Team:Paris Bettencourt/Notebook/Indigo
Week 27th June - 3rd July
Introduction
Inspired by the idea of removing wine stains, we have decided to give an artistic tone to our project. What if we can exploit the power of enzymes not only to digest wine stains, but also to create art? For this we are planing to find enzymes and micro-organisms which can digest indigo dye used to colour denim. Our ultimate goal is to have a tool which can create beautiful patterns by bleaching out the indigo colour in our favourite pair of jeans. To achieve this we are using the same strategies as the Enzyme search and Microbiology group. Thus, within the Mission indigo group we will try to find enzymes and micro-organisms, especially fungi, capable of digesting indigo.
Bacillus Pumillus laccase (bpul)
Within the iGEM's Registry of Standard Biological Parts registry we have found a candidate with great potential to digest indigo dye. Bacillus Pumillus laccase (bpul) has shown to be a good enzyme for digesting many phenolic compounds with structure similar to that of indigo, such as indigo carmine. Moreover, this enzyme will be investigated by the Enzyme search group for its ability to digest anthocyanins from wine stains. Documentation about this protein can be found in the iGEM parts registry under code: BBa_K863000. For more information check: http://parts.igem.org/Part:BBa_K863000
During the first week we have discussing and planing how to realize Mission Indigo sub-project. We have also transformed BBa_K863000 which contains His-tagged bpul into the E.coli DH5alpha strain in order to preserve the plasmid. The new strain was called DH5alpha-bpul and can be found in the glycerol stocks.
Protocols used for this are provided in the links bellow:
[link 1] [link2] [link 3]
Week 4th - 10th July
Week 11th - 17th July
Transforming E. coli BL21(DE3) strain with BBa_K863000
In the BBa_K863000 construct bpul laccase gene is under T7 promoter. Therefore, T7 polymerase is needed to transcribe bpul DNA sequence into mRNA which will then be used to produce bpul laccase.
We received the E. coli BL21 and BL21(DE3). Both strains have the same genetic background, but BL21 does not express the T7 polimerase and BL21(DE3) does.
This week we started by plating the BL21 and the BL21(DE3) strains and making electro-competent cells out of them. We have used this protocol: [link 5]
We minipreped the pbul plasmid from previously transformed DH5alpha-bpul using the ThermoScientific GENEJET Plasmid MIniprep kit #K0503. The protocol used is given in the link: https://tools.thermofisher.com/content/sfs/manuals/MAN0013117_GeneJET_Plasmid_Miniprep_UG.pdf
We electroporated 2uL of plasmid and 20uL of the electrocompetent cells. We plated on LB crm25, 200uL and 500uL (Jake told us that 99% of the cells would die due to overexpression of our protein)
Confluent growth observed on yesterday's plates (problem with antibiotic? - I do not believe so because the plate with the + control - interstudy + control with the GFP - shows that only cells carrying the plasmid were able to survive on the plate - See Figure)
Restricking the laccase plasmid ones just in case in new plates.
BL21(DE3) strain was transformed with BBa_K863000 using the protocol described in the following link: [link4]
Highlights
Transformation efficiency: 1–5 x 107 cfu/μg pUC19 DNA
BL21(DE3) strain genotype:
fhuA2 [lon] ompT gal [dcm] ΔhsdS
Induction of T7 polymerase production is achieved by adding IPTG to the growth media.
Week 18th -24th July
Denim Experiment
We want to find microbes that naturally digest indigo. In order to find them, we started with a simple experiment: we put pieces of denim in a M9 agar medium. We prepared M9 without glucose, so there is no other nutrient but denim. The medium was not sterilized, since our goal is to find whatever organisms that feeds on indigo. Pieces of denim were cut directly from a pair of jeans, and soaked in M9 media. They were then put in a square petri dish, later fill with M9. One piece was dip in media and put in a bottle with the lid not totally closed. At first, the plates were left open, for any microbes to contaminate it. Problem is, it dried out really fast. For the piece of denim in the bottle, it wasn’t a problem at all, and it stayed soaked.
Week 25th -31st July
Species isolation
First colonies were observed on denim 4 days after the start of the experiment. 6 different strains were identified and potential candidates for degradation of indigo on denim. These colonies grew on the piece of denim that was put in the bottle, as it wasn’t dry. The plates were emptied and done again, this time having the lid almost closed to keep moisture.
Samples were harvest and inoculated on LB plates, in order to isolate them. Plates were put at 30 °C for 24 to 48 hours. They grew well except for one for which a new sample was taken. A database was created with details on them. As half of the strains were fungi, the question on how to work with fungi was raised and investigated throughout our lab work.
Pleurotus ostreatus
During this week we investigated a potential candidate called Pleurotus Ostreatus. Indeed, a few plant pathogen mushrooms are described in literature as indigo-degrading organisms, the degrading enzyme being laccase. Pleurotus ostreatus is a mushroom that grow on decayed wood, but can also be cultured on straw containing medium or other similar media. It is actually an edible mushroom, used in cooking. We will probably work with it.
Week 1st - 7th August
Producing fully coper loaded bpul
bpul enzyme has 4 coper ions which help catalyze its reactions. In order to make sure we obtain fully coper loaded enzymes we have grown our BL21(DE3)-bpul strain in specific conditions:
- Overnight culture was diluted 1:50 and grown until exponential phase
- After reaching exponential phase 0.1 mM of IPTG was added to initiate bpul transcription
- 0.25 mM CuCl2 was added to meet the coper needs
- Cells were incubated for 24h at 25oC.
- They were grown 4h with shaking and then 20h without. [reference] it increases the yield of coper loaded enzymes
Full protocol description can be found here [link 6].
1L of cells BL21(DE3)-bpul was prepared alongside 1L of BL21(DE3) cells (without the bpul construct) which will be used as a positive control.
After 24h incubation cells were collected by centrifugation, washed with PBS and stored at -20 oC.
Later, protein extract was obtained by using B-per reagent with lysozyme and DNase-1 (protocol).
Preparing solutions
Stock solutions of chemical needed to perform the indigo degradation were performed. I have faced some troubles with dissolving non-polar indigo and ACS, but eventually I have succeeded and mixes are presented below:
- 100 mM Acetosyringone (ACS) stock solution - 196 mg of ACS was dissolved in small volume of ethanol, after which the water was added until 10 mL volume
- 50 mM indigo stock in DMSO - 39.34 mg of indigo dye was disolved in 3 mL of DMSO
- 1M CuCl2 stock solution - 2.689g of CuCl2 was dissolved in 20 mL of H2O
- 0.1M Porassium phosphate buffer 7.8 pH - 1.541g of K2HPO4 and 156.2mg of KH2PO4 in 100mL of H2O
Species isolation
Now that we isolated a few strains, we could start species identification. A 16S colony PCR was performed, using all bacterial strains that were grown on LB. After that a gel electrophoresis was performed to check if the PCR worked and gave good results, leading to the next step in identification: DNA purification of PCR products to send them for sequencing. As there was a suspicion of primers contamination only one sample was sent.
3 new strains appeared on denim pieces during the week. They were harvest and inoculated on LB plates, just like the first ones. After all samples were taken, the piece of denim that was in the bottle was washed with water, and ethanol to make sure no fungi remained as they can easily disperse and contaminate stuff. Sadly, it seems no color removing was observed; it could either be that it is a longer process, or it needs other factors to make it more effective.
P. ostreatus arrived on Thursday, waiting for it to grow!
Week 8th - 14th August
indigo degradation experiments
2 experiments of indigo degradation were performed, between which the protein extracts were frozen once (which might have potentially lowered the bpul efficiency). Before experiments proteins were extracted using B-per and quantified using Bradford reagent kit. First experiment has failed because of indigo precipitation, thus in a new media solution indigo was dissolved in 0.5% Tween80. Indigo degradation experiments were performed in the presence of ACS to help the degradation reaction.
Results
Figure 1. Curve after staining standard BSA solutions with Bradford reagent. The function of the obtained curve was used to quantify protein extracts from the table below. Control sample had 19.903 g/L of proteins. bpul sample had 12.636 g/L of proteins
| Control sample 10x dilution | 0.9074 |
|---|---|
| Control sample 100x dilution | 0.2484 |
| Control sample 100x dilution | 0.2543 |
| bpul sample 10x dilution | 0.6819 |
| bpul sample 100x dilution | 0.2055 |
| bpul sample 100x dilution | 0.20573/th> |
Figure 2. Figure shows the absorbance scan of different concentrations of indigo solutions. Here, stock solution of indigo dissolved in DMSO was used and mixed with water to get different concentrations. Later experiments failed because of precipitation of indigo in water
Figure 3. After failed experiment, we have decided to repeat it but with indigo dissolved just in water to avoid precipitation. 0.5% of Tween 80 was used to increase indigo solubility in water. The result on the figure shows saturated water-indigo solutions and x2 dilutions.
Figure 4. Successful indigo digestion experiment.
Species isolation
Throughout the last week we collected 9 samples on denim. However growing them in LB plates we could see that there was not one specie per sample, but rather two or even three different ones per colony we harvested. To go further in species isolation all different strains from the first plate were inoculated on a second plate, giving us not 9 but 16 candidate organisms. Growth rate varied between the 16 strains. After a few tries we were able to have 16 grown organisms on LB plates. Another round of 16S colony PCR was performed with 9 out of 16 strains.
Our first sequence came back, and after blasting, it seems to be an enterobacteria.
Our first experiment with denim was over, but it was not perfect: even though we had organisms growing on denim, this fabric is made of cotton tainted with indigo, so we can’t be sure that these organisms fed on indigo or on cellulose. So it’s time for our second experiment: cultivate these organisms on medium with indigo as the only carbon source.
Week 15th - 21st August
Species isolation
Looking at the results of the PCR from last week, it was obvious that the protocol we used was not suited for fungi. Indeed fungi are eukaryote hence having a 18S rRNA and not a 16S (we still tried the 16S colony PCR with fungi in case it works for some of them). We thus spent the week working on a protocol for DNA extraction and colony PCR for fungi. Luckily almost half of the species isolated from jeans are bacteria so we could carry a 16S colony PCR for them, going further in the identification of our candidates. DNA was then purified and sent for sequencing. We had a total of 11 samples from 6 different strains to work with, from which 9 were eligible for sequencing. Next step is making plates, with either indigo as the only carbon source or glucose + indigo. Each strains will be inoculated on a plate with indigo and a plate with a piece of denim.
Determining indigo solubility
In order to follow indigo degradation with our enzyme (bpul) we had to determine the function which would connect the absorbance measurements with indigo concentration. This was done by following Beer-Lambert law. 8 concentrations ranging from 20 mg/L to 0 mg/L of indigo dissolved in potassium phosphate buffer have been made. In total we had 4-plicates of buffer from which indigo solutions were made. Each indigo concentration has been measured 3 times to confirm result, at 3 different wavelengths (660nm, 670nm and 680nm) which match the peak of indigo absorbance. In total we had so in total we had 288 measurements (96 per wavelength). Measurements belonging to same concentrations and wavelength have been. From the averages 3 graphs (one per wavelength) with tread-lines and functions were made. One is shown below.
Week 22nd - 28th August
Week 29th August - 4th September
Week 11th - 17th July
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igem2016parisbettencourt@gmail.com 