"

From 2012.igem.org

Contents 1 WEEK 1 - June 1st-7th 2 WEEK 2 3 WEEK 3 4 WEEK 4 5 WEEK 5 6 WEEK 6 7 WEEK 7 8 WEEK 8

WEEK 1 - June 1st-7th

Friday

Today we started with a briefing from Mustafa where the main action points of the day were discussed. We had been planning the project of this year.We determined our parts that will be necessary:

• K124014 holin
• K559010 halorhodopsin+terminator(X2)
• K112806 endolysine/T4
• K112808 endolysine/holin/CMV/antiholin

These 4 parts was diluted and tagged. Then we coded these parts:

• C1:J23100 cons-promoter
• C2:CMV promoter
• P1:CL promoter
• H1:Holorodopsin
• P2:IPTG
• L1:endolsin
• L2:holin
• l3:eth+antiholin


We prepared 35 plates and 200 ml LB broth for next experiments.

• Transformation (L1,L2,L3) 

• C1,C2 streak
• Coloniese are incubated in liquid culture.(C1)

Saturday

• Coloniese are incubated liquid culture.( 1,5 ml AMP ) for :

C1(x2),C2,N1,L2,L3.

• We made this process 2 times because of high mistake risk for first times

Sunday

• Single colony isolation has made for C1-x,C1-y,C2,L1,L2 and L3.
• C1-x and C1-y are digested with EcoR1 and Pst1.
• We prepared liquid culture for C2,L1,L3 again.Because there was not enough DNA for digestion.

Monday

• Single colony isolation has been made for L1,L2 and L3.
• Digestion has been made with EcoR1 and Pst1 for L1,L3 and C2.
• electrophoresis was performed for C1-x,C2,L1,L2 and L3.
• After electrophoresis,results of L1,L3 was true but the others was wrong.

Tuesday

• Digestion has been made with Xba1 and Pst1 for L1 and L3.
• 34 mg/ml chloramphenicol included 49 tubes was prepared.
• Chloramphenicol included 17 plates was prepared.
• IPTG induceble promoter was coded as P2.
• Transformation has been made for H1,L1P2 and L3P2
• We prepared liquid culture for C1,C2,L2

Wednesday

• Single colony isolation has been made for C1,C2,L2
• We prepared liquid culture for C2 again.Because there was not enough DNA for digestion.
• C1 and L2 are digested.
• electrophoresis was performed for C1 and L2.
• The result of C1 was true but the result of L2 was wrong so a new digestion will been performed and after that electrophoresis will been performed for new L2 and old L2.
• We prepared liquid culture for P2L1,P2L3,C2,H1 and L2.

Thursday -We made isolation for P2L1,P2L3,H1,C2,L2 and then digestion has been made with EcoR1 and Pst1 for all of these parts.After that electrophoresis was performed for these parts. -The results of P2L1 and P2L3 might be true , we are not sure about this so we need some expert assistance and we need to sequence these part for being sure. Unfortunately the result of L2 was wrong again. -We prepared a new liquid culture for L2.

WEEK 2

Friday -In order of isoation, digestion and electrophoresis was performed to L2 and C2.By the way electrophoresis was performed 2 times for being sure but both of results were wrong. -Digestion has been made with Xba1 and Spe1 for H1. -Ligation was performed for P2 and H1 ,C1 and GFP.

WEEK 3

Wednesday - Transformation has been made for C2,L2,P2H1 and C1-GFP (unsuccessful) - We prepared 200 ml LB broth for next experiments.

Thursday - Transformation has been made for C2,L2,P2H1 and C1-GFP. -C2 and L2 (successful)

Friday - We prepared 200 ml LB broth and 15 plates for next experiments - Transformation was made for P2H1 and C1-GFP. - We prepared liqued culture for C2 and L2.

Saturday - Single colony isolation has been made for C2 and L2. -C2 and L2 are digested with EcoR1 and Pst1.

WEEK 4

Monday -electrophoresis was performed for C2 and L2.(Unsuccessful) -We prepared Tfb1 and Tfb2 for compatent cell. -We prepared liquid culture for C2 and L2.

Tuesday - Single colony isolation has been made for C2 and L2.
- electrophoresis was performed for C2 and L2. - C2 and L2 are digested with EcoR1 and Pst1.

WEEK 5

Monday - We added compotent cells to ampicilin included liquid culture and plate. -We added compotent cells to cloramphenicoli included plate. -We incubated cloramphenicol resistance bacteria to ampicilin included plate. -We added compatent cells to plate without antibiotic.

Tuesday -We prepared 15 CHL plates. -We tested compatent cell by RFB transformation. -We prepared liquid culture for preparetion of compatent cell.

Wednesday - Transformation was made for C2,L2 and C1-GFP.

Thursday -Ligation was performed for P2 and GFP. - We prepared liqued culture for L2 and C2. - We tested new compatent cell by RFB transformation. - We prepared kanamycin including plate.

Friday - Single colony isolation was made for C2 and L2. - C2 and L2 are digested with EcoR1 and Pst1. - electrophoresis was performed for C2 and L2.(Unsuccessful) - Ligation was performed for P2H1 with kanamycin. - We prepared kanamycin including plate. - We prepared liqued culture for P2GFP and L2. - Transformation was made for P2H1.

WEEK 6

Saturday -Isolation was made for L2 and P2-GFP.After that digestion was made with EcoR1 and Pst1 for these parts.Finally electrophoresis was performed to these parts.The result of P2-GFP was true but L2 was wrong. -We prepared 5 liquid culture for P2H1. -Digestion was made with EcoR1 and Spe1 for P2-GFP

Sunday --Isolation was made 5 coloni for 69h.But microcantifuge is broked. - We prepared liqued culture for J04450(400) - Transformation was made for J04450(500-700) -Our parts were renamed.

WEEK 7

Tuesday - Transformation was made for J04450(500-700) -Digestion was made J04450(500-700) EcoR1 and Pst-1. -We diluted Lac-1. - Transformation was made for Lac-1. - electrophoresis was performed for J04450(500-700) -Ligation was performed for 705-79h. - Transformation was made for 40h,705 and 79h.

Thursday -We made isolation from liquid culture of 40h for testing contamination.Then we made digestion with EcoR1 and Pst1. After that electrophoresis was performed . -Ligation was made for 40h and 70h.After that transformation was made for these parts. -We prepared 3 liquid cultures for each one of LacI,705 and 79h (totally 9 liquid cultures were prepared )

Friday -Isolation was made for LacI,705 and 79h.Then digestion was made with EcoR1 and Pst1 for 705 and 79h , with EcoR1 and Spe1 for LacI.After that electrophoresis was performed for all of these parts.But results werent good.

Sunday

• We prepared liqued cultures for Zt and K.
• Isolation was made 0,H and U.
• Digestion was made 0,H and U.
• Electrophoresis was performed for 0,H and U.
• Transformation was made W,P,Z,Alpha,Zt,K.
• We prepared compatent cell and AMP plates

WEEK 8

Monday

- We prepared liqued cultures for W,P,Z,Alpha,Zt and k. - Transformation was made 40hgx,40h and 70h. - Digestion was made u1,u2 and u3.(unsuccessful) - Electrophoresis was performed for u1,u2 and u3.