IGEM:Harvard/2006/NES Notebook

Introductory Materials

IGEM:Harvard/2006/Lab_Intro

New Reporters

Due to issues with T7 promoter, the following biobricks were rehydrated 6/14

• J04430

Constitutive (IPTG-inducable?) GFP

• J04450

Constitutive (IPTG?) RFP

• I13522

Constitutive GFP using TetR promoter. No experience; might not work.

• I6060

Constitutive (IPTG?) YFP. Apparently gives uneven YFP expression

Transformed with 2ul of DNA on 15ul bacteria; twice for each plasmid. Took 3 ul from each incubation of and added into two two mixed SOC cultures. Let's see what happens when LacI GFP, RFP, YFP are co-cultured.

One of each LacI plasmid type, GFP, RFP, YFP, LacI were cultured overnight on plates treated with 10mM IPTG.

Microscope Notes

Overlap between FITC and YGFP blocks prevents differentiation between YFP and GFP. RFP can be differentiated between YFP/GFP very well.

YFP/GFP fades ~3 hours after being taken out of the incubator after overnight incubation. RFP was still strong at +3h.

IPTG doesn't make an apparent difference in fluoresence.

Promoter Research

7/25/06 Made Glycerol cultures of

• J04430 (lac GFP)
• J04450 (lac RFP)
• I13522 (tet GFP)

We need to transfer these constructs onto pSB4A3

Construct Building

Digestion

• Digestions - Vary enzyme concentration

1. 8ul Midiprep KaiC DNA + .5 ul X+P enzyme + 2.5ul Buffer #3 + 2.5ul BSA + 11ul H2O -> 45C 6H
2. 8ul Midiprep KaiC DNA + 1 ul X+P enzyme + 2.5ul Buffer #3 + 2.5ul BSA + 10ul H2O-> 45C 6H
3. 8ul Midiprep KaiC DNA + 1 ul X+P enzyme + 2.5ul Buffer #3 + 2.5ul BSA + 10ul H2O-> 37C 6H
4. 8ul Midiprep Backbone DNA + .5 ul S+P enzyme + 2.5ul Buffer #2 + 2.5ul BSA + 11ul H2O-> 45C 6H
5. 8ul Midiprep Backbone DNA + 1 ul S+P enzyme + 2.5ul Buffer #2 + 2.5ul BSA + 10ul H2O-> 45C 6H
6. 8ul Midiprep Backbone DNA + 1 ul S+P enzyme + 2.5ul Buffer #2 + 2.5ul BSA + 10ul H2O-> 37C 6H
7. 8ul Midiprep Backbone DNA + 2.5ul Buffer #2 + 2.5ul BSA + 12ul H2O
8. 25ul H20
9. 8ul Midiprep KaiB DNA + .5 ul X+P enzyme to 25ul + 2.5ul Buffer #3 + 2.5ul BSA + 6ul H2O -> 45C 6H
10. 8ul Midiprep KaiB DNA + 1 ul X+P enzyme to 25ul + 2.5ul Buffer #3 + 2.5ul BSA + 5ul H2O-> 45C 6H
11. 8ul Midiprep KaiB DNA + 1 ul X+P enzyme to 25ul + 2.5ul Buffer #3 + 2.5ul BSA + 5ul H2O-> 37C 6H
12. 20ul H20 + 1.75ul Buffer #2 + 1.75ul Buffer #3 + 2.5ul BSA

Due to the ambiguous backbone lanes above, four digetss were redone with new backbone midiprep.

4II. Same as 4 above with new (8.22) backbone midiprep.

5II. Same as 5 above with new (8.22) backbone midiprep.

6II. Same as 6 above with new (8.22) backbone midiprep.

12. 8ul Midiprep Backbone cut once with S.

• Phosphatase

- 25ul Digest + 1ul Phosphatase 1H at 45

• Run on Gel - Create purified and unpurified samples

Add 12 ul of each to the gel.

Chose the most successful digest and run a gel extraction.

Ligation

• Ligation -
• "PCR Insert": DNA from GeneArt Plasmid cleaned up with PCR cleanup kit.
• "Gel Insert": DNA from GeneArt Plasmid cleaned up with Gel Extraction Kit.
• "XXX Backbone": DNA from BB_J0450 midiprep

1. KaiB PCR Insert + PCR Backbone: I:B + NEB Enz= 3:1
2. KaiB PCR Insert + Gel Backbone: I:B + NEB Enz= 3:1
3. KaiB Gel Insert + Gel Backbone: I:B + NEB Enz= 3:1
4. KaiB PCR Insert + PCR Backbone: I:B + NEB Enz= 6:1
5. KaiB PCR Insert + Gel Backbone: I:B + NEB Enz= 6:1
6. KaiB Gel Insert + Gel Backbone: I:B + NEB Enz= 6:1
7. KaiB PCR Insert + PCR Backbone: I:B + Roche Enz= 3:1
8. KaiB PCR Insert + Gel Backbone: I:B + Roche Enz= 3:1
9. KaiB Gel Insert + Gel Backbone: I:B + Roche Enz= 3:1
10. KaiC PCR Insert + PCR Backbone: I:B = 3:1
11. KaiC PCR Insert + Gel Backbone: I:B = 3:1
12. KaiC Gel Insert + PCR Backbone: I:B = 3:1
13. Uncut J04500

Reagants:

• Kaib PCR/Gel Insert: #10
• KaiC PCR/Gel Insert: #2 PC/GP
• PCR/Gel Backbone II: #5 II (8.22 Backbone) PC/GP

Mixtures: NEB

• 3:1 - 3ul:1ul DNA + 1ul ligase + 2 ul 10x buf + 13ul H2O
• 6:1 - 6ul:1ul DNA + 1ul ligase + 2 ul 10x buf + 10ul H2O

Roche

• 3:1 - 3ul:1ul DNA + 4ul H2O + 2ul 5x dilution buf + 10ul Ligation Buf + 1ul ligase
• 6:1 - 6ul:1ul DNA + 1ul H20 + 2ul 5x dilution buf + 10ul Ligation Buf + 1ul ligase

The Result of transformations: No Dice. Samples #1, #4, #7, #10, and #14 produced colonies, which I assume result from uncut backbone from the backbone PCR cleanup.

After corresponding with Perry: Ligation with: 2ul backbone, 8ul DNA + 10ul Ligation Buf + 1ul Roche ligase Using

• KaiC PCR/Gel Insert #2
• Kaib PCR/Gel Insert: #10
• Gel Backbone II #5 II (8.22 Backbone)
• Gel Backbone II #6 II (Top band)
• Gel Backbone II #6 II (Middle band)

Transform in 14ul cells

1. KaiC PCR + Gel Backbone #5 II at 21C
2. KaiC Gel + Gel Backbone #5 II at 21C
3. KaiC Gel + Gel Backbone #6 II (top) at 21C
4. KaiC Gel + Gel Backbone #6 II (middle) at 21C
5. KaiC Gel + Gel Backbone #5 II at 17C
6. KaiB Gel + Gel Backbone #5 II at 21C
7. 1ul Uncut J04500

A transformation of each of these was done with all 21ul of above ligation products 1-7 on 14ul cells. Additionally, 16ul of ligation 3 from 8.23 was plated on 14ul of cells.

Assay for construct

The following colonies were found

• Two colonies of 8.22 #5 (KaiB)
• Three colonies of 8.22 #8 (KaiB)
• One colony from 8.23 #3 (KiC ligated at 21 degrees)
• A full plate of colonies from 8.23 #5 (KaiC ligated at 17 degrees)

A colony PCR was performed and a LB culture started for the following colonies:

1. KaiB (8.22 #5) (Invitrogen PCR Mix)

2. KaiB (8.22 #5) (Qiagen PCR)

3. KaiB (8.22 #8) (Invitrogen PCR Mix)

4. KaiB (8.22 #8) (Qiagen PCR)

5. KaiB (8.22 #8) (Invitrogen PCR Mix)

6. KaiC (8.23 #3) (Qiagen PCR)

7-20. KaiC (8.23 #5) (Odd Samples Invitrogen PCR Mix; Even Samples Qiagen PCR Mix)

21. Uncut J04500, 40x dilution

22. Uncut J04500, 40x dilution

23. No DNA (Invitrogen PCR Mix)

23. No DNA (Qiagen PCR Mix)

24. H2O

Two different kits were used due to problems in 8.25 cyano PCR.

Second Round of Digests

8/27

• 16ul Midiprep KaiA DNA + 2 ul X+P enzyme + 5ul Buffer #3 + 5ul BSA + 20ul H2O-> 45C 6H -> 1ul Phosphatase for 20 seconds (mistake)
• 16ul Midiprep KaiB DNA + 2 ul X+P enzyme + 5ul Buffer #3 + 5ul BSA + 20ul H2O-> 45C 5H -> 1ul Phosphatase for 20 seconds (mistake)
• 16ul Midiprep KaiC DNA + 2 ul X+P enzyme + 5ul Buffer #3 + 5ul BSA + 20ul H2O-> 45C 5H -> 1ul Phosphatase for 20 seconds (mistake)
• 16ul Midiprep Backbone DNA + 2 ul X+S enzyme + 5ul Buffer #2 + 5ul BSA + 20ul H2O-> 45C 5H -> 1ul Phosphatase for 1H

Five additional transformations of the following with first-round digestion products:

• Ligation with: 2ul 2x backbone (5II), 8ul 2x KaiC Insert (digests 1+2 PC) + 10ul Ligation Buf + 1ul Roche ligase for 10 min at 21 degrees
• Ligation with: 2ul 1x backbone (5II), 8ul 1x KaiC Insert (digest 1 PC)+ 10ul Ligation Buf + 1ul Roche ligase for 10 min at 21 degrees
• Ligation with: 1ul 1x backbone *5II), 8ul 1x KaiB Insert (digest 10 GP) + 10ul Ligation Buf + 1ul Roche ligase for 10 min at 21 degrees
• .5ul uncut J04500
• Nothing

All 2x DNA vacuum centrifuged. All in 20ul of top10F' competant cells.