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From 2012.igem.org

Weeks

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16

Week 2: 18th June - 24th June

Monday, 18th June

• Inoculation
• Auxin and salkowski's test purchased

Tuesday, 19th June

Miniprep

• J23100 RFP: 101.9 ng/uL
  
• K515100 Auxin: 319.7 ng/uL
  

Misc

Liquid culture of aux and RFP

Wednesday, 20th June

Previous work :

Liquid culture of DH5a with mRFP and auxin plasmid

Main goals:

1. Auxin toxicity test
2. Bacterial integration assessment into the xenopus embryos

Experiments

1. Xenopus embryos present a protective membrane, we decided to realize our bacterial deposits experiments on xenopus with and without their membrane. Then, we could see if bacterias could enter into the embryos naturally.
2. On 96 plates, each plate contains 1 embryo:
   • Plate 1:
     • Line A & B = on embryos without protective membrane : 500 uL mRFP plasmid on DH5a deposite + 1mL LB + 1mL MMR medium
       
     • Line C : 1 2 3 : normal embryos : 500 uL mRFP plasmid on DH5a deposite + 1mL (LB + MMR medium)
       
     • Line E & F = embryos without protective membrane : 500 uL AUX plasmid integrated on DH5a deposit + 1mL LB + 1mL MMR medium
       
     • Line G & M1-6 = normal embryos : 500 uL AUX plasmid integrated on DH5a deposit + 1mL LB + 1mL MMR medium
       
     • Line M7-9 = normal embryos : controls
   • Plate 2 (normal embryos only):
     • Line A & B
     • Line C : LB/MMR medium
     • Line D : RFP 0.1uL
     • Line E : AUX 0.1uL
     • Line G & H : LB/MMR medium only (no embryos)
     
3. On 16 Plates each plate contains 3 tadpoles:
   • Line A : 0.5uL RFP bacteria
   • Line B1 : 0.3uL RFP bacteria
   
   

We put this plate on UV light 15min after the deposit:

Thursday, 21st June

Results from Wednesday's experiments:

+ = Alive; - = Dead

• Plate 1: All the embryos are dead except H7
• Plate 2:

  L\C	1	2	3	4	5	6	7	8	9	10	11	12
  A	-
  B	-
  C	-
  D	-	-	+	-	+	-	+	+	+	-	+	+
  E	+	-	-	+	-	+	-	-	-	-	+	+

• Plate 3:

  L\C	1	2	3	4
  A	-	+	+	+
  B	-	+	+	+

Comments:

• Plate 2: 50% of dead when we spread bacterias on embryos. All dead if we drown the embryos of bacteria.
• Plate 1: All dead except 1
• Plate 3: All the tadpoles are fluorescent except the control one.

Friday, 22nd June