Team:Fatih-Medical/Lab/Diary

Diary

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.

Week 9

Saturday

• J04500, K103006 were digestioned and carried out electrophoresis
• Transformations were made for XyIE,pCons and TEV Cleavage Site.
• Digestion was made for TEV N and TEV C from their previous isolated forms.Then electrophoresis was carried out for these parts.

Sunday

• Ligation of TEV N-C and placI was performed and transformed
• Ligation of K103006 and plac3 was performed and transformed
• Isolations of XylE,pCons and TEV Cleavage Site were performed
• Digestion,ligation and transformation of XylE,pCons and placI were performed.
• Presentation which would take place at Dışkapı on thursday was prepared and studied
• We planned to make a video clip of our team

Week 10

Monday

• Laboratory was organized
• Electrophoresis of OmpA-Pro was carried out but probably there was a contamination so the results weren’t what we expected
• Ligations of GFP and OmpA were performed

Tuesday

• Liquid culture of pCons and XylE were performed
• Digestion and electrophoresis of pLac and OmpA were performed
• The list of emergency items were written

Wednesday

• Transformations of J04500, K103006 and E0040 were performed
• Isolation and digestion were performed from liquid culture of pCons+XylE
• The results were correct. Streak was performed from liquid culture of pCons+XylE

Thursday

• Research day

Friday

• Research day

Saturday

• Research day

Sunday

• Research day

Week 11

Monday

• Research day

Tuesday

• Research day

Wednesday

• Research day

Thursday

• Research day

Friday

• Research day

Saturday

• Research day

Sunday

• Research day

Week 12

Monday

• Application was made to TUBITAK 2209-C for sponsorship activities

Tuesday

• Digestion of pLac+OmpA+500 was performed.Then electrophoresis was carried out for these parts.

Wednesday

• Digestions of pLac, OmpA and 500 were performed. Then electrophoresis was carried out for these parts.
• pLac and OmpA results were correct
• Meeting was carried out and procedures would be discussed again

Thursday

• Digestion procedure was changed
• Digestions of pLac and OmpA were performed. Then electrophoresis was carried out for these parts.The results were correct so gel extraction was performed for these parts.
• Although digestion and electrophoresis of 500 was performed again, the result was same; nothing.

Friday

• Research day

Saturday

• Research day

Sunday

• Research day

Week 13

Monday

• Research day

Tuesday

• Research day

Wednesday

• Research day

Thursday

• Liquid cultures of 500 from different dates was prepared.
• Ligation of 59M (pLac+OmpA) was performed

Friday

• Transformations of Ω, Ω2 and 59M were performed
• 4 digestions (two of them are control group) of Ω2 were performed

Saturday

• Research day

Sunday

• Research day