Team:Cambridge/LabBook/Week4

Monday

7. Experiment: Transformation of TOP10 cc (Ben, Emily, Bill, Hannah, Will & Anja)

Top10 cc taken out of -80°C freezer and tawed on ice. 1ml pipette tip cut with scissors sterilised with ethanol and flamed. Using cut pipette tip ~50μl of TOP10cc were transferred to 1.5ml Eppendorf tubes (3x)

1. 2μl of resuspended (in 10μl deionised water) BBa_J13002 (plasmiid with Tet^R represed PoPs/RIPs generator) was added.
2. 2μl of resuspended (in 10μl deionised water) BBa_I712019 (plasmid with firefly luciferase) was added
3. 1.5μl of pHK724 (plasmid containing lux4 gene) was addded

Ce]]s were held on ice for 30min. Heat shocked for 60s at 42°C (waterbath). As a control TOP10cc that had not been transformed with anything (no plasmid DNA added) were subjected to same treatment (as cells to be transformed). Put on ice for ~2min. Added 250μl pre-wardmed (in 37°C) SOC. Incubated at 37°C for Ph with rotation (Eppendorf tubes---> 12ml falcon tubes, tape over top). Plated 50micorl on pre-warmed (in 37°C incubator) LB agar plates with Amp (with blue L-shaped spreader). Grow colonies overnight at 37°2.

8.Experiment: Set up overnight culture of E.coli/pHK555 (Will & Anja)

Inoculated single bacterial colony (E.coli/pHK555) in 5ml LB in a 12ml falcon tube. Incubated at 37°C with rotation overnight. (Since it was difficult to see the colony sent from Jim Slock, we picked twice from where we anticipated colony to be and set up 2x5ml LB overnight cultures)

9.Experiment: Set up overnight cultures of W3110 hns 93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10 Tet^R, GM230 hns-205::Tn10 Tet^R (Ben & Anja)

Inoculated single bacterial colonies in 5ml SOB (i.e. 4 different cultures), incubated at RT with shaking overnight.

Tuesday

10. Experiment: Preparing chemically competent cells (W3110 hns 93-1, BW25113 Δhns::kan,W3110 hns-205::Tn10 Tet^R, GM230 hns-205::Tn10 Tet^R (Ben, Will, Paul, Bill, Emily, Hannah & Anja)

(followed protocol for 'TOP10 chemically competent cells cells' from OpenWetWare) Inoculated 0.5ml of the 4 bacterial strains (overnight cultures) in 85ml SOB each. Incubated at 37°C with shaking (180rpm). put on at 11:55am

Bacterial Strains:

ID	Type
(1)	GM230 hns-205::Tn10 TetR
(2)	BW25113 Δhns::kan
(3)	W3110 hns 93-1
(4)	W3110 hns-205::Tn10 TetR

OD₆₀₀ measurements:

Time	OD600 (1)	OD600 (2)	OD600 (3)	OD600 (4)
1:00pm	0.019	-0.008	-0.020	0.049
2:00pm	0.034	0.027	0.001	0.124
3:00pm	0.059	0.114	0.049	0.268
3:25pm	0.092	0.235	0.116	-
3:40pm	-	0.297	-	-
4:00pm	0.159	-	0.267	-
4:25pm	0.203	-	-	-
4:40pm	0.256	-	-	-

Cooled cells and (newly prepared) CCMB80 Buffer in ice bath for ~20 minutes. (4) was on ice for over an hour. (2) was on ice for ~40 minutes. (3) was on ice for ~40 minutes.

2 x 30-35ml of each of the four 85ml cultures were poured into 50ml falcon tubes. Centrifuged at 3000g at 4°C for 10 min. Discarded supernatant. Resuspended cell pellet in 20ml tube CCMB80 buffer (ice cold). On ice for 20 min. Centrifuged at 3000g at 4°C for 10 min. Discarded supernatant. Resuspended cell pellet in 5ml tube CCMB80 buffer. Incubated on ice for 20 min. Aliquoted 200μl portions into Eppendorf tubes colour-coded according to the above listed colour labels. Stored at -80°C

11. Experiment: Streaking out of bacterial cultures (E. coli/pHK555) (Paul & Anja)

On LB agar plates with Chloranphenicol: E. coli/pHK555 (from both overnight cultures) on two individual plates incubated at 37°C overnight.

12. Experiment: Set up overnight cultures (TOP10 with BBa-J13002, TOP10 with BBa_I712019) (Ben & Anja)

Results from transformations on 02/08/10

• Colonies grew for TOP10 cells transformed with BBa_J13002 (promoter + rbs) and those transformed with BBa_I712019 (firefly luciferase) :-)
• no cells grew for untransformed TOP10 cells plated on LB agar + Amp plates (neg control)
• small (but plenty) colonies grew for TOP10 cells transformed with pHK724, these were left in the incubator for another 24h, and then put at 4°C

Inoculated single bacterial (TOP10 with BBa-J13002 and TOP10 with BBa_I712019 individually) colony in 5ml LB, incubated at 37°C with shaking (180rpm) overnight

Wednesday

13. Experiment: Measuring competency (W3110 hns 93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10 Tet^R, GM230 hns-205::Tn10 Tet^R) (Theo & Hannah)

Followed protocol under 'TOP10 chemically competent cells' from OpenWetware

Competent cells from all 4 different strains taken out of -80°C freezer and thawed on ice. 1ml pipette tips cut with scissors dipped in ethanol and flamed. For each strain 50μl of cells were transferred to a separate 1.5ml Eppendorf tube. 1μl pUC19 (standard plasmid) was added to each 50μl of cells. Held on ice for 30 min. Heat shocked at 42°C for 60s (water bath). On ice for ~2min. 250μl SOC was added to each of 4 tubes. Each Eppendorf tube was put in a 12ml falcon with tape over the top and incubated at 37°C for 1h with rotation. 20μl of each of the 4 different transformed strains were plated on LB agar plages with Amp. Colonies were grown overnight at 37°C.

14. Experiment: Making competent and transforming E. coli/pHK555 (Peter & Paul)

50μl EZ Comp buffer transferred to Eppendorf tube and placed on ice. Single E. coli/pHK555 colony resuspended in EZ Comp buffer(2x single colony in 50μl EZ each, 1 streak of colonies in another 50μl EZ). Vortex if needed to suspend clumps. 3μl of pHK724 plasmid added to solution, mixed and incubated on ice for 20 min.

Heaat shocked at 42°C for 90s (water bath). Incubated at RT for 5 min. 1ml LB added. Eppendorf tubes put in 12ml falcon tubes with tape over top. Incubated at 37°C for 1h with rotation. Plated 100μl on LB agar plates with Cm and Amp. Incubated at 30°C for 48h.

15. Experiment BioBrick Standard Assembly (Emily & Bill, Paul & Anja)

Took overnight cultures of

1. TOP10 with BBa_J13002 (plasmid with promoter + rbs)
2. TOP10 with BBa_I712019 (plasmid with firefly luciferase)

Plasmid DNA Purification

following the "QIAprrep Spin Miniprep Kit using a Microcentrifuge" protocol.

Restriction enzyme digest

Prepared at RT in the listed order:

	promoter + rbs	luciferase
nuclease-free H2O	15μl	14μl
10x Fast Digest Buffer	2μl	2μl
Plasmid DNA	2μl	2μl
FD enxyme SpeI	1μl	1μl
FD enzyme XbaI	-	1μl

It was checked (Nanodrop!) that 2μl of plasmid DNA would not contain more than 1μg of DNA. The reaction mixtures were mixed gently (flick tube) and spun in the microcentrifuge for 15s (13,000 rpm). It was then incubated at 37°C (water bath) for 30 min.

Gel Electrophoresis

An E-gel EX Agarose 1% was mounted on the transilluminator. Nanodrop readings were taken for both restriction enzyme digests:

• Plasmid with promoter + rbs: 21.4 ng/μl => 5μl gives 100ng
• Plasmid with luciferase: 19.7ng/μl => 5μl gives 100ng

For each digest the following mixture was made up

• 3μl 6X orange loading Dye
• 5μl plasmid DNA (restriction digest)
• 12μl deionised H₂O

Gel was loaded according to the scheme below:

Easyladder II	promoter + rbs	promoter + rbs	luciferase	luciferase
10μl	20μl	20μl	20μl	20μl

Empty wells were filled with 20μl deionised H₂O. Gel was run and DNA gragments of appropriate sizes were observed:

• plasmid with promoter andd rbs => 2153b
• plasmid without luciferase => 3426b
• firefly luciferase => 1653b

The plasmid with promoter + rbs as well as te firefly luciferase bands were cut out of the gel (gel cut pipette tips) and DNA was extracted separately from the two gel pieces by following the QIAquick Gel Extraction Kit protocol.

Ligation

Fermentas 5X Rapid ligation buffer was taken out of -20°C fridge, thawed and mixed thoroughly prior to use. Nanodrop measurements of the linearised vector DNA as well as the insert DNA were taken:

• plasmid with promoter + rbs: 56.8ng/μl
• firefly luciferase: 29.6ng/μl

10-100ng of linearised vector DNA should be added to the ligation mix=>1μl. 3:1 molar exess of insert DNA should be added to the ligation mix.

(56.8ng.2153b) * 1653b * 3 = 131ng => 4.5μl

Added to a mirocentrifuge tube:

Plasmid with promoter and rbs	1μl
Firefly luciferase	4.5μl
5X Rapid ligation buffer	4μl
T4 DNA ligase	1μl
nuclease-free H2O	9.5μl
	20μl

The mix was vortexed and spun for 15s in the microcentrifuge (13,000 rpm). It was incubated at 22°C (RT) for 30 min and stored at 4°C.

16. Experiment: Set up new overnight cultures (TOP10 with BBa_J13002, TOP10 with Ba_I712019) (Peter & Anja)

Thursday

17. Experiment: Measuring competency (W3110 hns 93-1, BW25113 Δhns: kan, W3110 hns-205::Tn¹⁰ Tet^R, GB 230 hns-205::Tn¹⁰ Tet^R) (Theo & Peter)

After 24h the pUC19 transfmration from 04/08/10 had yielded:

W3110 hns 93-1	0 colonies/plate
BW25113 Δhns::kan	13 colonies/plate
W3110 hns-205::Tn10 TetR	1 colony/plate
GM230 hns-205::Tn10 TetR	10 colonies/plate (many v small ones-> prob result of degraded Amp)

pUC19 transformations were repeated following the protocl outlined on 03/08/10, with the exeption that not 20μl but 100μl of the transfomrd strains were plated on LB agar plates with Amp. As a control 100μl of the transformed strains were plated on LB agar plates without anti-biotics.

18. Experiment: Standard BioBrick Assembly (Emily, Bill & Anja)

Took oversight cultures of:

1. TOP10 with BBa_J13002 (plasmid with promoter + rbs)
2. TOP10 with BBa_I712019 (plasmid with firefly luciferase)

Plasmid DNA purification

following the "QIAprep Spin Miniprep Kit using a Microcentrifuge" protocol

Restriction enzyme digest

Prepared at RT in the listed order:

	promoter + rbs	luciferase
nuclease-free H2O	15μl	14μl
10x Fast Digest Buffer	2μl	2μl
Plasmid DNA	2μl	2μl
FD enxyme SpeI	1μl	-
FD enzyme PstI	1μl	1μl
FD enzyme XbaI	-	1μl
	20μl	20μl

It was checked (Nanodrop!) that 2μl of plasmid DNA would not contain more than 1μg of DNA. The reaction was mixed gentlyy, spun down and incubated at 37°C for 30 min

Gel electrophoresis

E-gel EX Agarose 1% was mounted on the transilluminator. Nanodrop readings were taken for both reaction enzyme digests

• plasmid with promoter + rbs 27.7ng/μl => 4μl give 100ng
• plasmid with luciferase 29.1ng/μl => 4μl give 100ng

Prepared following mixture for both digests

• 3μl 6X orange loading Dye
• 5μl plasmid DNA (restriction digest)
• 12μl deionised H₂O

Gel was loaded according to the scheme below:

Easyladder II	promoter + rbs	promoter + rbs	luciferase	luciferase
10μl	20μl	20μl	20μl	20μl

Empty wells were filled with 20μl deionised H₂O. Gel was run and DNA bands of length corresponding to plasmid with promoter + rbs (2153b) as well as firefly luciferase (16553b) were cut out the gel. DNA was extracted separately from the two gel pieces by following the QIAquick Gel Extraction Kit Protocol

Ligation

Fermentas 5X Rapid ligation buffer was taken out the -20°C freezer, thawed on ice and mixed thoroughly.

Nanodrop measurements

Plasmid with promoter + rbs	2.0ng/μl
Firefly luciferase	3.1ng/μl

10-100ng of linearised vector DNA should be used in ligation mix => 10μl (20ng). 3:1 molar excess of insert DNA should be added to ligation mix.

(20ng/2153b) * 1653b * 3 = 46ng => 15μl (46.5ng)

Added to a microcentrifuge tube:

Plasmid with promoter + rbs	10μl
Firefly luciferase	15μl
5X Rapid ligation buffer	8μl
T4 DNA ligase	2μl
Nuclease-free H2O	5μl
	40μl

Vortexed, spun briefly and incubated at 22°C (RT) fr 30 min

Transformation

TOP10 cc taken out of -80°C freezer and thawed on ice, 1ml pipette tip cut with scissors sterilised with ethanol and flamed. 50μl of TOP10cc were transferred to 1.5ml Eppendorf tube. 2μl of ligation reaction were added. Cells were held on ice for 30 min. Heat shocked for 60s at 42°C (water bath). Put on ice for ~2min. Added 250μl SOC. Incubated at 37°C for 1h with rotation. Plated 100μl on LB agar plates with Amp. Incubated overnight at 37°C.

19. Experiment: Set up overnight cultures (E. coli/pHK555 transformed with pHK724) (Theo & Anja)

2 cyan-glowing colonies of E. coli/pHK555 transformed with pHK724 had grown.

One of these was inoculated in 5ml LB and incubated at 30°C overnight.

20. Experiment: Isolation of pHK555 and pHK724 and transformation of TOP10, BW25113 Δhns::kan and GM230 hns-205::Tn10 Tet^R (Hannah & Will)

Took an E. coli/pHK555 and a TOP10/pHK724 colony and separately purified plasmids following the "QIAprep Spin Miniprep Kit using a Microcentrifuge" protocol. Nanodrop measurements. TOP10, BW25113 Δhns::kan and GM230 hns-205::Tn10 Tet^R cells were transformed with both plasmids together and separately. Control cells were left untransformed. Transformation was performed according to the protocol in experiment 18 (instead of ligation reaction, pHK555 (6μl) and pHK724 (7μl) were added to the commp cells. TOP10 cc transformed with both plasmids got 5μl of pHK724 only). Transformed cells were plated on different antibiotic-containing plates as listed in the table below:

	TOP10	BW25113 Δhns::kan	GM230 hns-205::Tn10 TetR
pHK555 &	Cm+Amp	Cm+Amp	Cm+Amp
pHK725	Cm || Amp	Cm || Amp	Cm || Amp
	No antibiotic	No antibiotic	No antibiotic
pHK555	Cm	Cm	Cm
	No antibiotic	No antibiotic	No antibiotic
pHK724	Amp	Amp	Amp
	No antibiotic	No antibiotic	No antibiotic
none	No antibiotic	No antibiotic	No antibiotic

21. Experiment: Set up overnight cultures (E. coli/pHK555 and TOP10/pHK724) (Hannah & Will)

Friday

22. Results: Measuring competency (W3110 hns 93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10 Tet^R, GM230 hns-205::Tn10 Tet^R

After 24h the pUC19 transformation from Thursday had yielded:

W3110 hns 93-1	1 colony/plate
BW25113 Δhns;:kan	~60 colonies/plate
W33110 hns-205::Tn10 TetR	6 colonies/plate (many tiny ones)
GM230 hns-205::Tn10 TetR	~30 colonies/plate (many tiny ones)

Control: All cells grew fine on LB agar plates without antiiotic.

23. Results: Transformation of ligated promoter + rbs and luciferase

After 24h no colonies weree observed on the LB agar + Amp plate.

24. Results: Overnight culture of glowing E. coli/pHK555 transformed with pHK724

The culture glowed (cyan) in the dark.

25. Results: Transformation of TOP10, BW25113 Δhns::kan and GM230 hns-205::Tn10 Tet^R with pHK555 and pHK724

		TOP10	bw25113 Δhns::kaan	GM230 hns-205
	Cm & Amp	No colonies	No colonies	No colonies
pHK555 &	Cm	Few (5)	Few, normal size	Few
pHK724	Amp	Many, small	Many, small	No colonies
	No antibiotic	Smear	Smear	Smear
pHK724	Amp	Many, small	Many, small	Many diff sizes
	No antibiotic	Smear	Smear	Smear
pHK555	Cm	Large colonies	Many, large	None
	No antibiotic	Smear	Smear	Smear
none	No antibiotic	Smear	Smear	Smear

No bacteria from any of the three strains took up both plasmids simultaneously, although when both the plasmids were added to a strain simultaneously there was growth on both of the individual resistance plates – i.e. some bacteria had taken up one and some the other but the probability that any would take up both was so low that no colonies were observed on the ChlAmp plate. No bacteria strains were killed by the heat shocking as smears of bacteria were seen on every plate without antibiotics added that also had a transformed strain plated. The takeup of plasmid 7 was successful in Invitrogen Top 10 strain and in the Red Strain.

In the Black strain, the results from the Black strain are less clear – when we added both plasmids there was no colony growth on the Ampicillin plate (plasmid 7 indicates ampicillin resistance) therefore no plasmid 7 was taken up, however we observed growth on the plate which had been transformed by only plasmid 7 then added to an Amp plate. The colony size was extremely variable though, and although there was no colour differences visible we hypothesise that it may have been contaminated during the procedure.

26. Experiment: Streaking out of E. coli/pHK555 trasnformed with pHK724 (Paul, Emily & Anja)

Streaked out on a LB agar plate with Cm and Amp

Used to write out 2x 'Cambridge', 'Sterilin' & 'iGEM 2010' on LB agar plates with Amp.

27. Experiment: of plasmids pHK724 & pHK555 from TOP10 & SLOCK 10 cells respectively (Will & Emily)

Followed "QIAprep Spin Miniprep Kit" protocl from QIAGEN 4 ml of overnight cultures from each (prepared by Will & Hanah) of TOP10/pHK724 and E. coli/pHK555 were used to extract plasmids.

Results

50μl of sultion was obtained, 1μl of which was used to NanoDrop

plasmid	Nucleic acid concn
pHK555	34.1ng/μl
pHK724	15.7ng/μl

plasmids were stored at 4°C

28. Experiment: Making long-term stocks of pHK555 (in Slock10) & pHK724 (in TOP10) (Peter & Emily)

Preparation of 40% glycerol solution

100% glycerol	40ml
Deionized sterilised water	60ml

For pHK555 & pHK724, separately:

• Added 1ml of 40% glycerol solution to cryogenic vial
• Added 1ml of sample culture to vial
• Gently vortexed vial to mix

Stored in -80°C freezer in 'Black-GM230 hns-205' storage box. Vials are labelled with stickers: '7' for pHK724, '5' for pHK555.

29. Experiment: Transformation of TOP10, Red strain and Black strain with plasmid pHK724. They have each already been transformed with plasmid pHK555 (Hannah & Theo)

Aim

We hope to see each colony glowing, with different brightnesses

Protocol

• Followed Jim Stock's protocol using his EX comp buffer
• Left growing in 30°C incubator over a few days

Results

Strain	Plasmid Added	Glowing	Colonies
TOP10 with pHK555	pHK724	Yes	Yes
Red strain with pHK555	pHK724	No	Yes
Black strain with pHK555	pHK724	No	Yes

Conclusion

Only TOP10 successful however colonies seen on both of red and black strain plates.

Further Work

Plate out red and black strain colonies - possible that they had been left to incubate for too long and glowed then stopped glowing.

Sunday

30. Experiment: Various

• Wrote out in Slock strain, placed in 30°C incubation overnight
  • iGEM 2010
  • Sterilin
  • Cambridge
  • We got Glowth

• Made up the ~3ml of Slock growth strain to 200ml, added antibiotics (A+C)

• Inoculated 5ml of LB with
  • W3110 hns-205::Tet^R (+T)
  • GM230 hns-205::Tn10 Tet^R (+T)
  • pHK555 trans 724 (glowth in Slock) (A+C)
  • Top10 trans promoter + rbs (A)
  • Top10 trans luciferase (A)
  • Top10 ligation (A)