The step by step of our lab processes
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
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
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
Pay iGEM
Order plastic ware, enzymes, kits and chemicals
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
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
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
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
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
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.
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
gene and primer synthesis order with IDT finally accepted
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
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
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)
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
(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:
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.