From 2009.igem.org

Meetings Calendar

Formal Lab Session - 21st September 2009

Team Newcastle 2009 iGEM 21-09-09 IMG 1276.JPG

Overview

Stochastic Switch Team -

Metal Sensor Team -

Sporulation Tuning/Chassis Team -

Stochastic Switch Team

Team Newcastle 2009 iGEM 21-09-09 IMG 1281.JPG

Today we digested the sac minipreps with EcoRI and XbaI. It seems clear that there was something wrong with the minipreps (such as poor procedure) as there seemed to be little or no DNA in the sample.

Did a gel fragment clean up on the sspb fragment cut from the gel, this was put in the 'fragments' box in the freezer ready for later.

50ul of ara+pSB1AT3 plasmid was cut with SpeI and PstI in order to insert the sspb fragment after the operator. Rather than doing a gel fragment prep a PCR clean up (kit) was done on the digest to save time.

We ligated the ara + sspb fragments using Chris' method:

2ul Ligase buffer
2.5ul Vector DNA
14.5ul insert DNA
1ul ligase

= 20ul

This was left in the fridge overnight to improve the ligation conditions.

Metal Sensor Team

Introduction

Practical Outline

Team Newcastle 2009 iGEM 21-09-09 IMG 1273.JPG

Here are the list of tasks which need to be accomplished by the end of the day:

Run analysis digests of the 5 midi-preps of cotC, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3 (midi-prepped in the 11/09/09 lab session and digested in the 18/09/09 lab session)

Team Newcastle 2009 iGEM 21-09-09 IMG 1295.JPG

- If successful, double digest 40ul of pMUTIN4 with BamHI and HindIII and place under DNA gel electrophoresis; excise band produced and extract DNA
- If unsuccessful, inoculate 5ml of LB with E. coli containing pMUTIN4 (for mini-preps and midi-preps)
Check PCR reaction (amplification of cotC-GFP-smtA) on gel and clean up if successful
- If successful, digest with BamHI and HindIII
Should the pMUTIN4 DNA extraction be a success and the cotC-GFP-smtA BioBrick be successfully amplified (by PCR) and extracted, ligate the two fragments together.
- Reactions include:
  - vector + insert + ligase
  - vector + ligase
  - vector and NO ligase
Inoculate 3ml of LB with TPA2 cells (for production of competent DH5-alpha E. coli cells)

Procedure

Analysing midi-prep digests

Team Newcastle 2009 iGEM Geldoc 2009-09-21 13hr 11min.jpg

Lane 1 = blank
Lane 2 = DNA HindIII ladder
Lane 3 = cotC midi-prep digest
Lane 4 = kinA midi-prep digest
Lane 5 = pGFP-rrnB midi-prep digest
Lane 6 = pMUTIN4 midi-prep digest
Lane 7 = pSB1AT3 midi-prep digest.
Lane 8 = PCR reaction 1
Lane 9 = PCR reaction 2
Lane 10 = blank
Lane 11 = pMUTIN4 double digest (10ul)
Lane 12 = DNA HindIII ladder

Double digesting pMUTIN4 midi-prep DNA

Double digest of 10ul of pMUTIN4 DNA was conducted in the following manner:

10ul of pMUTIN4 DNA
1ul of FastDigest BamHI
1ul of FastDigest HindIII
2ul of restriction buffer
6ul of sterile distilled water

The digests were allowed to commence for 1 hour under 37C incubation. Samples of this digest were then analysed through DNA gel electrophoresis and can be seen in the GelDoc photograph above.

Cleaning up cotC PCR product

The cotC_GFP_smtA BioBrick was cleaned up using GenElute's PCR clean-up kit and it's protocol was adhered to.

Excising digested pMUTIN4 from gel

Firstly 40ul of pMUTIN4 was digested using BamHI and HindIII in the following manner:

40ul of pMUTIN4 DNA
4ul of FastDigestBamHI
4ul of FastDigestHindIII
8ul of buffer
24ul of sterile distilled water

Incubated for 1 hour under 37C in waterbath. DNA was then all placed into agarose gel and DNA gel electrophoresis was allowed to commence for 20 minutes under 70 volts. Band was excised and cleaned up
Band weighed 0.351 grams so the amount of gel solubilization solution used was 1053ul.

Quantification of cotC and pMUTIN4

cotC = 26.6ng/ul pMUTIN4 = 23.6ng/ul

Sporulation Tuning/Chassis Team

Team Newcastle 2009 iGEM 21-09-09 IMG 1275.JPG

Introduction

We have already got the double clone,the next thing need to do is to ligate this sleB:cwlJ fragment with pMutin4 plasmid to get the pspac sequence from pMutin4. pMutin4 contains HindIII and BamHI restriction sites, but our double clone sequence didn't contain those sites. If we want to ligate double clone fragment with pMutin4 backbone, we need PCR our double clone fragment and add HindIII and BamHI restriction sites.We designed cwlJsleB primers here.
We digest pMutin4 with HindIII and BamHI to get our plasmid backbone.
Midi Prep L1 and L2 transformation strains to get large amount of plasmid.
Our kinA synthesized part was arrived, we need to construct this gene into our integrate plasmid and transform it into B.subtilis.

Experiment procedure

PCR doulbe clone fragment

Prepare buffer mix and Taq

 Reaction buffer  10ul x 4 = 40ul
            dNTP   2ul x 4 =  8ul
 --------------------------------
                             48ul
 12ul for each reaction

 GoTaq(5U/ul)   0.25ul x 2 =  0.5ul
 ddH2O                        4.5ul
 ----------------------------------
                                5ul
 2.5ul for each reaction

Exp. PCR experiment for gene cwlJ:sleB
No.	Water (ul)	PCR Mix (ul)	Forward Primer (ul)	Reverse Primer (ul)	Template (ul)	Polymerase used (ul)	Total Volume (ul)
1	28.5	12	cwlJsleBForward 2.5ul	cwlJsleBReverse 2.5ul	pSB1AT3:cwlJ:sleB 2ul	GoTaq 2.5ul	50
2	30.5	12	cwlJsleBForward 2.5ul	cwlJsleBReverse 2.5ul	0ul	GoTaq 2.5ul	50

PCR procedure: STD50

95C 2min -->[95C 30S --> 50C 30S --> 75C 1min] 30 runs --> 75C 5min --> store at 4C

Prepare for Midi Prep

Add 50ml LB+Amp+Tet liquid medium into a flask, get 200ul cell mixture from Mini prep culture.
Culture L1 and L2 overnight.

Digest pMutin4 plasmid for ligation

Prepare the digest reaction.

 dd H2O                     7ul
 10X fast digest buffer     7ul
 Fast HindIII               3ul
 Fast BamHI                 3ul
 pMutin4 plasmid           50ul
 ------------------------------
                           70ul

Incubate 1 hour at 37 degree.

Digest kinA fragment

Team Newcastle 2009 iGEM 21-09-09 IMG 1282.JPG

Digest pMK-RQ vector to get kinA fragment.(Jess has done this part)
Run big well gel to seperate kinA and pMK-RQ backbone.
Use gel extraction kit to purify kinA fragment.

Conclusion

When we cut pMK-RQ with EcoRI and NheI, both pMK-RQ back bone and kinA fragment are about 2k bp. To get the purified kinA fragment, we need to run the gel for a longer time to seperate the two bands. The other way is to introduce the third enzyme which can cut the backbone into 2 pieces and keep kinA's integrability.