Team:Exeter/lab book/gibs/wk3

Operon Construction  |  Glycobase

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9am

Carried out 3A BioBrick assembly of pBAD weak-RBS (BBa_K764018) and pBAD strong-RBS (BBa_K764019)

N.B. All volumes and concentrations from protocol halved. Used volumes of water and DNA shown below.

Upstream

Plasmid DNA:

(1) Pbad Weak - 2.37 µl

(2) Pbad Strong - 3.83 µl

MilliQ water:

(1) Pbad Weak =18.88 µl

(2) Pbad Strong =17.42 µl

Downstream

RBS (BBa_B0034) - 6.46 µl

MilliQ water - 14.79 µl

Destination plasmid

Linear plasmid DNA - 20 µl

MilliQ water - 1.25µl

Incubated 36 °C for 10 minutes

Heat inactivated at 80 °C for 20 minutes

Ligation as from protocol, with full volumes and concentrations.

DNA stored at -20°C prior to afternoon transformation

2.45pm

Resuspended DNA of synthesised genes according to IDT resuspension protocol (Alex B. and Becca then transformed it.)

Using 3A assembled DNA (BBa_K764019) competent cells were transformed.

2 µl of DNA from Pbad S-RBS added to 25 µl top10 competent cells

Spread plates of 20 and 100 µl made

**Wednesday 25.7.12**

9.45 am

Biobricking attempt 2

To make Pbad Strong-RBS (BBa_K764019)

From 3A BioBrick assembly protocol

Upstream

Pbad Strong (BBa_K206000) - 3.8 µl

MilliQ water - 38.7 µl

Downstream

RBS BBa_B0034 - 6.5 µl

MilliQ water - 6.7 µl

Destination plasmid pSB1T3 - tetracycline resistance (n.b. this construct was later transferred to a chloramphenicol resistant vector to be sent to the registry)

Linear plasmid DNA - 20 µl

MilliQ water - 22.5 µl

Incubated 36 °C for 10 minutes

Heat inactivated at 80 °C for 20 minutes

Ligation as in protocol

Stored at -20°C prior to afternoon transformation

Standard assembly run in parallel to 3A

Upstream:

pBAD Strong plasmid DNA: 15.3 µl

EcoRI-HF: 1 µl

SpeI: 1 µl

10X NEBuffer 2: 5 µl

100X BSA: 0.5 µl

MilliQ water: 17.2 µl

Downstream:

RBS (BBa_B0034) plasmid DNA (500ng): 25.8 µl

EcoRI-HF: 1 µl

XbaI: 1 µl

10X NEBuffer 2: 5 µl

100X BSA: 0.5 µl

MilliQ water: 6.7 µl

Digested for 1 hour at 36 °C

DNA cleanup from enzymatic reactions using microcentrifuge and Quiagen column.

(In the meantime, Freddie ran the upstream fragments on an 8% low-melting agarose gel, but lack of bands meant standard assembly could not be completed. We later discovered the sequences we had were wrong, and in reality the sequences were too short to appear on a gel.)

For RBS plasmid (post-digestion)

1.40 µl digest + 120 µl QG buffer + 40 µl isopropanol mixed and transferred to column (n.b. RBS 2 may not have been adequately mixed before being transferred to column)

2.Centrifuged for 1 minute, 13000 rpm, room temp.

3.Flow through discarded

4.500 µl buffer QG added to column

5.Centrifuged for 1 minute, 13000 rpm, room temp.

6.Flow through discarded

7.750 µl buffer PE added to column

8.Centrifuged for 1 minute, 13000 rpm, room temp.

9.Flow through discarded

10.Centrifuged for 1 minute, 13000 rpm, room temp.

11.Moved column to fresh 1.5 ml eppendorf

12.30 µl water added

13.Left to stand for 1 minute

14.Centrifuged 13000 rpm, 1 minute, room temp.

15.DNA stored at -20 °C after analysis with a nanodrop spectrophotometer

3A assembled pBAD strong-RBS construct was transformed

2 µl DNA added to competent cells

Spread plates of 20 µl and 100 µl made on LB(tetracycline) agar

**Thursday 26.7.12**

Suspended VF2 and VR sequencing primers (according to manufacturer’s instructed volumes) and prepared 50 µl aliquots for later

Resuspended BBa_I0500 (large pBAD promoter - selected as alternative to pBAD strong due to sequence match with Gibson primers)

2 µl of BioBrick DNA transformed according to protocol

Spread plates of 20 µl and 100 µl made on LB(kanamycin) plates

Plates incubated at room temperature over weekend

Transferred colonies from plates prepared 25.7.12 into LB(tetracycline) broth (1:1000)

**Friday 27.7.12**

9 am

Supernatant discarded

Miniprep according toMiniprep of 3A assembled plasmid (from 25.7.12)

Step 10 50µl milli-Q water added in place of elution buffer

Centrifuged 1 minute, 13000 rpm, room temperature

5 µl water added

Centrifuged 1 minute, 13000 rpm, room temperature

Column discarded, DNA concentration measured with nanodrop and DNA stored in eppendorf at -20 °C

(n.b. digest gel was run for analysis, but is considered irrelevant due to inconclusive results from small fragment size and because BBa-I0500 was decided to be more useful for this part of the project)

Streak plates made from dregs of overnight culture (see above)