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 5H -> 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 -> 6ul Phosphatase for 1H

to produce

• KaiA/B/C 1: PCR Cleanup, eluted in 30ul of 80C 1x EB. -->Worked Well
• KaiA/B/C 2: PCR Cleanup, eluted in 90ul of 80C 1/3x EB and vacuum centrifuged to 1x. -->Worked Poorly

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.

Ligation

1. 8ul KaiA + 2ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase
2. 8ul KaiB + 2ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase
3. 8ul KaiC + 2ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase
4. 5ul KaiA + 5ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase
5. 5ul KaiB + 5ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase
6. 5ul KaiC + 5ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase
7. 2ul KaiA + 8ul J04500 + 10ul Ligation Buf + 1ul Roche ligase
8. 2ul KaiB + 8ul J04500 + 10ul Ligation Buf + 1ul Roche ligase
9. 2ul KaiC + 8ul J04500 + 10ul Ligation Buf + 1ul Roche ligase
10. 8ul GP KaiA + 2ul J04500 + 10ul Ligation Buf + 1ul Roche ligase
11. 8ul GP KaiB + 2ul J04500 + 10ul Ligation Buf + 1ul Roche ligase
12. 8ul GP KaiC + 2ul J04500 + 10ul Ligation Buf + 1ul Roche ligase

From each ligation, two plates were made:

• 2ul ligation mix + 14ul Top10F' cells
• 16ul ligation mix + 14ul Top10F cells.

Samples 10, 11, and 12 were plated on Carb plates, as gel purification removes the need to select against uncut GeneArt plasmids

Results

• 12 hours later, no Kan plates had colonies except for the positive control.
• 12 hours later, all carb plates except for the 2ul #12 plate had colonies. The Carb positive control had an prder of magnitude more cells than the experimental plates. Experimental plates had between 500 and 1000 colonies. Contamination from uncut GeneArt plasmid is feared. However, there is reason to believe that the Carb plates would do better as the Top10f' kit describes Kanomycin selection as "decreasing" transformation efficiency.

The colonies seen on the Carb plates were digested, re-ligated GeneArt plasmid. The gel purification did not eliminate all cut backbone, and the Carb selection did not remove the religated form.

Third Round of ligations

8.31 Ligated and transformed

1. 6ul GP KaiA + 2ul 8.22 GP backbone + 2ul DNA dilution Buffer + ligation mix (2ul h20, 10ul ligation Buf, 1ul Roche ligase) -> 2ul on cells
2. 6ul GP KaiB + 2ul 8.22 GP backbone + 2ul DNA dilution Buffer + ligation mix -> 2ul on cells
3. 6ul GP KaiC + 2ul 8.22 GP backbone + 2ul DNA dilution Buffer + ligation mix -> 2ul on cells
4. 6ul of 8.22 ligation #8 on cells
5. 6ul of 8.22 ligation #8 on cells
6. 2ul of 8.30 ligation #10 on cells
7. 2ul of 8.30 ligation #11 on cells
8. 2ul of 8.30 ligations #12 on cells

Replicating Peng's results

1. 6ul PCR KaiA + 2ul Peng's 8.30 backbone digest + ligation mix -> 2ul on cells
2. 4ul PCR KaiB (all I had left) + 2ul Peng's 8.30 backbone digest + 4ul h20 + ligation mix -> 2ul on cells
3. 6ul PCR KaiC + 2ul Peng's 8.30 backbone digest + ligation mix -> 2ul on cells

Al ligations were run for 45 min at 25C and then overnight at 15C. Transformed cells were plated on Kan plates

Searching for Kan resistant bacteria on colonies from amp plate

Many colonies from 8.30 AMP plates #10 #11 and #12 were restreaked on Kan plates, selecting against re-ligated GeneArt plasmids in hopes of identifying an insert-holding plasmid.

Result: two different streaks produced colonies for both #10 (KaiA) and #11 (KaiB). A colony PCR was started for 4 colonies of each streak. Noteto self: KaiA and KaiB labels switched on PCR and in LB cultures. Label A2.4 may have mixed lb culture.

Duplication of glycerol stocks

• GeneArt KaiA
• GeneArt KaiB
• GeneArt KaiC
• BioBrick BB_J04500