Notebook

The step by step of our lab processes

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

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

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 rhaeticusAF1 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

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.

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

Attempt Goldengate – fail

Transform K. rhaeticus AF1……FAIL

1% agarose gel of miniprep of new parts

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

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 SapI 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

(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:

Gel of PCR from genomic DNA of K. medellinensis:

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:

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 BsaI

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.

Golden gate to insert TU1 and TU2 into pOdd1 and pOdd2 (using SapI)

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

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!!!!!!!

culture AF1 for HPLC

typical task list for the day:

October 2022: second week

Transform K. rhaeticus AF1 with the Unicamp_brazil plasmid BBa_K4435305.

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

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

Construct parts BBa_K4435306, BBa_K4435307 and BBa_K4435314 (select red colonies).

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