Team:Orleans/Notebook

Notebook

2019
july
22 july- 28 july
Reception of our Bacteria, Cupriabidus Metallidurans CH34 and Escherichia Coli S17-1, the growth (on plate and liquid culture) for glycerol stock.

After receiving the Cupriavidus Metallidurans CH34 and Escherichia Coli S17-1 bacteria, the received bacterial pellets were suspended in BM3 and subsequently placed in a continuous culture of these two strains.
Glycerol stocks are the realized to preserve these strains.

These bacteria were also spread on Petri dishes to ensure that they were free of contamination and that the cells were viable. First determination of Metals in station sludge by ICP for Pb and Cd in supernatant and dry matter.

The various sludge samples received from the wastewater treatment plant, including Tamburg sludge (T) and excess sludge (BE), were analysed by ICP to determine their cadmium and lead concentration. For each sludge, a sample was dried in the oven at 105°C over night and a supernatant sample after settling was taken.
All these samples are acidified by adding 6mL of HNO3 to 65% and 3mL of HCl to 37%.
The dried samples are mineralized by heating in a microwave oven, then all samples are filtered on 0.22µm cellulose membranes.
These samples are then measured by ICP.

Determination of sludge dryness

The dryness of the different samples of sewage sludge was determined by weighing for each sludge a sample before and after drying in an oven at 105°C over night.
The calculation of the dryness is as follows:
Results for the dryness calculation :

Results of dryness :

august
29 july - 4 august
Receipt of bacteria

C. Metallidurans was cultured to amplify it and perform genomic DNA purification using the genejet genomic DNA purification kit.

PCR on C. Metallidurans genome

PCR amplification on the metallidurans genome to amplify the sequences bordering the targeted genes in 3' using the primers designed in June.

Solutions prepared for PCR (50µL):
10 µL Tp Phusion HF 5X
5 µL dNTP
5 µL Fwd primer
5 µL Rev primer
2.5 µL Plasmid (1ng/mL, 0.1ng/mL or 0.01ng/mL)
22 µL H2O (QSP)
0.5 µL DNA pol Phusion

Purification of PCR products by the genejet gel extraction kit and DNA cleanup micro kit

Migration of PCR products to agarose gel

These gels show the correct functioning of all the PCRs of the 3' inserts.
Purification by Plasmidic Miniprep of plasmids pSB1C3 and double digestion by the restriction enzymes EcoR1 and Spe1
Continuation of 3' PCR (CzcD and CzcA genes)

Digestion of Biobrick in pSB1C3 after amplification in DH5alpha and miniprep of this plasmid
19 august - 25 august
PCR amplification of 5' inserts

Receipt of plasmids pCM184 and pBBR1MCS-2 from Addgen and amplification in DH5α
Miniprep plasmid of these plasmids then migration on gel
Here the PCR Amplifications of the 3' inserts all worked, however, only the 5' inserts of the pbrR and pbrT genes and CzcA were correctly amplified

Double digestion pBBR1MCS-2 and pCM184 from DH5α.


Purification by plasmid Miniprep of pSB1C3, double digestion by EcoRI and SpeI of the biobrick and purification on gel


Purification by plasmid Miniprep, double digestion (by the same restriction enzymes) and purification on gel of digested pBBR1MCS-2


PCR on Colony of construction pCM184 - insert 3'


Digestion of plasmid pCM184 and 5' inserts (pbrR, pbrT, czcA) and Ligation in pCM184 Then Transformation S17-1

PCR on colonies from previous transformations
26 august - 1 september
Digestion of the plasmid pBBR1MCS-2 with the restriction enzymes SpeI and EcoRI Digestion by the same restriction enzymes and purification of biobrick on gel Transformation of S17.1 by the construction pBBR1MCS-2 + Biobrick
september
2 september - 8 september
PCR on colonies from the previous transformation

Double digestion by the restriction enzymes BamHI and EcoRI and Pan ligation in pBBR1MCS-2 + Biobrick
Addition of the Pan developer to the construction pBBR1MCS-2 - Biobrick It was digested by the restriction enzymes HindIII and EcoR1. Similarly, the construction pBBR1MCS-2 - Biobrick was purified by the GeneJET Plasmid Miniprep Kit, and digested by the same restriction enzymes as Pan The digested promoter was then linked to the construction digested by the T4 ligase.

Migration of the construction pBBR1MCS-2 + Biobrick + pan before and after digestion by EcoRI The ligation product was then introduced by transformation into E. Coli S17.1 to amplify it.
9 september - 15 september

Colony PCR on the last transformation The colonies obtained were then tested by PCR on colonies to select the bacteria transformed with the new construction pBBR1MCS-2 - Pan - Biobrick.
16 september - 22 september

colony PCR on the biobrick after electroporation (failed) on C. Metallidurans CH34
30 september - 6 october

second electroporation + colonie miniprep (2 negatives / 2 positifs)


Realization of the final gel showing the different constructions realized