Team:TU Delft-Leiden/Project/Notebook/LabjournalLandmine

Landmine Labjournal

This labjournal will give more information on the cloning and characterisation experiments performed within the landmine detection module.
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Content page notebook

1st of July

Grow on shakeflasks the strains received from Belkin lab (J::GFP, F::GFP, J::lux, F::lux, FB2::lux, FB2A1::lux) and the host E. coli strain C43(DE3). F is the promoter from the yqjF gene from Escherichia coli. J is the promoter from the ybiJ gene from Escherichia coli. FB2 and FB2A1 are improved versions of the yqjF promoter generated via mutagenesis.

2nd of July

Miniprep Belkin samples (J::GFP, F::GFP, J::lux, F::lux, FB2::lux, FB2A1::lux)
Glycerol stock of Belkin samples (same as above) and the host C43(DE3)

16th of July

Miniprep + Glycerol Stocks of mKate2.
Measured the concentration of the isolated DNA with nanodrop:
• mKate2 39.2 ng/ul

22th of July

PCR ybiJ from Belkin ybiJ:lux	Amount	Use
Template: ybiJ:lux [233 ng/ul]	75 ng	0.35 ul
FW ybiJ (5 uM)	5 pM	2.5 ul
RV ybiJ (5 uM)	5 pM	2.5 ul
dNTP (10 mM)		1.5 ul
pFx		0.2 ul
Buffer	10x	5 ul
MgSO4 (50 mM)		1 ul
Enhancer		5 ul
MilliQ		32 ul
Total volume		50 ul

Miniprepped the samples cultivated on 15.07.2014, except for the strain AYCE189. Measured the concentration of the isolated DNA with nanodrop:
PAYC002 122.8 ng/ul
rr12y(rii)g 52.6 ng/ul
PAYC003 187.0 ng/ul
rrjt12(11)g 32.3 ng/ul
PAYC005 27.8 ng/ul
PAYC008 22.3 ng/ul
PAYC006 37.0 ng/ul
PAYC007 22.3 ng/ul
I5023 22.5 ng/ul
C640 14.3 ng/ul
mKate 39.2 ng/ul
eGFP 165.1 ng/ul
Rhamnose 47.7 ng/ul
Cultivated the samples again in shakeflasks. Made glycerolstocks of all the cultivated samples from 15.07.2014.

Test the competency of C43(DE3):
30 ul competent C43 cells + 100 ng pUC19 (AmpR)(190 ng/ul), plated in duplo on Cm (neg. control), Amp (pos. control) LB plates and on LB plate without antibiotics
30 ul competent C43 cells plated in duplo on Cm (neg. control), Amp (neg. control) LB plates and on LB plate without antibiotics (pos. control)

17th of July

Results of the plates for positive and negative control of C43
• C43 + pUC19(AmpR) + Amp plates Much growth
• C43 + pUC19(AmpR) + Cm plates No growth
• C43 + pUC19(AmpR) + without antibiotic Much growth
• 2x diluted C43 + pUC19(AmpR) + Amp plates Single colonies
• 2x diluted C43 + pUC19(AmpR) + Cm plates No growth
• 2x diluted C43 + pUC19(AmpR) + without antibiotic Much growth
• C43 + no plasmid + Amp plates No growth
• C43 + no plasmid + Kan plates No growth
• C43 + no plasmid + Cm plates No growth
We decided to not dilute the competent cells for the future experiments.
CFU for pUC19(AmpR): 1120 colonies on the plate.
Test the competency of BL21(DE3)
• 30 ul competent BL21 cells + 100 ng pUC19 (AmpR)(190 ng/ul), plated in duplo on Cm (neg. control), Amp (pos. control) LB plates and on LB plate without antibiotics
• 30 ul competent BL21 cells plated in duplo on Cm (neg. control), Amp (neg. control) LB plates and on LB plate without antibiotics (pos. control)
• 2x diluted 30 ul competent BL21 cells + 100 ng pUC19 (AmpR)(190 ng/ul) plated in duplo on Amp (pos. control) LB plates

18th of July

Results of the plates for positive and negative control of BL21(DE3)
• BL21 + pUC19(AmpR) + Amp plates Much growth
• BL21 + pUC19(AmpR) + Cm plates No growth
• BL21 + pUC19(AmpR) + without antibiotic Much growth
• 2x diluted BL21 + pUC19(AmpR) + Amp plates Single colonies
• BL21 + no plasmid + Amp plates No growth
• BL21 + no plasmid + without antibiotic Much growth
• BL21 + no plasmid + Cm plates No growth

22th of July

Prepared antibiotics, 500ul in each eppendorf cup. Saved in ‘Antibiotics’ box in the freezer with other iGEM materials. 1000x stock solutions.
• Chloramphenicol diluted in EtOH
34 mg/ml in EtOH -> 0,391 gram diluted in 11,5 ml EtOH
• Kanamycin diluted in H2O
10 mg/ml in H2O -> 0,095 gram diluted in 9,5 ml H2O
• Ampicillin diluted in H2O
100 mg/ml in H2O -> 0,92 gram diluted in 9,2 ml H2O
Pre-culture DH5-alpha has been prepared, 100 mL LB medium is cultivated and placed in the shaker. Overnight at 37 degrees and 180 rpm.

23th of July

Made competent cells of DH5a and saved in the freezer (-80).

28th of July

The samples asked from Exeter iGEM team were prepared and sent to them. The samples requested are: - BBa_K1022115 , Kanamycin resistant - BBa_K1022105 , Chloramphenicol resistant - BBa_K112808 , Ampicillin resistant Transformation of pUC19 in BL21 and DH5a as a control • 30ul + 100ng pUC19 (190 ng/ul) o Plated 50ul and 100 ul on Cam, Amp and plates without antibiotics • 30ul competent cells o Plated 50ul and 100 ul on Cam, Amp and plates without antibiotics

29th of July

Cristy and Anne Results of the DH5a and BL21 plates: • BL21 + pUC19(AmpR) + Amp plates Much growth, big single colonies • BL21 + pUC19(AmpR) + Cm plates No growth • BL21 + pUC19(AmpR) + without antibiotic Much growth • BL21 + no plasmid + Amp plates No growth • BL21 + no plasmid + without antibiotic Much growth • BL21 + no plasmid + Cm plates No growth • DH5a + pUC19(AmpR) + Amp plates Much growth, little single colonies • DH5a + pUC19(AmpR) + Cm plates No growth • DH5a + pUC19(AmpR) + without antibiotic Much growth • DH5a + no plasmid + Amp plates No growth • DH5a + no plasmid + without antibiotic Much growth • DH5a + no plasmid + Cm plates No growth Tomek and Esra: making trace elements solution 2 attempts

30th of July

Esra Making the M4 minimal medium Trace Elements Solution (third attempt) + buffer (exact contents will be updated) -Changed adding order -Adding order 1- EDTA, 2- Mg, 3-Mn, 4-NaCl, 6-CoCl etc. -Added number 5- (FeCl diluted in 22.5 mL HCl) at the end!! -All added except FeCl (in HCl) -> no precipitations!! All is diluted well! Inoculated the transformed iGEM registry constructs Resistance Transformation Grown in (ml LB): Cam DH5a + k823017 5ml & 10 ml Cam DH5a + k808000 5ml & 10 ml Kan DH5a + I20260 5ml & 10 ml Cam DH5a + 80017 5ml & 10 ml Amp DH5a + J231100 5ml & 10 ml Amp DH5a + pUC19 5ml & 10 ml

20th of August

Janna Made medium M4 with different carbon sources. 1. 400 ml 40 mM D/L-lactate M4 342 ml MilliQ 10 ml Buffer 40 x 4 ml 0.1 M CaCl2 40 ml 0.4 M D/L-lactate 4 ml Trace elements 100 x 2. 400 ml 40 mM glycerol M4 342 ml MilliQ 10 ml Buffer 40 x 4 ml 0.1 M CaCl2 40 ml 0.4 M Glycerol 4 ml Trace elements 100 x 3. 400 ml 40 mM glucose M4 342 ml MilliQ 10 ml Buffer 40 x 4 ml 0.1 M CaCl2 40 ml 0.4 M Glucose 4 ml Trace elements 100 x

21th of August

Janna Tested competent cells BL21 culture 1, BL21 culture 2 and C43. I used pUC19 as test DNA (ampR). Made the following combinations: 1. 30 μl BL21.1 with 1 μl MilliQ 2. 30 μl BL21.1 with 1 μl pUC19 3. 30 μl BL21.2 with 1 μl MilliQ 4. 30 μl BL21.2 with 1 μl pUC19 5. 30 μl C43 with 1 μl MilliQ 6. 30 μl C43 with 1 μl pUC19 There were no colonies on the plates, so the cells are not competent. Cristy: Made competent cells of CsgB (approximately 30 eppendorfs containing 100ul competent cells). OD600: 0,567 and 0,589

22th of August

Joan Prepare samples for Melbourne iGEM team: • pET23b - Ulp1-His6 (AmpR) • BBa_K1022107:pcI-Ulp in pSB1C3 col2 • BBa_K1022113:pBAD-Ulp-TT in pSB1C3 col2

26th of August

Chloramphenicol diluted in EtOH 34 mg/ml in EtOH -> 0,3912 gram diluted in 11,5ml EtOH. Divided in aliquots of 500 ul. Refilled stock with 22 new cups. Janna Making M4 with D/L-lactate. Same protocol as 20/8, only filter sterilized the whole bottle after making it.

28th of August

Janna Transformation of pUC19 in C43+ET20 as test Made the following transformations: 0.5 ul pUC19 plasmid (concentration of 100.2 ng/ul) added to 30 ul of C43+ET20 competent cells 1 ul MilliQ added to 30 ul of C43+ET20 competent cells as a negative control Followed the transformation in home-made competent cells protocol and made six plates with each 100 ul: pUC19 in C43+ET20 (ampR and camR): Ampicillin - some growth Chloramphenicol - some growth Amp and Cam - lots of growth MQ in C43+ET20 (camR): Ampicillin - no growth Chloramphenicol - some growth Amp and Cam - no growth This means the cells are competent, but the efficiency is low.