, since it was not possible to observe the growth of transformed strains on the antibiotic plates. This scenario led us to question the antibiotic concentrations adopted in the published protocol and perform experiments to test the resistance of *K. rhaeticus* AF1 to various concentrations of the antibiotics Kanamycin, Ampicilin and Chloramphenicol. With the results of this experiment, we interpreted the bacterial response to different antibiotics and learned to adapt the protocol efficiently. From there, we rethought the design for the transformation, establishing optimized protocols for retesting, which allowed us to successfully transform *K. rhaeticus* AF1 for the first time. It is important to note that we successfully transformed *K. rhaeticus* AF1 with novel backbones constructed in this project.

# Design new level 1 and level 2 backbones with selection markers compatible with *Komagataeibacter*

Level 2 plasmids from iGEM’s kit that have compatible origin of replication for *Komagataeibacter* encode for the spectinomycin resistence marker which has not being reported as functional for *K. rhaeticus*. Furthermore all level 1 plasmids with ori compatible with *K. rhaeticus* encoded KanR marker, limiting the options of plasmid combinations. Therefore we designed and constructed level 1 and level 2 plasmids with AmpR and CmR, markers compatible with *Komagataeibacter*.

![](https://static.igem.wiki/teams/4435/wiki/pages/engineering-success/eng-1.png)

# Design new level 2 backbones with adapters compatible with assembly of 4 TUs derived from pOdd1-4

Our strategy for the assembly of multiple transcriptional units for concomitant overexpression of all genes encoding key enzymes involved in BC synthesis, requires the level 1 assembly of the promotor, RBS, CDS and terminator in pOdd1-4. Inserts from pOdd plasmids can then be assembled in compatible level 2 plasmids. Unfortunately, the iGEM distribution kit did not provide level 2 plasmids that could combine pOdd1-4 with a backbone encoding K. rhaeticus compatible ori. Thus, we designed adaptors for the replacement of prefix and suffix from BBa\_J428366, BBa\_J428367, BBa\_J428369, BBa\_K4435304, BBa\_K4435305, BBa\_K4435311, and BBa\_K4435312 with an adapter suitable for GoldenGate assembly of pOdd1-4 using the type IIs enzyme *Sap*I. Thus far we confirmed the correct replacement of cloning regions of 6 out of 8 plasmids. It is important to note that 5 of the newly engineered plasmids were derived from marker swap plasmids we constructed in stage 9.

![](https://static.igem.wiki/teams/4435/wiki/pages/engineering-success/eng-2.png)

# Design of a container for light induction and design of molds for custom BC production

An astonishing application of bacterial cellulose is its potential as a scaffold for cell culture. To better evaluate BC suitability for this purpose we designed an assay to grow fibroblasts over BC sheets. The results showed good adhesion of human cells to the novel substrate but exposed the possibility that BC membranes might interfere with cell development and differentiation. After some modifications in the protocol BC production protocol and using human melanoma cells, we still identify steps that could be improved, such as the production of BC with more consistent thickness. This process drove us to design 3D molds for BC production in the adequate format and thickness for cell cultivation. We built Gcode, 3D printed the molds, and tested the production of BC hexagons of defined dimensions. Those were used in a new assay for cell culture, showing improved results, but suggesting new aspects that can be further improved.

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

# Overview

We faced the challenge of developing a project ranging from characterization of non-standard growth media, through BC cultivation, hardware engineering, computer simulation, strain design, plasmid engineering, transformations, and applications of our final product (BC). Due to the broadness of the project and the limited time for its execution, we developed multiple fronts in parallel. Here we present some of the highlights of our work.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/fluxograma.svg)

# Establishment of *K. rhaeticus* AF1 transformation protocol

A consistent transformation protocol is crucial for any strain engineering project. Thus, we invested much work in establishing the ideal conditions for *K. rhaeticus* AF1 electroporation. Initially, we based our attempts on the strategies used for the transformation of *K. rhaeticus* iGEM (Goosens, Vivianne J., et al., 2021), however, all of our experiments failed. . Other literature sources were also used in an attempt to carry out the transformation, but none of them resulted in success.

After troubleshooting parameters such as preparation of competent cells, plasmid selection markers, plasmid replication origins, the intensity of electroporation, cuvette brands, cell density, and many other parameters, we decided to change the suggested antibiotic concentration on our HS agar plates. After all of our frustrating attempts, we finally established a consistent protocol for *K. rhaeticus* AF1 transformation.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-1.png)

Figura 1. Efficiency of **Komagataeibacter rhaeticus** AF1 tested with different concentrations of antibiotics.

Optimized *Komagataeibacter rhaeticus* AF1 transformation protocol by electroporation ([protocol](https://docs.google.com/document/d/1RZtNi85U2spozimZ5CDCeTo409KxxFF08jsHbn2zFRY/edit?usp=sharing))

# Toolbox

## Construction of broad species spectrum plasmids with compatible selection markers

(further details in the parts section)

*Komagataeibacter* is a non-conventional chassis in which traditional *E. coli* replication origins are not functional. Hence, plasmid replication requires broad-spectrum origins such as RK2, pBBR1 or RSF1010. Within iGEM´s 2022 distribution kit there were 6 plasmids with such requirements, namely the GoldenGate level 1: BBa\_J428346, BBa\_J428347, and BBa\_J428349 (KanR), and the GoldenGate level 2: BBa\_J428366, BBa\_J428367, and BBa\_J428369 (SpecR). This posed a potential problem to our design strategy as, to the best of our knowledge, there is no report of successful use of the SpecR marker in *K. rhaeticus*. Thus, we design a strategy to replace the KanR and SpecR marker from level 1 and 2 plasmids (respectively) with both AmpR and CmR. This was accomplished by designing the primers JUMP\_mF and JUMP\_mR to amplify BBa\_J428346, BBa\_J428347, BBa\_J428349, BBa\_J428366, BBa\_J428367 and BBa\_J428369 excluding the KanR and SpecR selection markers (for details, see parts section). In parallel, we designed primers AmpR\_F and AmpR\_R to amplify the Ampicillin resistance cassette from BBa\_J428385 and primers CmR\_F and CmR\_R to amplify the chloramphenicol resistance cassette from BBa\_J428357. Employing digestion with *Sap*I and ligation with T4 DNA ligase, we succeeded in assembling new level 1 and level 2 Komagateibacter compatible plasmids with different marker options.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-2.png)

Figure 2. Amplification of plasmid backbones with JUMP-mF and JUMP-mR: A.BBa\_J428346, B.BBa\_J428347, C.BBa\_J428367, D.BBa\_J428349, E.BBa\_J428366 and F.BBa\_J428369.

Thus, we succeeded in constructing the following plasmids:

| New backbones | Details | Internal Code | Validation |
| --- | --- | --- | --- |
| BBa\_K4435301 | amplification of BBa\_J428346 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 2K to AmpR | phenotype |
| BBa\_K4435302 | amplification of BBa\_J428347 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 2M to AmpR | phenotype |
| BBa\_K4435303 | amplification of BBa\_J428349 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 4A to AmpR |  |
| BBa\_K4435304 | amplification of BBa\_J428366 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 6K to AmpR | phenotype |
| BBa\_K4435305 | amplification of BBa\_J428367 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 6M to AmpR | phenotype |
| BBa\_K4435308 | amplification of BBa\_J428346 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 2K to CmR |  |
| BBa\_K4435309 | amplification of BBa\_J428347 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 2M to CmR | phenotype |
| BBa\_K4435310 | amplification of BBa\_J428349 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 4A to CmR |  |
| BBa\_K4435311 | amplification of BBa\_J428366 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 6K to CmR | phenotype |
| BBa\_K4435312 | amplification of BBa\_J428367 with JUMP\_mF and JUMP\_mF; amplification of antibiotic resistance marker; Golden gate cloning with *Sap*I | 6M to CmR |  |
| BBa\_K4435315 | add ODD 4 TUs adapter, derived from BBa\_J428366: pJUMP42x-2A SpecR Type IIS Level 2 vector. Origin RK2 (broad-host-range); | 6K\_red | phenotype |
| BBa\_K4435316 | add ODD 4 TUs adapter, derived from BBa\_J428367: pJUMP43-2A SpecR Type IIS Level 2 vector. Origin pBBR1 (medium-copy, broad-host-range) | 6M\_red | phenotype |
| BBa\_K4435317 | add ODD 4 TUs adapter, derived from BBa\_J428369: pJUMP45-2A SpecR Type IIS Level 2 vector. Origin RSF1010 (Broad-host-range) | 8A\_red | phenotype |
| BBa\_K4435306 | add ODD 4 TUs adapter to BBa\_K4435304 | 6K to AmpR\_red | phenotype |
| BBa\_K4435307 | add ODD 4 TUs adapter to BBa\_K4435305 | 6M to AmpR\_red | phenotype |
| BBa\_K4435313 | add ODD 4 TUs adapter to BBa\_K4435311 | 6K to CmR\_red | phenotype |
| BBa\_K4435314 | add ODD 4 TUs adapter to BBa\_K4435312 | 6M to CmR\_red |  |

Phenotipic validation: Image illustrating *E. coli* strains harbouring novel plasmids and streaked onto plates with the indicated antibiotics.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-8.png)

Figure 3. *E. coli* transformed with swapped markers plasmids growing in media containing antibiotics.

Our final proof of success is the transformation of *Komagataeibacter* with part BBa\_K4435305, which has the new antibiotic resistance cassette and shows it’s efficiency in *Komagataeibacter rhaeticus* AF1 (5 and 10 mg/L ampicillin allowed background growth of cells electroporated but without plasmids; 400 mg/L - the recommended ampicillin concentration in the literature - exceeded the tolerable levels for the AF1 strain; 25 mg/L of ampicillin did not allow the growth of cells without plasmids but selected positive transformants). All the new plasmids with swapped markers are await for sequencing.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-3.png)

Figure 4. Different concentrations of antibiotics used in bacteria transformation.

Our engineering strategy for the replacement of bcsZ or bcsA promotors requires the genomic integration of cassettes with include: 1 kb of homology to the region 5´the integration locus, recyclable antibiotic resistance marker (AmpR flanked by FRT recombination sites), transcriptional unit encoding the LexRO transcriptional repressor under the control of strong to medium promotors, and a final module encoding the LexRO binding region (operator) followed by RBS and 1 kb of homology to the region 3´the integration locus. This design requires level 2 assembly of 4 transcriptional units (basic TUs assembled in pOdd1 to pOdd4 plasmids).

In parallel, we also cloned in level 0 plasmids genes (from *K. rhaeticus* and *K. medellinensis*) encoding key proteins in the pathway that converts glucose to BC (awaiting sequencing results). These will be assembled into transcriptional units TU1 to TU4 in pOdd1 to pOdd4 plasmids. Concomitant overexpression of 4 different proteins requires their assembly into level 2 *Komagataeibacter* compatible plasmids.

Unfortunately, the available backbones did not meet our needs, which requires the presence of *Sap*I restriction with ends complementary to 5´of TU1 (pOdd1) and 3´of TU4 (pOdd4), thus we designed primers AdapterTU\_reD\_F and AdapterTU\_reD\_R and amplified the red reporter from BBa\_J04452. PCR products were assembled by GoldenGate using *Bsa*I and T4 DNA ligase with level 2 plasmids. Successful assembly yielded red colonies and original unedited plasmids yielded green colonies.

![](https://static.igem.wiki/teams/4435/wiki/registry-parts/level-2-red-adapter-colonies.png)

Figure 5. LB Amp plates with green colonies indicating the transformation with the original cloning site (replacement not sucessfull), and red colonies showing the sucessfull introduction of pOdd1-4 compatible level 2 adaptor.

New backbones (Type IIs compatible):

| New backbones | Source | Internal Code | Validation |
| --- | --- | --- | --- |
| BBa\_K4435315 | add ODD 4 TUs adapter, derived from BBa\_J428366: pJUMP42x-2A SpecR Type IIS Level 2 vector. Origin RK2 (broad-host-range) | 6K\_red | phenotype |
| BBa\_K4435316 | add ODD 4 TUs adapter, derived from BBa\_J428367: pJUMP43-2A SpecR Type IIS Level 2 vector. Origin pBBR1 (medium-copy, broad-host-range) | 6M\_red | phenotype |
| BBa\_K4435317 | add ODD 4 TUs adapter, derived from BBa\_J428369: pJUMP45-2A SpecR Type IIS Level 2 vector. Origin RSF1010 (Broad-host-range) | 8A\_red | phenotype |
| BBa\_K4435306 | add ODD 4 TUs adapter to BBa\_K4435304 | 6K to AmpR\_red | phenotype |
| BBa\_K4435307 | add ODD 4 TUs adapter to BBa\_K4435305 | 6M to AmpR\_red | phenotype |
| BBa\_K4435313 | add ODD 4 TUs adapter to BBa\_K4435311 | 6K to CmR\_red | under construction |
| BBa\_K4435314 | add ODD 4 TUs adapter to BBa\_K4435312 | 6M to CmR\_red | phenotype |

## Cloning the basic parts

Considering the BC synthesis pathway we were able to identify key enzymes involved in cellulose production, specially when Glucose is the main carbon source for this biosynthesis. Trough our modeling, we confirm which main enzymes could be overexpressed and help in the production of cellulose.

After glucose (GLC) import we see this conversion into D-glucose-6-phosphate (G6P) by glucokinase (GK), which is then converted into D-glucose-1-phosphate (G1P) by UTP-glucose-1-phosphate uridylyltransferase (galU). From that UDP-glucose (UDPG) is produced by hosphoglucomutase (pgm), which requires UTP by nucleoside diphosphate pyrophosphorylase (ndp) production. UDPG works as a substrate for cellulose synthesis by bcsA, which requires cyclic diguanylic acid (C-di-GMP) produced by diguanylate cyclase (dgc).

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-5.png)

Figure 6. Bacterial cellulose production pathway. Adapted from [Hur et al., 2020](https://doi.org/10.4014/jmb.2006.06026)

In order to obtain experimental results consistents with the modelling prediction, identifying the limiting enzyme in the pathway, we amplified genes encoding GK, galU, pgm and ndp from genomic DNA of *K. rhaeticus* AF1 and *K. medellinensis*, along with the synthesis of dgcI. These are being cloned into BBa\_J428381, BBa\_J428382, BBa\_J428383, and BBa\_J428384, under the control of the strong constitutive promotor BBa\_K4435012, RBS BBa\_K4435015 and terminator BBa\_K4435017 (given us level 1 composite parts BBa\_K4435120 to BBa\_K4435127).

The constructs were transformed into *E. coli*, which grew efficiently, proving the antibiotic resistance and, consequently, the right assembly of the plasmids. Some of it’s resistant colonies were isolated, plasmid prepared, and analyzed by agarose gel electrophoresis. The band sizes show many candidates were sent for sequencing. Further colonies are being selected for constructs where we have not selected plasmids of the correct size in our first attempt (only 1 colony was selected per plate in our first screen). We are confident that we have level 0 plasmids for all our basic parts (awaits confirmation).

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-6.png)

Figure 7. Example of gel electrophoresis of undigested (supercoilled) plasmids with candidate level 0 parts.

Parts listed below will be assembled into transcriptional units (TU1 to TU4) in pOdd 1 to 4 plasmids for further level 2 assembly into BBa\_K4435306 or BBa\_K4435307. These can then be transformed into *K. rhaeticus* and BC production yields are quantified.

Level 0 (Type IIs compatible):

| Level 0 Part | Short name | Origin and protein cloned |
| --- | --- | --- |
| BBa\_K4435004 | Kr\_galU\_TU1 | *K. rhaeticus* AF1 galU\_UTP-glucose-1-phosphate uridylyltransferase |
| BBa\_K4435005 | Kr\_pgm\_TU2 | *K. rhaeticus* AF1 pgm\_Phosphoglucomutase |
| BBa\_K4435006 | Kr\_ndp\_TU3 | *K. rhaeticus* AF1 ndp\_Nucleoside diphosphate pyrophosphorylase |
| BBa\_K4435007 | Kr\_GK\_TU4 | *K. rhaeticus* AF1 GK\_Glucokinase |
| BBa\_K4435008 | Km\_galU\_TU1 | *K. medellinensis* galU\_UTP-glucose-1-phosphate uridylyltransferase |
| BBa\_K4435009 | Km\_pgm\_TU2 | *K. medellinensis* pgm\_Phosphoglucomutase |
| BBa\_K4435010 | Km\_ndp\_TU3 | *K. medellinensis* ndp\_Nucleoside diphosphate pyrophosphorylase |
| BBa\_K4435011 | Km\_GK\_TU4 | *K. medellinensis* GK\_Glucokinase |
| BBa\_K4435003 | dgc1\_TU4 | dgc1\_diguanylate cyclase |

# Hardware

## Hardware - dark vs light

Aiming to improve our cellulose production and detach dark (no BC production) and light (bcs induction) phases of the process, we designed and built a two step bioreactor. As our project proposes, *Komagataeibacter rhaeticus* AF1 should have a period of growth, in the absence of light, followed by activation of it’s bcs operon by luminous stimulus. To predict the best moment to start the induction of cellulose production, resulting in a higher amount of final product, we comply our math model. The conditions of presence or privation of light and even the change-over them can be respected in the developed hardware. Experimental tests demonstrate the the efficiency of bioreactor by the exponencial growth of *E. coli* and suggested a big condition advantage for obligate aerobic organisms.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-9.png)

Figure 8. Bioreactor produced in the lab.

The operation of the bioreactor is simple, it works with (a) the culture medium with genetically modified KAF1 where as soon as it reaches a maximum OD, without harming the colony, it will be inserted into our closed system (b) through a hose that connects (a) and (b). And thus we activate the blue light that is contained in its lid. In this way we can fully control the cellulose production. All materials in the bioreactor are sterile.

# Glucose consumption by *Komagataeibacter rhaeticus* AF1

## HS medium vs agroindustrial residues

### 1. standard media (HS)

*Komagataeibacter* was grown in liquid HS media, which has D-glucose as its sugar source. We asked ourselves if the bacteria use this glucose as a substrate for cellulose production. So, in order to evaluate glucose consumption, we used a quantification technique based on High Performance Liquid Chromatography (HPLC).

For this analysis, we injected 5 samples collected from different days of bacterial growth, to compare their sugar elution profile to a standard sample of monosaccharides and short-chain carbohydrates. This way it was possible to identify and quantify the sugars present in our sample by integrating their corresponding peaks.

The media samples’ elution profiles showed peaks that matched with glucose and, the short-chain sugar made of three units of glucose, cellotriose. It is possible to see glucose’s concentration decrease, as well as the cellotriose’s concentration increase as the growth days go by in the chromatograms. This observation shows that the glucose monomer is consumed by the bacteria and glucose polymers are being formed, as glucose decrease coincides with cellotriose’s increase (Figure 1).

The chromatograms obtained are available [here](https://static.igem.wiki/teams/4435/wiki/pages/results/carbohydrate-quantification-protocol-1.pdf)

![](https://static.igem.wiki/teams/4435/wiki/pages/results/carbohydratequantification.svg)

Figure 9. Glucose and cellotriose concentrations through the days of *Komagataeibacter*’s growth in HS liquid media.

### 2. Agroindustrial residue media

We also wondered about glucose concentration in the alternative agroindustrial residue media, in a way to predict bacteria growth. So we measured it in the HPLC as well. The chromatogram in figure shows a concentration of 3.1836 g/L of glucose and the presence of other carbohydrates that were not identified, as they are not in the standard sample of monosaccharides and short-chain carbohydrates.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/glucosequantificationorangemedia.svg)

Figure 10. Agroindustrial residues media chromatogram for sugar quantification.

# BC production in defined shapes

## 3D structures

With the goal of producing BC membranes that could coat complex 3-dimensional structures, we investigated the requirements for unfolding the surface of different objects onto 2D. By using the perimeter of this 2D object as a guide, we devised a strategy to 3D print molds for the production of BC sheets. As proof of principle, we constructed molds representing the iGEM logo and the surface of a sphere and used to produce BC sheets.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-11.png)

Figure 11. Cellulose grow in 3D molds for cell cultivation

---

## Scaffold for cell culture

An exceptional application of BC is its use as a scaffold for cell culture, once it forms colloidal level dispersions in an aqueous medium and shows strong and crystalline structure, boosting cell culturing experiments.(Madhushree Bhattachary et al., 2012). In previous studies, bacterial cellulose has already been tested and shown to be convenient as a scaffold for hard tissues (eg. bone and cartilage). Aiming to evaluate the adherence of distinct cell lines in BC and their efficiency as scaffolds we perform a vast number of assays during our project. First of all, we used the lineage of human skin fibroblast NIH3T3 and reached a praiseworthy cell adhesion in all the groups. In the figure below we can see cells under brightfield microscopy, with a morphology slightly different from the expected pattern, but adhered to the blanket as expected.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-7.png)

Figure 12. Fibroblast adherence in cellulose sheet colored with DAP (blue for nucleus) and Phalloidin (green for actin) revealling it`s morphology

The second batch of tests was with the SK-MEL-28 strain of human melanoma, in which we obtained good adhesion to bacterial cellulose, however, morphologically, they do not appear to undergo cell differentiation, since they have a phenotype similar to that found when the cell does not is adhered to the substrate. In addition to these points, we could also observe that cell adhesion is greater in thinner cellulose when compared to thicker cellulose.

In view of the results obtained, we were able to identify some points that are amenable to the improvement of our design of experiment, as a way of standardizing the blankets used for cultivation, minimizing the effects of variable parameters. In this scenario, we rethink our design and proposed the use of a 3D mold ([hardware](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D)) that allows the standardization of BC production in optimal size and thickness for cell culture. After the growth of these new cellulose sheets, we ran an assay with C2c12 mouse myoblasts, which shows the efficiency of cell adherence and differentiation rate. The standardization of the sheets seems to be an effective improvement for cell culture as we can see from the pictures. Some observations allowed during these experiments propose the requirement of thinner sheets.

![](https://static.igem.wiki/teams/4435/wiki/pages/results/results-12.png)

Figure 13. Myoblasts adhesion in cellulose sheets

# References

* Helander, Anders, Asgeir Husa, and Jan-Olof Jeppsson. "Improved HPLC method for carbohydrate-deficient transferrin in serum." Clinical chemistry 49.11 (2003): 1881-1890.
* Muir, Jane G., et al. "Measurement of short-chain carbohydrates in common Australian vegetables and fruits by high-performance liquid chromatography (HPLC)." Journal of agricultural and food chemistry 57.2 (2009): 554-565.
* Helander, Anders, Asgeir Husa, and Jan-Olof Jeppsson. "Improved HPLC method for carbohydrate-deficient transferrin in serum." Clinical chemistry 49.11 (2003): 1881-1890.
* Muir, Jane G., et al. "Measurement of short-chain carbohydrates in common Australian vegetables and fruits by high-performance liquid chromatography (HPLC)." Journal of agricultural and food chemistry 57.2 (2009): 554-565.
* Fricke, P.M., Klemm, A., Bott, M. et al. On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases. Appl Microbiol Biotechnol 105, 3423–3456 (2021).
* Hur DH, Choi WS, Kim TY, Lee SY, Park\* JH, Jeong\* KJ. Enhanced Production of Bacterial Cellulose in *Komagataeibacter xylinus* via Tuning of Biosynthesis Genes with Synthetic RBS. J. Microbiol. Biotechnol. 2020;30:1430-1435.
* Madhushree Bhattacharya, Melina M. Malinen, Patrick Lauren, Yan-Ru Lou, Saara W. Kuisma, Liisa Kanninen, Martina Lille, Anne Corlu, Christiane GuGuen-Guillouzo, Olli Ikkala, Antti Laukkanen, Arto Urtti, Marjo Yliperttula, Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture, Journal of Controlled Release, Volume 164, Issue 3, 2012,Pages 291-298
* *Komagataeibacter* Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria Vivianne J. Goosens, Kenneth T. Walker, Silvia M. Aragon, Amritpal Singh, Vivek R. Senthivel, Linda Dekker, Joaquin Caro-Astorga, Marianne L. A. Buat, Wenzhe Song, Koon-Yang Lee, and Tom Ellis ACS Synthetic Biology 2021 10

{% endblock %}