IARA

NOTEBOOK

WEEK 1

Monday - Jul. 22

Day one!!! Today we’re going to visit the lab and get started with E.coli biofilm quantification with crystal violet.

iGEMmers:

Vinícius
Beatriz
Amanda
João Paulo
Juliana

Today’s steps:

Biofilm initial analysis: preparation of staining and washing solutions: crystal violet and 30% acetic acid
Synthesis of bacterial growth media for biofilm analysis: LB and LB+agar
Preparation of pre-inoculation medium: liquid LB
All bottles containing media were autoclaved

USED PROTOCOLS:

Biofilm quantification with crystal violet - staining and washing solutions preparation

We finished our day with pre-inoculated cells and negative control of biofilm growth incubated at 37 ºC in the shaker.

Wednesday - Jul. 24

Today we could verify the biofilm formation in our 96-well using the spectrophotometer

iGEMmers:

Amanda - morning and afternoon

Beatriz - morning and afternoon

João Paulo - morning and afternoon

Juliana - morning

Raquel - morning

Vinicius - morning and afternoon

Today’s steps:

Analyse the biofilm formation and consequently the cell growth in the 96-well plate

96-well plate preparation scheme.

We think it is important to highlight our preliminary results on biofilm quantification: the wells containing biofilm producing cells showed a darker purple stain after the dye addition, but also some of the control wells have showed some growth, indicating contamination.

Plate reading:

Scheme representing the blank addition in order to realize the absorbance measurements.

96-well plate image after the biofilm growth procedure. It is possible to notice that some of the control wells showed biofilm growth, which indicates contamination

WEEK 2

Wednesday - Aug. 21

Preparation day! We planned and selected useful vectors for amplification by E.coli DH5α cloning, then prepared and sterilized LB culture media providing material for following experiments

iGEMmers:

Amanda
Juliana
Lucas
João Paulo

Today’s steps:

LB media preparation;
DGC’s vectors amplification
Material autoclaving.

Thursday - Aug. 22

First day E.coli DH5α transformation. The vectors inserted in bacteria are pSB1K3 (iGEM vector), pACYCDuet, pETSUMO(ydeH) e pETSUMO(wspR)

iGEMmers:

Amanda
Juliana
João Paulo
Raquel

Today’s steps

Antibiotic containing plate preparation;
Concentration measurement of stock vectors ;
Resuspension of iGEM’s kit DNA
Bacterial transformation using:

pSB1K3 (vetor iGEM): kanamicina

pACYCDuet: cloranfenicol

pETSUMO (wspR): kanamicina

pETSUMO (ydeH): kanamicina

Friday - Aug. 23

We assembled results from our previous experiment of bacterial transformation, a.k.a., the previous day.

iGEMmers:

Amanda
Beatriz
Luana

Today’s steps:

Transformed bacteria plate checking;
Lab organization.

chloramphenicol plates - Control e pACYCDuet

kanamycin - Control, pSB1K3::RFP, pETSUMO(wspR) and pETSUMO(ydeH)

WEEK 3

Tuesday - Aug. 27

Following last week’s procedures, we kept doing bacterial transformations and cultures. Also, we pre-inoculated the bacteria that were on hold in our fridge since friday, Aug 23rd

iGEMmers:

João Paulo
Lucas
Nayla

Today’s steps:

Cell isolation

Preparation of 4 cell cultures for isolation being DH5α transformed with pSB1K3::RFP, pACYCDuet, pETSUMO (ydeH) e pETSUMO (wspR)

Wednesday - Aug. 28

We found a procedure mistake in yesterday’s protocol, so the plasmidial DNA extraction from our stationary culture is not reliable anymore and must be redone. In the afternoon, we prepared antibiotic stock solutions and redid the cultures

iGEMmers:

Amanda
João Paulo
Juliana
Luana

Today’s steps - Morning (Invalidated due to unavailable selective media: 27.ago.019)

plasmidial DNA extraction (miniprep);
concentration measurement of obtained DNA

VETOR	[ ] ng/uL	260/280	260/230
pSB1K3::RFP	125.4	1.83	1.66
pACYCDuet	37.5	1.87	1.77
pETSUMO(wspR)	81.1	1.70	1.14
pETSUMO(ydeH)	59.9	1.94	1.88

Concentration values of obtained DNA

Today’s steps - Afternoon

antibiotic stock solutions preparation;
Transformed bacteria isolation (selective media).

Thursday - Aug. 29

Transformed cells were stocked in glycerol and plasmidial DNA was extracted from the 4 pre-inoculated cultures of DH5α containing the following plasmids: PSB1K3::RFP, pACYCDuet, pETSUMO(wspR) e pETSUMO(ydeH)

iGEMmers:

Luana
Nayla

Today’s steps:

Glycerol stock of transformed cells;
Mini-prep, plasmidial DNA extraction;
DNA concentration measurement (NanoDrop).

VECTOR	[ ] ng/uL
pACYCDuet	247.56
pETSUMO(wspR)	89.96
pETSUMO(ydeH)	79.23

Values of the DNA concentration measurements performed with the Nanodrop

WEEK 4

Wednesday - Sep. 11

PCR preparation day! We needed to isolate the “MermAID” and the “secretion machinery” synthetic DNA fragments. We resuspended primers and synthetic DNA, planned the PCR steps in the thermal cycler using a temperature gradient. We used the Q5® High-Fidelity 2X Master Mix da NEB reaction kit.

iGEMmers:

Amanda
Juliana
Lucas
Raquel

Today’s steps:

Morning

Primer and DNA resuspension (MermAID synthetic DNA and secretion machinery);
Primer aliquoting.

Afternoon

Synthetic DNA resuspension;
Thermocycler parameters adjustment.

Primers data

Primers list

Synthetic DNA	Mass (ng)	Buffer volume TE (ul)
MermAID 1	1000	100.0
MermAID 2	784	78.4
MermAID 3	750	75.0
MermAID 4	1000	100.0
Sec mach 1	100	100.0
Sec mach 2	100	100.0
Sec mach 3	100	100.0
Sec mach 4	100	100.0
Sec mach 5	100	100.0
Sec mach 6	100	100.0

PCR adjustment parameters

Bio-Rad T100 Thermal Cycler

Friday - Sep. 13

First PCR! The “MermAID” synthetic DNA and the “secretion machinery” DNA fragments were amplified using our NEB kit (Q5® High-Fidelity 2X Master Mix). We later prepared the agarose+BrET gel for PCR validation. Now that we’ve separated the Twist Bioscience adapter ends, we may start the genetic circuits assembly using the Gibson’s method

iGEMmers:

Amanda
Beatriz
Juliana
Nayla
Raquel

Today’s steps:

Morning

PCR preparation

Afternoon:

PCR;
agarose+BrET gel preparation;
run the PCR products in the gel.

WEEK 5

Tuesday - Sep. 17

We repeated the gel verification protocol of our first PCR due to diffuse DNA bands. We also renewed our agar medium stock, which was left to autoclave and resuspended our primers. We also carried the E.coli transformations with the DGCs YdeH, WspR and DGCO.

iGEMmers:

Juliana
Lucas
Luana

Today’s steps:

Morning

LB+agar medium preparation
PCR verification of A1, A2 e A3 fragments

Afternoon

bacterial transformation with DGCs

Wednesday - Sep. 18

iGEMmers:

Amanda
Juliana
Lucas
Raquel
Nayla
Luana

Today’s steps:

Afternoon

Secretion machinery PCR product purification (Parts M1 to M5)
Isolation of DGCs transformed bacteria

Thursday - Sep. 19

At first, we intended to continue our experiments on the DGC transformed cells, staining cultures with crystal violet. In the previous lab session we isolated the transformed cells, now we should plaque and observe cell behaviour in function of time and [IPTG]. But, as soon as we arrived at the lab, we realized we’ve forgotten our transformed bacteria control culture, i.e., transformed with an empty plasmid. Then we repeated the PCR protocols for A1, A2 and A3 parts, this time adding DMSO due the spread bands we got last time.

iGEMmers:

Nayla
Luana

Today’s steps:

PCR of A1, A2, A3 fragments

Saturday - Sep. 21

iGEMmers:

Amanda
Beatriz
Juliana
Luana
Nayla
Raquel
Raissa

Today’s steps:

Amplification of A1, A2 and A3 fragments in a temperature gradient
0.8% agarose+BrET gel fabrication for product extraction, validation and purification
Isolation of DGC transformed cells (pre-inoculum)
Control competent cells preparation: BL21 transformed with petsumo
A1, A2 purification

A3 fragment didn’t presented a 300kb band then it couldn’t be purified

WEEK 6

Sunday - Sep. 22

iGEMmers:

Amanda
Juliana
Raquel
Raissa

Today’s steps:

Glassware cleaning and preparation
Biofilm quantification experiment - step 2

cell dilution
96 well plates preparation

Preparation of LB medium in 5 ml tubes
Kanamycin stock solution preparation at 100 mg/ml, to be diluted till 50 μg/ml when used

Monday - Sep. 23

iGEMmers:

Luana
Giane

Today’s steps:

Crystal violet solution at 0.1% preparation (to be used later)
non-competent E. coli BL21plated
96 well plates staining for biofilm evaluation

WEEK 7

Monday - Sep. 28

iGEMmers:

Luana
João Paulo
Tandy
Amanda
Nayla

Today’s steps:

Acetic acid solution preparation
96-well plates washed after 24h with 30% acetic acid
Solutions were transferred to a clean 96-well to take measurements in the spectrophotometer

Tuesday - Oct .1

Today we took the absorbance spectrum of our 96-well plates after 48h incubation (continuing last week’s experiment) - André "Polegs" and Rafa helped us out. We also transformed our bacteria with all of the available DGCs in order to repeat the biofilm quantification, we also cloned our empty pETSUMO.

iGEMmers:

Amanda (Morning and afternoon)
Juliana (Morning)
Raquel (Morning and afternoon)
Polegs (Morning)

Today’s steps:

Morning

Biofilm formation analysis by spectrophotometry

Afternoon

Raissa has given us new guidelines for our afternoon shift so we performed the transformation of BL21 with all of the DGCs looking for new results in biofilm quantification, but as soon as we got the vectors we realised that we almost had ran out of empty pETSUMO vectors and besides we didn’t know its volume or concentration. We asked for help and Helo (Raquel’s advisor) told us to dilute the material and proceed with the NanoDrop measurement:

[ ] ng/uL	Average (ng/uL)	[empty pETSUMO]FINAL
53.6	55.7	111.4 ng/uL
52.7
60.8

After the concentration measurement, we calculated the necessary volume for a final mass equal to 50 ng to carry out the transformation protocol. Then we decided to proceed with the empty pETSUMO cloning in DH5α due to the low quantity of the stored vector. The table below shows the concentrations of vectors and how much of each plasmid we used:

pETSUMO	[ ] ng/uL	Volume for 50 ng (uL)
wspr	107.6	0.5
YdeH	66.6	1
yddv	125.3	0.5
vazio	111.4	0.5

OUR PRIMERS ARRIVED!

We then resuspended the DNA and aliquoted for PCR protocols in the next day.

Wednesday - Oct .2

Today we proceeded with the vectors PCR, then made a verification and a purification gel.

iGEMmers:

Amanda (Morning/Afternoon)
Juliana (Morning/Afternoon)
Lucas (Afternoon)
Raquel (Morning/Afternoon)

PROTOCOLS

DNA resuspension - iGEM protocol
Bacterial transformation protocol

Today’s steps:

Morning

PCR of 3 vectors:

M1: assembly 1
P: Linearize pSB1K3
D: open Duet to insert merChina

Vector	Name	Length (pb)	Primers	Tm (ºC)	Thermo cycler	Line
pSB1K3	P	2204	41/42	64.4	64.4	F
pACYDuet	M1	3648	21/22	69.9	70.1	C
pACYDuet	D	3647	43/44	71.9	71.9	A

Obs.: This time we’ve used Tm indicated in EXXTEND’s form and not Benchling’s.

Afternoon

To pursue the gold medal, we should've started assembling the China Team parts at the same time as ours. However, we didn’t have enough DNA to proceed with a PCR of 2 to 3 ng of material per well, then we have to clone this parts first. So we transformed DH5α with China’s part, BBa_K346004, which was later resuspended.

WEEK 8

Sunday - Oct .6

Today the secretion machinery and MermAID were inserted into competent DH5α after the NEB reaction

iGEMmers:

Amanda
Juliana
Giane

USED PROTOCOLS:

Bacterial transformation

Today’s steps:

Afternoon

This afternoon we transformed DH5α with NEB reaction (using all of the 20 uL) that we completed last friday (04.10.19), we inserted MermAID fragments and secretion machinery sequence, located in pSB1K3 plasmid, we also made a control culture. The chosen antibiotic is kanamycin.

Tuesday - Oct .8

We ran an electrophoresis procedure for the secretion machinery, prepares LB media (with and without agar), then inoculated cells for transformation. Also, we prepared Petri dishes to cultivate our transformed bacteria. Later, we did a electrophoresis of plasmids containing YdeH and the original merChina.

iGEMmers:

Amanda (morning)(afternoon)(night)
Nayla (afternoon)(night)
JP (afternoon)(night)
Raquel (morning)(afternoon)(night)
Juliana (afternoon)(night)
Luana (night)

USED PROTOCOLS:

1 - LB medium recipes

3 - preparation of antibiotic containing plates

4 - cell inoculation

13 - BrET agarose gel protocol

14 -purification of PCR product in agarose gel (Promega protocol)

Today’s steps:

Morning

We carried out a PCR of our transformed colonies with MermAID and secretion machinery parts.

Afternoon

Agarose gel preparation for colonies PCR verification after NEB reaction of MermAID and secretion machinery. The image below shows the resultant bands:

We chose the colony based in MermAID best result because the final gel lacked a 5 kb band, where the secretion machinery should be. As the results were not according to the expected we must repeat the PCR in order to guarantee the correct linking of parts.

Then we made 3 LB+agar media, 500 ml of medium separated in 3 containers, we also prepared 28 tubes with 5 ml of liquid LB. Everything was autoclaved.

Night

Petri dishes were filled with medium and NEB product transformed cells were pre-inoculated. The image below shows a scheme of the plates division:

The pre-inoculum was done in LB medium + kanamycin in 8 different test-tubes and a extra tube for the control sample.

We ran an electrophoresis to purify the parts we aim to characterize, i.e., YdeH and merChina.

Wednesday - Oct .9

Moving on, we made a miniprep with the pre-inoculated cells, te ones transformed with NEB product and IARAα. SOC medium was prepared to start the biofilm quantification protocol and characterize our DGCs.

iGEMmers:

Amanda
Juliana
Raquel

Today’s steps:

Morning

The pre-inoculum M3 opened inside the shaker and A3 hasn’t grown. So we proceeded the miniprep with the other samples (M2, M4, M5, M6, M7 and A6), following step consisted in repeat A3 inoculation using the pizza dish.

After the miniprep, we measured the concentration of the resultant DNA with the NanoDrop. Proceeding with the experiments, we then diluted 10 times this DNA which led to a final concentration of 11,4 ng/uL, 1 uL of this solution was used in a 50 uL PCR procedure.

Thursday - Oct. 10

The MermAID, colonie A3, got its miniprep redone the previous night. The lab team autoclaved LB filled tubes and empty tubes and later made new measures in the NanoDrop. We then measured the OD and pre-inoculated cells for 24-well plates.

iGEMmers:

Amanda
Juliana
Raquel
Paulina
Lucas
Nayla

Today’s steps:

Gibson assembly reagents quantities calculations
Actual DNA assembly

We calculated the concentrations to link Assemble 1, i.e., MermAID and Duet, and put merChina in Duet vector.

The math for this step is pretty simple as we are only working with two fragments, a basic crossed relation is enough between our DNA concentration and a arbitrary mass value in ng corresponding to an approximation of NEB reagents, by that we may find the necessary volume of solution. We repeated these calculations for all the four parts.

Iaraα:
Our MermAID sample (Iaraα) had a concentration of 3,6 ng/uL

Choosing almost at random an initial value of 50 ng (after checking the if the picomol value was between 0,03 and 0,2) and knowing that NEB proportion is 1 vector: 2 insert. The same procedure was followed to analyse 25 ng of Duet.

Obs: the sum of the picomol values of all fragments must be between 0,03 and 0,2 at the end of the dilutions.

Next, we confirmed that the calculated values fitted the variation, but the required volume to make the reaction happen would cost us 3 NEB reactions at once due to the low concentration of Iaraα.

Dessa forma, fomos testando diferentes massas que nos permitissem pegar um volume menor de amostra, para se adequar no volume final necessário (duet + iara) = 10uL

Following this logic, we checked for different masses, searching for results that would allow us to use a smaller volume of our NEB kit but still fit the final volume of Duet + Iaraα which ir 10 uL.

Iara𝛂:

3,6 ng ------------------ uL

34,02 ng ------------------- x = 9,45 uL

Duet (M1):

30,93 ng ---------------- uL

17, 01 ng --------------- x = 0,55 uL

9,45 uL + 0,55 uL = 10 uL

So to the next assemble, we needed to insert our old DGCs and merChina in the Duet vector.

Base pairs and concentrations are as follows:

MerChina - 364 bp - 145,8 ng/uL

Duet - 3647 bp - 34,96 ng/uL

However, due to merChina’s high concentration, we diluted it by a factor of 10 and proceed with the calculation described above.

Duet Iara:
Mer China:
Duet China:

After finishing calculations, we united the NEB products, incubated them for 40 min in the thermocycler at 50oC. Even though, 40 min is not the recommended incubation time for NEB, we chose to follow Ana Eliza’s suggestion, since that she had already worked several times with NEB reactions and got some experience from it.

Friday - Oct. 11

iGEMmers:

Raquel (morning)
Paulina (morning)
Lucas (night)
Luana (night)

Today’s steps:

Redo the SOC medium in correct concentrations
Transformation of DH5α with MermAID Iaraα + Duet and transformation of BL21 with MachSec (secretion machinery) + pSB1K3.
Verification of our colonies of transformed bacteria (MachSec, merChina, Iaraα)

we didn’t observed a relevant growth

Lavagem da placa de teste de DGCs com PBS para análise posterior
Washed our DGCs test plates (24-well) with PBS buffer for future analysis

Saturday - Oct. 12

iGEMmers:

Nayla
Nico

Today’s steps:

Preparation of 30% acetic acid and crystal violet solutions for further use
After 24h of incubation in agitation at 37oC, the 96-well plate was removed from the incubator for analyses

Abs at 590 nm after staining with crystal violet

Preparation of LB medium for competent cells protocol
Gibson assembly procedure: calculation of volumes and concentrations for 3 reactions for linking Iaraα and Duet
Preparo das soluções de Hg em SOC e LB para que seja feita a curva de calibração das concentrações
Hg dilution in SOC and LB media to calibrate a absorbance curve in function of concentration
Transformation of DH5α with Iaraα using Leo’s adapted protocol

WEEK 9

Sunday - Oct. 13

iGEMmers:

Nayla
Paulina

Today’s steps:

Normalization of cell quantity for DGCs testing plates
MachSec: Cell growth is still not enough and we must incubate this pre-inoculum longer
PCR: remove the chimera from MermAID
Preparar para uso futuro:
Preparation for further use:

erlenmeyers filled with culture media, SOC and LB (4 x 100ml)

Protocolo de células competentes para MaqSec
Proceeded to competent cells protocols for MachSec:

Kanamycin resistant cells
final quantity: 20 microtubes of 50 uL each

Monday - Oct. 14

iGEMmers:

Luana
Raquel
Amanda

Today’s steps:

A3 fragment was amplified alternatively with Phusion restriction enzyme and then purified:

DNA bands located at 900 bp in the verifying gel

DNA material had its concentration checked

Tuesday - Oct. 15

iGEMmers:

Raquel
Amanda
Paulina
Juliana

Today’s steps:

Three chimera samples, α, &beta and &gamma, had its concentration measured for PCR amplification
We won’t be able to proceed to link our vector with Gibson assembly reaction, first we must repeat the pre inoculation step
Pré-inóculo, miniprep, PCR de outras colônias transformadas, das quais isolam-se a quimera
Using the previously separated (supposedly transformed) colonies, from which we got fragments A10, A11, A13, A14, we isolated the chimera:

carried on to pre-inoculation, miniprep and PCR
the colonies will be cultured in “pizza” plates

Development of 4 tests:

biofilm formation in 24-well plates, incubated for 24h and 48h

analysis were carried following Raissa’s protocolo for crystal violet staining
biofilm is visible after being treated with the dye!

coconut fiber experiment set:

the fiber was shaped into small bunches (approximately cubes) using the wells of a 96-well plate to format it
each cube weighted around 60 ug
the 32 cubes produced were applied into two 24-well plates filled with SOC medium for DGCs experiment, all done in triplicates

Last chimera amplification attempt:

we will be using this morning’s pre-inocula miniprep

Hg disk diffusion test (MermAID in action!):

preparation of 3 different concentrations of HgCl2: 20 mg/ml, 200 ug/ml, 20 ug/ml
all of our MermAID samples available were tested (A10, A11, A12, A13, A14)

Wednesday - Oct. 16

iGEMmers:

Nayla
Amanda
Juliana
JP

Today’s steps:

Right in the morning we went for the last attempt to amplificate de MermAID in ideal conditions, using DMSO and BSA (not enough time for another PCR!)

PCR Results did not displayed a significant result
We then abandoned this goal, we were not able to isolate the chimera from Iaraα

Moving on, we set plates ready for measuring biofilm formation of DGCs static and agitation samples, both 24h and 48h plates

biofilm quantified with spectrophotometry

In the afternoon, we again pre-inoculated cells to repeat the DGCs quantification experiment, but this time we added coconut fiber

Saturday - Oct. 19

iGEMmers:

Amanda
Raquel
Paulina
Juliana
Nayla
Luana
João Paulo

Today’s steps:

First thing in the morning, we prepared new media bottles (LB and LB + agar) for the next steps

pre-inoculum of another transformed culture, indicated as A3
new Petri dishes for Hg disk diffusion tests with this last bacteria

We analyzed the previous Hg disk diffusion test (performed with A10 to A14 fragments) and results were not the expected and MUST be repeated
By the end of the morning we begun mapping the cell growth in different Hg concentrations, aiming to compare results with UFAM 2014 iGEM Team
New disk diffusion test: Hg solutions were set at concentrations equal to: 0, 200, 1000, 2000, 10000, and 40000 ug/mL

triplicate samples + two control plates (with and without cells) were cultured with DH5α transformed by A3 fragment
results will be analyzed tomorrow morning

Sunday - Oct. 20

iGEMmers:

Amanda
Raquel
Paulina
Juliana
Nayla
Luana

Today’s steps:

5 A.M.: DH5α transformed by A3 fragment were plated to complete the Hg disk diffusion test, so we can evaluate Iaraα resistance; cultures were placed in the incubator at 7 am.
We also started the growth curve of our Iara-alpha with different mercury concentrations. We measured for 6 hours.
We started to map our bacterial growth curve in Hg solutions of different concentrations: 0, 7.5 ug/mL, 20 ug/mL, 200 ug/mL, 2000 ug/mL
Also in the morning we prepared a 72h DGCs experiment combined with coconut fiber