Sbb14-Kelvin Li

Organism

• Thermotoga maritima MSB8
  • ATCC number: 43589D-2
  • PubMed accession number (Nucleotide database): AE000512
  • genome (Genbank format): File:Thermotoga maritima MSB8.gb

Translation table

Bacterial, Archaeal and Plant Plastid Code

  AAs  = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG
Starts = ---M---------------M------------MMMM---------------M------------
Base1  = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG
Base2  = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG
Base3  = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG

valyl-tRNA synthetase

• Protein Sequence:

MAELSTRYNPAEIETKWYRYWEEKGYFTPKGVGEKFSIVIPPPNITGRIHMGHALNITLQDIVVRYKRMK
GYDVLWVPGEDHAGIATQNAVEKFLLQTQGKTREEIGREKFLEITWEWANKYRREIREQIKALGASVDWT
RERFTLDEGLSRAVRKVFVELYRKGLIYRGKYIVNWCPRCKTVLSDEEVEHKEHKSKLYYVKYPVKDSDE
YIVVATTRPETMLGDTAVAVHPEDERYKNFVGKTLILPLVGREIPVVADKYVDPKFGTGAVKVTPAHDPN
DYLIAQRHNLPMIEIFDDNARINENGGKYKGLDRYEAREKIVKDLEEQGFLVKIEDYTHSVGHCYRCDTV
IEPKLSDQWFVSTKPLAKRAIEAVENGEIRFFPERWTKVYLNWMYEIRDWCISRQLWWGHRIPVWYCQDC
GHLNVSEEDVEKCEKCGSTNLKQDEDVLDTWFSSALWPFSTLGWPEETEDLKRYYPTDLLVTGFDIIFFW
VARMIMMGYEFMNDKPFSHVYIHQLVRDKYGRKMSKSLGNGIDPLEVIDEYGADPMRFTLAILAAQGRDI
KLDPRYFDAYKKFANKIWNATRFVLMNLEDYKEVPLENLKTVDKWILTRLNKTVEEVTNALENYDFNIAA
RTIYNFFWDDFCDWYIEASKPRLKTEERNLVQTVLVKVLDASLRLLHPFMPFLTEELWQKLPVAGESITI
AKWPEIERELIDETAEKEFTRLMNMVRGVRNVRAEMNLPQSQRVKVYIKGYEVTEEEELLLKTLGNIEEV
SFVNEKPPKTATAYVEEEIEAYVDLGGLIDFEKEKERLKQIMEKIQKEIDRLEKKLANKDFVEKAPEEVV
EETKEKLNTNRERLARLESILRDLE

threonyl-tRNA synthetase

• Protein Sequence:

MKIKVKLPDGKEKEYDRGITPAEIAKELGIKKAIGAVVNGELWDLKRPIENDCELRLVTLEDPEAPEFYR
HTMAHILAQAVMRLYGKENVKLGIGPTIENGFYYDFDIKNGRLTEEDLPKIEQEMKKIIKENLPIERKEI
SKEEARELFRDQPYKLELIEEIEGNRVTIYRQGEFVDLCRGPHLPSTGIVKHFKLLSVSGAYWRGSEKNP
MLTRVYGTAFAKKEDLDNYLKFLEEAQRRDHRKLGPQLELFMLNTEYAPGMPFFLPKGVVVLNELMKFSR
ELHRERGYQEIFTPLIMNEQLWKISGHWDHYAENMYFIEKDEERYAVKPMNCPGHILVYKSRTVSYRDLP
LRFFEFGRVHRYERSGVLHGLMRVRSFTQDDAHIFCTPDQIEEEILGVLDLINTIYSQFGFTYRVELSTM
PEDHMGDEAIWEKATTALKNALERAGLSYKVNEGEGAFYGPKIDFHIRDSIGREWQCATIQLDFMMPEKF
NVTYIGPDNKEHRAVMIHRAIYGSLERFFGILIEHFAGAFPTWLAPIQVAVIPISEKHNDGAEKVARRIS
QEGFRVFFDNRRETLGYRIRQAQTQKIPYMIIIGDKELESGKISVRTRTGKEIKDVDPEHFVETLRNEVL
SRKLELSMEG

Construction files

Plasmid: pBAD OLED (a compatible derivative of pBAD/myc-His A)

Two references for pBAD/myc-His_A. [1] [2]

Construction of threonyl-tRNA synthetase part

SOE'ing of one NcoI internal restriction site.

PCR externF/internR on genome      (241 bp, A)
PCR internF/externR on genome      (1731 bp, B)
PCR externF/externR on A + B       (1944 bp, pcrproduct)
Digest pcrproduct                  (NcoI/EcoRI, L, pcrdigest)
Digest pBAD                        (NcoI/EcoRI, 4053+41, L, plasmiddigest)
Ligate pcrdigest and plasmiddigest (5981 bp, finalplasmid)
---------------------------------------------
>externF  ThrRS_NcoI_Fwd
ccagtccatggctaagataaaggtgaagcttccag
>externR  ThrRS_EcoRI_Rv
ccagtgaattcttatccctccattgaaagttcg
>internF  ThrRS_SOE-NcoI_Fwd
ctacagacacacGatggcacacatcctc
>internR  ThrRS_SOE-NcoI_Rv
gaggatgtgtgccatCgtgtgtctgtag
>pBAD     pBAD myc-His A
>genome   thermotoga maritima MSB8 genome

Construction of valyl-tRNA synthetase part

SOE'ing of one EcoRI internal restriction site.

PCR externF/internR on genome      (2176 bp, A)
PCR internF/externR on genome      (468 bp, B)
PCR externF/externR on A + B       (2161 bp, pcrproduct)
Digest pcrproduct                  (NcoI/EcoRI, L, pcrdigest)
Digest pBAD                        (NcoI/EcoRI, 4053+41, L, plasmiddigest)
Ligate pcrdigest and plasmiddigest (6653 bp, finalplasmid)
---------------------------------------------
>externF    ValRS_NcoI_Fwd
ccagtccatggcagaactctcgacgagatac
>externR    ValRS_EcoRI_Rv
ccagtgaattctcattctagatctctgaggatg
>internF    ValRS_SOE-EcoRI_Fwd
cgcggagaaggaGttcaccaggctcatg
>internR    ValRS_SOE-EcoRI_Rv
catgagcctggtgaaCtccttctccgcg
>pBAD       pBAD myc-His A
>genome     thermotoga maritima MSB8 genome

2014-02-27

• Miniprepped parent plasmid from E. coli using protocol at Template:SBB-Protocols_Micro3.
  • Tube label: KL 4, KL pBad OLED grp 4
• Stored in freezer.

2014-03-04

• Received dry oligos.
• Spun tubes for approximately 30 seconds.
• Made stock solutions. Added water according to the table below, such that the concentration of each stock soution is 100μM.

Label	Quantity (nanomoles)	ddH2O added
ThrRS_NcoI_Fwd	28.5nmol	285μL
ThrRS_EcoRI_Rv	30.9nmol	309μL
ThrRS_SOE-NcoI_Fwd	33.9nmol	339μL
ThrRS_SOE-NcoI_Rv	38.5nmol	385μL
ValRS_NcoI_Fwd	35.8nmol	358μL
ValRS_EcoRI_Rv	32.7nmol	327μL
ValRS_SOE-EcoRI_Fwd	29.0nmol	290μL
ValRS_SOE-EcoRI_Rv	33.5nmol	335μL

• Vortexed and spun tubes.
• For each stock solution, created dilutions to 10μM consisting of:
  • 1μL oligo stock
  • 10μL ddH₂O
• Setup expand PCRs according to protocol in "PCR in Practice -> Basic PCR for cloning". Labeled tubes:

PCR tube label	Oligo 1	Oligo 2
T A, 4KL	ThrRS_NcoI_Fwd	ThrRS_SOE-NcoI_Rv
T B, 4KL	ThrRS_SOE-NcoI_Fwd	ThrRS_EcoRI_Rv
V A, 4KL	ValRS_NcoI_Fwd	ValRS_SOE-EcoRI_Rv
V B, 4KL	ValRS_SOE-EcoRI_Fwd	ValRS_EcoRI_Rv

• Stored oligo stock and PCR setups in freezer.

2014-03-06

Thawed and ran PCRs created on 2014-03-04. Used 2K55 program.

2014-03-07

The original SOE'ing PCR protocol is at Template:SBB-Protocols_PCR3.

• Retrieved finished PCR products (total of four tubes) from 2014-03-06.
• Spun tubes for approximately 15 seconds.
• Setup 1% agarose gel with 10 wells and 1x TAE buffer solution.
• For each of the four PCR products, mixed the following in a new tube:
  • 1μL 6x loading dye
  • 6μL PCR product
• Stored original PCR product tubes in freezer.
• Spun new, PCR-dye mix tubes for approximately 10 seconds.
• Loaded one gel well for each PCR-dye mix.
• Started gel at approximately 13:15. Ended at approximately 13:40.
  • Gel ran at 150 volts.
• Visualized gel with long blue light (called "trans UV" on machine)

• Cut out my four bands and put into two 1.5mL tubes, labelled as follows:

combined 1.5mL tube label	PCR product 1 original tube label	PCR product 2 original tube label
VA+VB, 4KL	V A, 4KL	V B, 4KL
TATB, 4KL	T A, 4KL	T B, 4KL

Note that the two tubes each contain two distinct PCR products.

• Added 650μL ADB buffer to each tube.
• Melted bands by partially submerging tubes in a warm water bath.
• Stored the two tubes in freezer. These are the templates for the third (and last) SOE'ing PCR step.

2014-03-11

• Retrieved two tubes from freezer from 2014-03-07: VA+VB, 4KL and TATB, 4KL.
• Finished remainder of Zymo gel purification: Arking:JCAProtocols
  • Skipped adding of 250μL isopropanol, even though the tube TATB, 4KL contains DNA that is less than 300bp.
  • Tranferred to Zymo column, in collection tube.
  • Spun at full speed for one minute. Discarded waste.
  • Added 200μL Zymo wash buffer
  • Spun at full speed for 30 seconds. Discarded waste.
  • Again added 200μL Zymo wash buffer.
  • Again spun at full speed for 30 seconds. Discarded waste.
  • Spun at full speed for 2 minutes to dry.
  • Eluted DNA with 50μL ddH₂O into fresh Eppendorf tubes: 1 minute full speed spin.
• Retrieved oligo stock from freezer; created two 10μM dilutions with two oligos in each:

Mixed dilution tube label	Stock Oligo 1	Stock Oligo 2	ddH2O
T oligo dilute	ThrRS_NcoI_Fwd (1μL)	ThrRS_EcoRI_Rv (1μL)	18μL
V oligo dilute	ValRS_NcoI_Fwd (1μL)	ValRS_EcoRI_Rv (1μL)	18μL

• Setup PCR reactions:

PCR tube label	Mixed oligo dilution tube	Template tube	10x Expand buffer ("2")	2mM dNTP's	Expand polymerase ("1")
T_PCR, 4KL	T oligo dilute (2μL)	TATB, 4KL (24μL)	3.3μL	3.3μL	0.5μL
V_PCR, 4KL	V oligo dilute (2μL)	VA+VB, 4KL (24μL)	3.3μL	3.3μL	0.5μL

• Ran on 4K55 program.
• Discarded mixed oligo dilutions (T oligo dilute and V oligo dilute).
• After PCR completes, the products in tubes T_PCR, 4KL and V_PCR, 4KL correspond to pcrproduct in construction files.

2014-03-13

• Retrieved the two PCR products from 2014-03-11: T_PCR, 4KL and V_PCR, 4KL
• Ran analytical gel at 160 volts in the two right-most lanes:

DNA	Lane	ddH2O	6x loading dye
T_PCR, 4KL (2μL)	second from right-most	6μL	1μL
V_PCR, 4KL (2μL)	right-most	6μL	1μL

Started at 10:55 AM and ended 11:47 AM.

• Visualized gel under long blue:

• Stored tubes in freezer.

2014-03-18

• Performed regular Zymo cleanup of tubes T_PCR, 4KL and V_PCR, 4KL.
  • Used 180μL ADB buffer and eluted with 33μL ddH₂O.
• Setup 30μL digestion.
  • Placed both tubes on ice.
  • In each tube, added:

15μL	DNA
3μL	10x NEB buffer "2"
1.5μL	EcoRI
1.5μL	NcoI
9μL	ddH2O

• Started digestion at 11:00 AM. Ended 12:00 PM. Held at 37°C throughout.
• Performed Zymo cleanup with 30μL ddH₂O elution.
  • Labeled tubes: T dig, 4KL and V dig, 4KL
• Stored tubes in freezer.

2014-03-20

• Made 10-well 1% agarose gel with:

1 gram	agarose powder
100mL	1x TAE buffer
10μL	GelGreen dye

• Retrieved tubes T dig, 4KL and V dig, 4KL from freezer.
• Prepared tubes for loading into gel:
  • 30μL digestion product
  • 3μL 6x loading dye
• Loaded 3 wells for each tube (6 wells total). Each well was loaded with 10μL of contents.
• Ran gel at 155 volts
  • Started 11:00 AM
  • Stopped 11:48 AM
• Cut bands as shown below:

• Sorted bands into two tubes (one for each DNA)
• Melted bands in 650μL ADB buffer at 51°C
• Performed regular zymo cleanup
  • Eluted each DNA into 15μL ddH₂O
• Stored tubes in freezer

Cleaned-up digest tube label	Original digest tube label
T pure, 4KL	T dig, 4KL
V pure, 4KL	V dig, 4KL

2014-04-01

• Retrieved mini-prepped vector plasmid from freezer.
  • tube label: KL 4, KL pBad OLED grp 4
• Prepared two identical 30μL digests (referred to as "1" and "2") on ice:
  • 15μL plasmid
  • 3μL 10x NEB Buffer "2"
  • 9μL ddH₂O
  • 1.5μL EcoRI
  • 1.5μL NcoI
• Incubated at 37°C
  • Started digests "1" and "2" at 10:17 AM and 10:31 AM, respectively.
  • Stopped both at 11:50 AM
• Made 8-well gel
  • 0.5g agarose powder
  • 50mL 1x TAE buffer
  • 5μL GelGreen dye
• Added 3μL 6x gel loading dye to each completed digest tube.

Lane	Contents
1	5μL ladder
2, 3	15μL per well, digest "2"
4, 5	15μL per well, digest "1"
6, 7, 8	Tae's stuff

• Ran gel at 160 volts
  • Started at 12:00 PM
  • Stopped at 12:30 PM
• Imaged under long blue light:

• Cut out my 8 bands (four large-fragment bands; four small-fragment bands)
  • Placed all large-fragement bands into new tube labeled KL big plasmid
  • Placed all small-fragment bands into new tube labeled KL small plasmid.
• Added 600μL ADB buffer to each tube.
• Heated tubes to 55°C and vortexed to melt bands.
• Placed tubes in freezer.

Note: I expected one 4kb fragment and possibly one 40bp fragment from the digest gel purification. However, I could not clearly determine the sizes of the bands due to insufficient separation in the gel. Therefore I saved both sizes of fragments.

2014-04-03

• Retrieved vector plasmid digest fragments KL big plasmid and KL small plasmid from freezer.
• Finished Zymo cleanup on each tube; eluted with 30μL ddH₂O.
• Created 10-well gel consisting of:
  • 5g agarose powder
  • 50mL distilled H₂O (this was accidental)
  • 5mL 10x TAE buffer
  • 5μL GelGreen dye
• Loaded wells as follows:

Lane	Contents
1	5μL ladder
2	2μL KL big plasmid + 6μL ddH2O + 1μL loading dye
3	2μL KL small plasmid + 6μL ddH2O + 1μL loading dye

• Ran gel at 160 volts
  • Started 10:58
  • Stopped at 11:55
• Imaged gel under trans UV light:

• Stored big and small plasmid digest fragments in freezer with labels KL big plasmid and KL small plasmid.

Note: I still could not clearly read the sizes of the large and small digest fragments. Upon comparison with my teammates' digest gels, I determined that the large fragment should be correct. The small fragment tube KL small plasmid will be discarded.

It also seems a bit odd that the large fragment lane in the gel shows several fainter bands.

2014-04-08

• Retrieved plasmid digest KL big plasmid and insert digests T pure, 4KL and V pure, 4KL from freezer.
• Setup ligation two ligation reactions, one for each insert:

6.5μL	ddH2O
1μL	T4 DNA ligase buffer
1μL	plasmid digest KL big plasmid
1μL	insert digest T pure, 4KL or V pure, 4KL
0.5μL	T4 DNA ligase

• Mixed contents, let sit on benchtop.
  • Started 10:14
  • Ended 10:44
• Began transformation:
  • Obtained one 200μL cell aliquot; kept on ice.
  • Added 30μL KCM to cell aliquot.
  • To each finished ligation tube, added 70μL cell mixture; discarded cell mixture.
  • Let the two tubes sit on ice for 10 minutes.
  • Heat shocked between 46°C and 50°C (used IncuBlock, which does not have precise temperature control)
    • Started 10:55
    • Ended 10:58
  • Added 100μL LB media, put on IncuBlock to incubate
    • Began 11:17
    • Ended 12:11
    • Temperature varied between 36°C and 41°C.
• Plated cells onto ampicillin plates:
  • 100μL of T construct on plate T 4KL
  • Over 100μL of V construct on plate V 4KL
• Discarded empty ligation reaction tubes
• Put plates into 37°C incubator for overnight incubation
  • Started 12:23

Notes: If the colonies don't turn out well, then controls will be run. Negative control: transform the digested vector plasmid alone (should not get any colonies). Positive control: transform original vector plasmid (should get colonies).

2014-04-10

• Retrieved T construct and V construct plates. Got no colonies. Discarded plates.
• Running positive control
  • Obtained 200μL cell aliquot
  • Added 30μL KCM
  • Mixed 70μL cell mixture and 2μL pBAD OLED plasmid (borrowed from Chris Coates) into new tube.
  • Heat shocked at 42°C for 3 minutes
  • Added 100μL LB media
  • Incubated at 37°C starting 11:15, ending 12:10
  • Plated

2014-04-11

• Retrieved positive control plate. Imaged under EPI white light:

• Sealed plate with parafilm and stored in refrigerator.

Note: positive control worked. So next step is to retry ligation with equal mass of insert and vector (i.e. equal brightness of gel bands).

2014-04-15

• Retrieved positive control plate.
• Put 4mL LB/ampicillin liquid media in each of two new test tubes.
  • Labeled tubes 4KL pick 1 and 4KL pick 2
• Picked two colonies and put in respective tubes:

• Stored test tubes in warm room.
• Re-sealed plate with parafilm and stored in refrigerator.
• Conducted inventory check:

label	contents
TA, TB, VA, VB	SOE PCR intermediate products for T and V constructs
T pure, V pure	T and V construct digests
T pdt, V pdt	unknown (!)
4KL 2014-04-10 (+)-control	colonies with pBAD OLED plasmid (originally miniprepped by Chris Coates)

2014-04-17

• Retrieved test tubes 4KL pick 1 and 4KL pick 2 from refrigerator.
  • Tubes are cloudy
• Miniprepped 2mL of cells from each tube and obtained two 50μL tubes of DNA.
  • Labeled plasmid DNA tubes: 4KL OLED pBad colony 1 and 4KL pBad OLED colony 2
• Donated test tubes of cells to Christy Truong